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Supplemental Material Seroprevalence of SARS-CoV-2 antibodies among Forcibly Displaced Myanmar Nationals in Cox's Bazar, Bangladesh 2020: a population-based cross-sectional study Mahbubur Rahman Institute of Epidemiology Disease Control and Research (IEDCR) DhakaBangladesh Samsad Rabbani Khan Institute of Epidemiology Disease Control and Research (IEDCR) DhakaBangladesh A S M Alamgir Institute of Epidemiology Disease Control and Research (IEDCR) DhakaBangladesh David S Kennedy Ferdous Hakim Research and Publication World Health OrganizationDhakaBangladesh Egmond Samir Evers Nawroz Afreen Institute of Epidemiology Disease Control and Research (IEDCR) DhakaBangladesh Ahmed Nawsher Alam Institute of Epidemiology Disease Control and Research (IEDCR) DhakaBangladesh MdSahidul Islam Research and Publication World Health OrganizationDhakaBangladesh Debashish Paul Rijwan Bhuiyan Coordination Center Ministry of Health and Family Welfare Cox's, BazarBangladesh Raisul Islam Adneen Moureen IEDCR Field Laboratory, World Health Organization Cox's, BazarBangladesh M Salimuzzaman Institute of Epidemiology Disease Control and Research (IEDCR) DhakaBangladesh Mallick Masum Billah Institute of Epidemiology Disease Control and Research (IEDCR) DhakaBangladesh Ahmed Raihan Institute of Epidemiology Disease Control and Research (IEDCR) DhakaBangladesh Sharif Mst Khaleda Akter Research and Publication World Health OrganizationDhakaBangladesh Sharmin Sultana Institute of Epidemiology Disease Control and Research (IEDCR) DhakaBangladesh Manjur Hossain Khan Institute of Epidemiology Disease Control and Research (IEDCR) DhakaBangladesh Kai Von Harbou Mohammad Mostafa Zaman Research and Publication World Health OrganizationDhakaBangladesh Tahmina Shirin Institute of Epidemiology Disease Control and Research (IEDCR) DhakaBangladesh Sabrina Meerjady Flora Directorate General of Health Services (DGHS) DhakaBangladesh Supplemental Material Seroprevalence of SARS-CoV-2 antibodies among Forcibly Displaced Myanmar Nationals in Cox's Bazar, Bangladesh 2020: a population-based cross-sectional study 10.1136/bmjopen-2022-0666532 WHO Emergency Sub-Office, World Health Organization, Cox's Bazar, Bangladesh Weighted and test adjusted prevalence Primarily we used WANTAI total antibody SARS-CoV-2 ELISA kits to estimate the seroprevalence, which has some limitations and could result in false positives and false negatives estimates. In order to validate the test result, we used Kantaro IgG ELISA test kit. We took the following approach to adjust the seroprevalence of WANTAI by KANTARO kits. Let = ( + ) represent the population prevalence of antibodies to SARS-CoV-2 and let = ( + ) be the proportion of participants who test positive in WANTAI. Let, = ( + | + ) be the sensitivity of the test (the probability of testing positive in WANTAI given an individual is positive in KANTARO), and let, = ( − ) be the specificity of the test (the probability of testing negative in WANTAI given an individual is negative in KANTARO). We can write the expected fraction of positive tests, p, as follows: = + (1 − )(1 − ) Therefore, the test adjusted estimated seroprevalence is expressed using following formula [1]- = −(1− ) (1− ) …………………………………………….. (1) Of 3,446 total samples in our study, we found 2,090 positives for WANTAI. Again, we used KANTARO quantitative test to those WANTAI positive samples and we found 290 negatives. Since we didn't test any negative WANTAI samples, and we assumed that all negative samples in WANTAI are also negative in KANTARO. A confusion matrix is displayed below- Bootstrapping for confidence interval We calculated confidence intervals on our weighted sample prevalence estimates based on a non-parametric highest density interval (HDI) bootstrap [2]. Our bootstrap procedure resamples data from our actual datasets for sensitivity, specificity, and prevalence. We use the following steps-▪ First, we draw a single bootstrap sample (i.e. with replacement) from the beta distribution of the sensitivity data. For this sample, we calculate the mean value of sensitivity. Let represent the value for the jth iteration of this procedure. ▪ First, we draw a single bootstrap sample (i.e. with replacement) from the beta distribution of the specificity data. For this sample, we calculate the mean value of specificity. Let represent the value for the jth iteration of this procedure. ▪ For our weighted estimate, we calculate weighted prevalence of WANTAI in the bootstrap sample . Let represent this value for the jth iteration of this procedure. ▪ We then calculate the test adjusted prevalence of antibodies to SARS-CoV-2 for the jth bootstrap sample using the equation (1) ▪ Repeat the steps above for j=1...1,000,000 bootstrap samples. ▪ In our tables, we report the highest density interval (HDI) of the distributions of over the 1,000,000 bootstrap samples as the lower and upper ends of the 95% bootstrap confidence intervals. The confidence intervals for the test-adjusted estimates were derived using bootstrap sampling, with 1,000,000 parametric bootstrap samples for each estimate, using the "adjPrevSensSpecCI" function of the "bootComb" R package. References Figure 2 : 2Test adjusted over weighted seroprevalence among respondent occupation, FDMN population, Bangladesh December 2020 Note: Blue dot represents the prevalence and error bar indicating confidence interval. Table : :Confusion Matrix between WANTAI and KANTARO where 'p' denoting the positive and 'n' denoting the negative test result.Sensitivity, = 1800 1800+0 = 1.00, and specificity, = 1356 290+1356 = 0.8238 Therefore, overall seroprevalence, = 0.574−(1−0.8238) 1−(1−0.8238) = 0.4829 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) BMJ Open doi: 10.1136/bmjopen-2022-066653 :e066653. 1 . 1Chimeddorj B, Mandakh U, Le LV, Bayartsogt B, Deleg Z, Enebish O, Altanbayar O, Magvan B, Gantumur A, Byambaa O, Enebish G. SARS-CoV-2 seroprevalence in Mongolia: Results from a national population survey. The Lancet Regional Health-Western Pacific. 2021 Dec 1;17:100317. 2. Bendavid E, Mulaney B, Sood N, Shah S, Bromley-Dulfano R, Lai C, Weissberg Z, Saavedra-Walker R, Tedrow J, Bogan A, Kupiec T. Covid-19 antibody seroprevalence in santa clara county, Supplemental Table 1. Responses at household, individual and laboratory testing levels, FDMN seroprevalence survey, Bangladesh December 2020 Note: FDMN, Forcibly Displaced Myanmar Nationals *Not included as sample as they do not qualify 1 as sample for the Survey †Household response rate (%)=[RCx100]/[RC+VH+RefI] ‡Individual response rate (%)=[Cx100]/[C+II+U+R+RI] §Laboratory testing response rate (%)=[Cx100]/[C+Rej] ¶ Overall response rate (%)=HRR*IRR*LTRR/(100*100) 1 The American Association for Public Opinion Research. 2016. Standard Definitions: Final Dispositions of Case Codes and Outcome Rates for Surveys. 9th edition. AAPOR. Supplemental Table 2: test adjusted over weighted SARS-CoV-2 antibody prevalence by sex across covariates, FDMN population, Bangladesh 2020 FDMN, Forcibly Displaced Myanmar Nationals *P<0.05 † Among those aged 6 years or more ‡ Among those aged 18 years or more BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Supplemental Figure 1. FDMN camps in Ukhiya and Teknaf of Cox's Bazar district, Bangladesh December 2020 Note: FDMN, Forcibly Displaced Myanmar Nationalscalifornia. International journal of epidemiology. 2021 Apr;50(2):410-9. 3. Henrion MY. bootComb-an R package to derive confidence intervals for combinations of independent parameter estimates. BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) BMJ Open doi: 10.1136/bmjopen-2022-066653 :e066653. Responses All n % At household level Roster completed (RC) 4398 70.9 Vacant house/ No HH respondent available (VH) 152 2.5 House not found* 1112 17.9 Refused interview (RefI) 540 8.7 Total 6202 100 Household response rate (HRR) † 86.4 At individual level Completed (C) 3678 83.6 No one eligible* 181 4.1 Unavailable (U) 297 6.8 Refused (R) 242 5.5 Total 4398 100 Individual response rate (IRR) ‡ 87.2 At laboratory testing level Laboratory testing completed (C) 3446 93.7 Sample rejected by laboratory (Rej) 232 6.3 Total 3678 100 Laboratory testing response rate (LTRR) § 93.7 Overall response rate ¶ 70.6 Available from: https://www.aapor.org/AAPOR_Main/media/publications/Standard- Definitions20169theditionfinal.pdf (Accessed on 3 September 2019) BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) BMJ Open doi: 10.1136/bmjopen-2022-066653 :e066653. Variable Weighted prevalence, % (95% CI) Weighted & kit adjusted prevalence, % (95% CI) Overall Female Male Overall Female Male Age, years All 57.4 (55.1-59.7) 57.4 (54.2-60.6) 57.4 (54.2-60.6) 48.3 (45.3-51.4) 48.3 (44.2-52.3) 48.3 (44.2-52.3) 1-17 49.4 (45.6-53.2) 48.6 (43.0-54.3) 50.2 (45.1-55.2) 38.6 (33.8-43.4) 37.6 (30.7-44.7) 39.5 (33.2-45.8) 18-94 65.5 (63.2-67.8)* 65.4 (62.4-68.2)* 65.7 (62.0-69.3)* 58.1 (55.2-61.1)* 58.0 (54.3-61.6)* 58.4 (53.8-62.9)* 18-49 64.9 (62.3-67.4) 65.1 (61.9-68.1) 64.7 (60.4-68.8) 57.4 (54.1-60.6) 57.6 (53.6-61.4) 57.1 (51.9-62.3) 50-94 68.5 (63.0-73.5)* 67.2 (59.5-74.2)* 69.5 (61.7-76.3)* 61.8 (55.1-68.0)* 60.2 (50.9-68.9)* 63.0 (53.7-71.4)* Education † (n=3,160) No formal education 61.7 (58.8-64.5) 61.4 (57.9-64.8) 62.1 (57.0-67.0) 53.5 (49.8-57.1) 53.1 (48.8-57.4) 54.0 (47.7-60.1) Primary above 61.8 (58.3-65.2) 59.0 (53.0-64.8) 63.8 (59.5-67.9) 53.6 (49.2-57.9) 50.2 (42.9-57.4) 56.1 (50.8-61.1) Camp location Teknaf 54.8 (48.9-60.6) 59.7 (51.0-67.7) 49.4 (41.3-57.6) 45.1 (37.8-52.2) 51.1 (40.6-60.9) 38.6 (28.7-48.7) Ukhiya 58.0 (55.5-60.4) 56.9 (53.5-60.3) 59.1 (55.5-62.5)* 49.0 (45.8-52.2) 47.7 (43.3-51.9) 50.4 (45.8-54.6)* Smoking ‡ (n=2,392) No 67.5 (64.9-70.0) 65.3 (62.2-68.2) 71.8 (67.1-76.1) 60.5 (57.3-63.7) 57.9 (54.0-61.5) 65.8 (60.1-71.2) Yes 59.1 (53.9-64.2)* 66.5 (55.6-75.9) 57.5 (51.6-63.2)* 50.4 (43.9-56.6)* 59.3 (46.5-71.2) 48.4 (41.2-55.5)* BCG vaccination No 58.8 (52.7-64.7) 58.9 (50.0-67.2) 58.7 (50.2-66.7) 50.0 (42.5-57.3) 50.1 (39.4-60.4) 49.9 (39.5-59.7) Yes 57.2 (54.7-59.6) 57.2 (53.8-60.6) 57.2 (53.7-60.6) 48.0 (44.8-51.2) 48.0 (43.8-52.4) 48.0 (43.6-52.3) Any relevant symptoms No symptom 55.3 (52.3-58.2) 53.3 (49.1-57.4) 57.3 (53.2-61.4) 45.7 (41.9-49.5) 43.3 (38.1-48.5) 48.2 (43.1-53.3) Any symptom 61.2 (57.7-64.6)* 64.6 (59.9-69.1)* 57.5 (52.3-62.5) 52.9 (48.5-57.2)* 57.0 (51.2-62.6)* 48.4 (42.0-54.6) Any comorbidity No comorbidity 56.5 (54.0-58.9) 56.1 (52.6-59.5) 56.9 (53.5-60.2) 47.2 (43.9-50.4) 46.7 (42.3-51.0) 47.7 (43.4-51.9) Any comorbidity 65.2 (59.2-70.7)* 67.3 (59.8-74.0)* 62.4 (52.4-71.4) 57.8 (50.4-64.5)* 60.3 (51.4-68.7)* 54.4 (42.4-65.5) Note: BMJ Open doi: 10.1136/bmjopen-2022-066653 :e066653. 12 2022; BMJ Open , et al. Rahman M

Tuberous Sclerosis Complex: Prenatal Diagnosis Using Ultrasound and Magnetic Resonance Imaging-A Report of Two Cases 2023 Eduardo Félix Martins Santana Department of Woman Health Israeli Faculty of Health Sciences Albert Einstein São PauloBrazil Ana Maria Faria Esteves Service of Gynecology and Obstetrics Municipal University Hospital of São Bernardo do Campo São Bernardo do CampoBrazil Daniella Guerra Delorenzo Service of Gynecology and Obstetrics Municipal University Hospital of São Bernardo do Campo São Bernardo do CampoBrazil Celso Hygino Department of Fetal Medicine Clínica de Diagnóstico por Imagem (CDPI) Rio de JaneiroBrazil Heron Werner Department of Fetal Medicine Clínica de Diagnóstico por Imagem (CDPI) Rio de JaneiroBrazil Edward Araujo Júnior Department of Obstetrics School of Medicine Federal University of São Paulo (EPM-UNIFESP) 156 apto. 111 Torre Vitoria05089-030Paulista, São Paulo, São PauloCEPBrazil Indian, Brazil PhDAraujo Júnior araujojred@terra.com.br. Rua Belchior Tuberous Sclerosis Complex: Prenatal Diagnosis Using Ultrasound and Magnetic Resonance Imaging-A Report of Two Cases J Radiol Imaging 33202310.1055/s-0042-1758196Address for correspondence Edward Introduction Prenatal diagnosis of tuberous sclerosis complex (TSC) can show rhabdomyomas, subependymal nodules, cortical tubers, and renal angiomyolipomas, which are the major features of TSC. [1][2][3] Ultrasound, being a noninvasive and repeatable examination, can be used to detect multiple nodules or a hyperechogenic myocardium, usually from the 20th week of pregnancy. 3,4 Currently, fetal magnetic resonance imaging (MRI) can identify brain lesions and sometimes even renal lesions, thus enabling parental counselling and postnatal management with target therapy. 5 Subependymal nodules and cortical tubers are T1-hyperintense and T2-hypointense, the former being more easily identified on fetal imaging. 5,6 However, subcortical lesions are more commonly detected in postnatal MRI. 7 In this article, we present two cases of prenatal diagnosis of TSC using both ultrasound and MRI. Abstract Tuberous sclerosis complex (TSC) is a multiple system neurocutaneous syndrome with a genetic disorder caused by different mutations in TSC1 or TSC2. Usually, TSC causes tumors in the heart, brain, kidneys, eyes, and lungs. However, tumors can also develop in any other organs. The prenatal diagnosis of TCS is based on the identification of fetal cardiac tumors by ultrasound and brain subependymal nodules, usually identified by fetal magnetic resonance imaging (MRI). We present two case reports of the prenatal diagnosis of TCS using both ultrasound and MRI, which were confirmed by clinical and radiological methods in the postnatal period accordingly. showed a typical brain subependymal nodule (►Fig. 2). She underwent a cesarean section due to breech presentation at 39 weeks' gestation and delivered a male newborn weighing 3,250 g. Apgar scores at the 1st and 5th minutes were 9 and 10, respectively. Postnatal clinical and imaging diagnoses confirmed the same findings seen in prenatal examinations. Genetic tests have not been performed to date. Case 2 This case involved a G1-P0 32-year-old pregnant woman, with no family history of TSC. Ultrasound at 32 weeks of gestational age showed an echogenic mass in the fetal heart, consistent with a rhabdomyoma, and hyperechoic lesions in the fetal brain (►Fig. 3). Fetal MRI at 35 weeks of gestational age showed typical brain periventricular and subependymal nodules as well as a cardiac rhabdomyoma (►Fig. 4). She underwent a cesarean section at 38 weeks' gestation due to low placental implantation and delivered a male weighing 3,100 g. Apgar scores at the 1st and 5th minutes were 9 and 10, respectively. Postnatal clinical and prenatal imaging diagnoses confirmed the same findings. Genetic tests have not been performed to date. Discussion After the diagnosis of TSC, a multidisciplinary approach, depending on the most affected systems or organs, is required. Testing for subclinical features with clinical examination, MRI, echocardiogram, electrocardiogram, chest computed tomography (CT), and blood workup needs to be carried out accordingly. 8 Most rhabdomyomas are benign, but they can lead to the obstruction of the cardiac flow depending on the size or location. Sometime this may be the only feature present in the prenatal period, with others developing later in life. They also have a high rate of spontaneous regression up to the age of 5 years. 5 Prenatal diagnosis allows the children to be tested even before the onset of epilepsy symptoms, leading to easier surveillance or targeted treatment. 5 Early findings can make parents aware of the risk of development of autism spectrum disorders and more specifically, of the early signs of the disorder. 9 Some brain lesions that suggest neurodevelopmental diseases can be detected on a third trimester ultrasound, despite this not being a routine exam for low-risk pregnancies in most countries. 10 Signs of renal impairment during the prenatal period are rare, and the reported cases showed cystic formations (unilateral or even diffuse polycystic involvement), accompanied by other findings in the fetal brain or heart. 5 In our case reports, we observed the brain periventricular and subependymal nodules on fetal MRI. Sonigo et al 11 assessed eight fetuses with multiple cardiac tumors, five of which showed hyperintense subependymal and cortical nodules on Tl-weighted images. They concluded that MRI was a valuable tool for diagnosing TSC in fetuses with multiple cardiac tumors. Age does not compromise a good imaging assessment, but it facilitates the diagnosis of TSC by MRI. Most subependymal nodules detected in the fetus and neonate are noncalcified. If TSC is clinically suspected in the first 3 months of life, imaging should not be delayed. TSC nodules in fetus and neonates are hyperintense on T1-weighted images and hypointense on T2-weighted images. The low degree of myelination helps to identify white matter abnormalities, which become less visible as myelination progresses. Since nodules calcify in childhood, they are more easily detected on CT and MRI susceptibility weighted images. 12 Chen et al 13 demonstrated that ultrafast MRI can detect small subependymal nodules in fetuses with cardiac tumors. The accuracy of ultrasound in identifying brain periventricular/ cortical nodules was low. However, we identified a typical brain hyperechogenic cortical nodule on ultrasound in case 2. Conclusion In summary, we presented two case reports of prenatal diagnosis of TSC using both ultrasound and MRI. Ultrasound is the first-line screening method for TSC by the identification of cardiac tumors, and MRI is important for identifying typical brain subependymal nodules accordingly. Fig. 1 1Case 1. Ultrasound image showing a cardiac rhabdomyoma (arrow) in the right atrium (26 weeks). Fig. 2 2Case 1. (A) Axial T2-weighted magnetic resonance image showing a brain subependymal nodule (arrow). (B) Sagittal T2weighted image showing a typical brain subependymal nodule (arrow). Fig. 3 3Case 2. (A) Ultrasound image showing a cardiac rhabdomyoma (arrow) in the interventricular septum (35 weeks). (B) Ultrasound image showing a brain cortical nodule (arrow). This case involved a primiparous (G1-P0) 35-year-old pregnant woman with no family history of TSC. Ultrasound at 24 weeks of gestational age showed an echogenic mass in the left ventricle of the fetal heart, consistent with a rhabdomyoma (►Fig. 1). Fetal MRI at 26 weeks of gestational ageCase Report Case 1 Keywords ► prenatal diagnosis ► tuberous sclerosis complex ► ultrasound ► magnetic resonance imaging 10 Mongrain V, van Doesburg NH, Rypens F, et al. A case report of severe tuberous sclerosis complex detected in utero and linked to a novel duplication in the TSC2 gene. BMC Neurol 2020;20(01): 324 11 Sonigo P, Elmaleh A, Fermont L, Delezoíde AL, Mirlesse V, Brunelle F. Prenatal MRI diagnosis of fetal cerebral tuberous sclerosis. Pediatr Radiol 1996;26(01):1-4 12 Baron Y, Barkovich AJ. MR imaging of tuberous sclerosis in neonates and young infants. AJNR Am J Neuroradiol 1999;20(05): 907-916 13 Chen CP, Liu YP, Huang JK, et al. Contribution of ultrafast magnetic resonance imaging in prenatal diagnosis of sonographically undetected cerebral tuberous sclerosis associated with cardiac rhabdomyomas. Prenat Diagn 2005;25(06):523-524 Indian Journal of Radiology and Imaging Vol. 33 No. 1/2023 © 2022. Indian Radiological Association. All rights reserved. Prenatal Diagnosis of Tuberous Sclerosis Santana et al. Indian Journal of Radiology and Imaging Vol. 33 No. 1/2023 © 2022. Indian Radiological Association. All rights reserved. Prenatal Diagnosis of Tuberous Sclerosis Santana et al. Conflict of InterestNone declared. Prenatal diagnosis of tuberous sclerosis complex using fetal ultrasonography and magnetic resonance imaging and genetic testing. C C Wang, C Y Wang, Y J Lai, T Y Chang, H Y Su, Taiwan J Obstet Gynecol. 5701Wang CC, Wang CY, Lai YJ, Chang TY, Su HY. Prenatal diagnosis of tuberous sclerosis complex using fetal ultrasonography and mag- netic resonance imaging and genetic testing. Taiwan J Obstet Gynecol 2018;57(01):163-165 Maternal and fetal tuberous sclerosis: do we know enough as an obstetrician?. N Sharma, S Sharma, J L Thiek, S S Ahanthem, A Kalita, D Lynser, J Reprod Infertil. 1802Sharma N, Sharma S, Thiek JL, Ahanthem SS, Kalita A, Lynser D. Maternal and fetal tuberous sclerosis: do we know enough as an obstetrician? J Reprod Infertil 2017;18(02):257-260 Maternal and fetal tuberous sclerosis complex: a case report questioning clinical approach. Ö S Onay, A Ç Sağlık, P Köşger, Onay ÖS, Sağlık AÇ, Köşger P, et al. Maternal and fetal tuberous sclerosis complex: a case report questioning clinical approach. . Turk J Pediatr. 6202Turk J Pediatr 2020;62(02):332-337 . G L Cotaina, G E Lázaro, M I Jiménez, C R Savirón, P D Lerma, Diagnosis of cardiac rhabdomyoma in the first trimester of pregnanCotaina GL, Lázaro GE, Jiménez MI, Savirón CR, Lerma PD. [Diag- nosis of cardiac rhabdomyoma in the first trimester of pregnan- . Ginecol Obstet Mex. 8403Ginecol Obstet Mex 2016;84(03):180-185 Diagnosis of tuberous sclerosis complex in the fetus. P Dragoumi, F O&apos;callaghan, D I Zafeiriou, Eur J Paediatr Neurol. 2206Dragoumi P, O'Callaghan F, Zafeiriou DI. Diagnosis of tuberous sclerosis complex in the fetus. Eur J Paediatr Neurol 2018;22(06): 1027-1034 Fetal and maternal manifestations of tuberous sclerosis complex: value of fetal MRI. R Goel, N Aggarwal, M E Lemmon, T Bosemani, Neuroradiol J. 2901Goel R, Aggarwal N, Lemmon ME, Bosemani T. Fetal and maternal manifestations of tuberous sclerosis complex: value of fetal MRI. Neuroradiol J 2016;29(01):57-60 Prenatal and postnatal diagnosis of rhabdomyomas and tuberous sclerosis complex by ultrafast and standard MRI. Y Zhou, S Z Dong, Y M Zhong, A M Sun, Indian J Pediatr. 8509Zhou Y, Dong SZ, Zhong YM, Sun AM. Prenatal and postnatal diagnosis of rhabdomyomas and tuberous sclerosis complex by ultrafast and standard MRI. Indian J Pediatr 2018;85(09): 729-737 Diagnosis of tuberous sclerosis complex in a patient referred for uncontrolled hypertension and renal dysfunction: a case highlighting the importance of proper diagnostic work-up of hypertensive patients. P A Sarafidis, A Bikos, C Loutradis, J Hypertens. 3510Sarafidis PA, Bikos A, Loutradis C, et al. Diagnosis of tuberous sclerosis complex in a patient referred for uncontrolled hyper- tension and renal dysfunction: a case highlighting the importance of proper diagnostic work-up of hypertensive patients. J Hyper- tens 2017;35(10):2109-2114 Genetic syndromes, maternal diseases and antenatal factors associated with autism spectrum disorders (ASD). A Ornoy, L Weinstein-Fudim, Z Ergaz, Front Neurosci. 10316Ornoy A, Weinstein-Fudim L, Ergaz Z. Genetic syndromes, mater- nal diseases and antenatal factors associated with autism spec- trum disorders (ASD). Front Neurosci 2016;10:316 Case 2 (A) Axial T2-weighted magnetic resonance image (half Fourier single-shot turbo spin-echo [HASTE]) at 35 weeks of gestational age showing a brain periventricular low-signal-intensity nodule (arrow). (B) Coronal T2 image (HASTE) at 35 weeks of gestational age showing brain periventricular low-signal-intensity nodules (arrows). (C) Sagittal T2-weighted image (HASTE) at 35 weeks of gestational age showing a brain subependymal low-signal-intensity nodule (arrow). (D) Sagittal T1-weighted image at 35 weeks of gestational age showing a brain subependymal and cortical high-signal-intensity nodule (arrows). (E): Axial T2-weighted image showing a cardiac rhabdomyoma in the interventricular septum. Fig, 435 weeks) (arrowFig. 4 Case 2 (A) Axial T2-weighted magnetic resonance image (half Fourier single-shot turbo spin-echo [HASTE]) at 35 weeks of gestational age showing a brain periventricular low-signal-intensity nodule (arrow). (B) Coronal T2 image (HASTE) at 35 weeks of gestational age showing brain periventricular low-signal-intensity nodules (arrows). (C) Sagittal T2-weighted image (HASTE) at 35 weeks of gestational age showing a brain subependymal low-signal-intensity nodule (arrow). (D) Sagittal T1-weighted image at 35 weeks of gestational age showing a brain subependymal and cortical high-signal-intensity nodule (arrows). (E): Axial T2-weighted image showing a cardiac rhabdomyoma in the interventricular septum (35 weeks) (arrow).

Supplemental material Neutrophils protect against Staphylococcus aureus endocarditis progression independent of extracellular trap release Severien Meyers Center for Molecular and Vascular Biology Department of Cardiovascular Sciences KU Leuven LeuvenBelgium Marleen Lox Center for Molecular and Vascular Biology Department of Cardiovascular Sciences KU Leuven LeuvenBelgium Sirima Kraisin Center for Molecular and Vascular Biology Department of Cardiovascular Sciences KU Leuven LeuvenBelgium Laurens Liesenborghs Center for Molecular and Vascular Biology Department of Cardiovascular Sciences KU Leuven LeuvenBelgium Caroline P Martens Center for Molecular and Vascular Biology Department of Cardiovascular Sciences KU Leuven LeuvenBelgium Liesbeth Frederix Center for Molecular and Vascular Biology Department of Cardiovascular Sciences KU Leuven LeuvenBelgium Stijn Van Bruggen Center for Molecular and Vascular Biology Department of Cardiovascular Sciences KU Leuven LeuvenBelgium Marilena Crescente Department of Life Sciences Manchester Metropolitan University ManchesterUnited Kingdom Dominique Missiakas Department of Microbiology University of Chicago ChicagoILUSA Pieter Baatsen EM-Platform of the VIB Bio Imaging Core and VIB Center for Brain and Disease Research KU Leuven LeuvenBelgium Thomas Vanassche Center for Molecular and Vascular Biology Department of Cardiovascular Sciences KU Leuven LeuvenBelgium Peter Verhamme Center for Molecular and Vascular Biology Department of Cardiovascular Sciences KU Leuven LeuvenBelgium Kimberly Martinod Center for Molecular and Vascular Biology Department of Cardiovascular Sciences KU Leuven LeuvenBelgium Supplemental material Neutrophils protect against Staphylococcus aureus endocarditis progression independent of extracellular trap release Correspondence to: Kimberly Martinod, PhD. Department of Cardiovascular Sciences, KU Leuven. 49 Herestraat, bus 911, 3000 Leuven, Belgium. +32 56 24 62 65 kim.martinod@kuleuven.be Running title: NETs, staphylocoagulase and S. aureus endocarditisStaphylococcus aureusneutrophil extracellular trapsneutrophilsinfective endocarditiscoagulases Persistent ID / URL The following parameters were determined: a = 0.05 b = 0.20 From previous experiments, the MRSA strain of S. aureus (USA300) is expected to give endocarditis in 30% of the mice. A relevant increase to 70% is estimated to be relevant (previously observed in experiment 3). Hence, 58 mice will be needed, based on the Fisher exact test (29 mice in the neutrophil depletion group and 29 in the control group). From previous experiments, the clinical strain of S. aureus is expected to give endocarditis in 50% of the mice. A relevant increase to 90% is estimated to be relevant (previously observed in experiment 3). Hence, 48 mice will be needed, based on the Fisher exact test (24 mice in the neutrophil depletion group and 24 in the control group). From previous experiments, S. epidermidis is expected to give endocarditis in 5% of the mice. A relevant increase to 45% is estimated to be relevant (previously observed in P189/2017). Hence, 42 mice will be needed, based on the Fisher exact test (21 mice in the neutrophil depletion group and 21 in the control group). Significance was reached with a lower number than calculated. Therefore not all animals were used. DOI [to be added] Experiment 2: neutrophil-selective PAD4 knockout mice infected with different bacterial strains and via different delivery models ( Figure 5, Figure S4) Sample Size: Please explain how the sample size was decided Please provide details of any a prior sample size calculation, if done. The following parameters were determined: a = 0.05 b = 0.20 For the experiments with the clinical strain, 48 mice will be needed. From previous experiments, the clinical strain is expected to give endocarditis in 50% of the mice. A relevant increase to 90% is estimated to be relevant (previously observed in P189/2017). Hencef, 48 mice will be needed, based on the Fisher exact test (24 mice in the neutrophil depletion group and 24 in the control group). For the experiments with USA300 (tail vein), 56 mice will be needed. From previous experiments, USA300 is expected to give endocarditis in 20% of the mice. A relevant increase to 60% is estimated to be relevant (previously observed in P189/2017). Hence, 56 mice will be needed, based on the Fisher exact test (28 neutrophil specific PAD4 null mice and 28 PAD4 flowed mice). For the experiment with USA300 (catheter), 40 mice are needed: From pilot studies and P040/2017 the group in which the valves are damaged or inflamed is expected to develop endocarditis in 30% of the cases. A reduction to 5% is estimated to be relevant. Hence, 40 mice are needed, based on the Fisher exact test (20 neutrophil specific PAD4 null mice and 20 control mice) The following parameters were determined: a = 0.05 b = 0.20 From pilot studies and project P040/2017 the group in which the valves are inflamed is expected to develop endocarditis in 30% of the cases. A reduction to 5% is estimated to be relevant. Hence, 20 mice per group are needed (40 mice in total). No possible difference could be observed. Therefore the experiment was ceased earlier. The following parameters were determined: a = 0.05 b = 0.20 From pilot studies and project P040/2017 the group in which the valves are inflamed is expected to develop endocarditis in 30% of the cases. A reduction to 5% is estimated to be relevant. Hence, 20 mice per group are needed (40 mice in total). More mice were needed than initially calculated to have enough endocarditis lesions for quantification analyses. Due to seasonal breeding difficulties the sample size of 18 was not reached in the MRP8Cre + xPAD4 fl/fl group. The following parameters were determined: DOI [to be added] a = 0.05 b = 0.10 The highest proportion observed in other studies is 60% and a reduction of 50% is assumed to be relevant. The smallest relevant effect is D=0.5. Hence, according to the Fisher exact test with 90% power, 21 mice will be needed per group, with 2 genotypes tested, yielding a total of 42 mice. Significance was reached with a lower number than calculated. Therefore not all animals were used. DOI [to be added] Inclusion Criteria Male mice between 8 to 14 weeks old. Exclusion Criteria Mice were excluded from the experiment when they died during the surgical procedure (mostly directly after histamine infusion) or when the aortic valve region was not properly sectioned. Randomization Groups were randomized in different cages. So different groups were housed in the same cage. Blinding Blinding was performed by a third researcher. The researcher that performed the sectioning and analyses was blinded during the whole experiment. After microscopic analyses the researcher was unblinded. Supplemental methods Fluorescence image acquisition and quantitative image analyses Brown-Hopps Gram and MSB staining To determine the proportion of mice that developed endocarditis, we performed a Figure 3B. Figure 3D. Supplemental figures and figure legends Experiment 6 :Sample 6neutrophil isolation from neutrophil-selective PAD4 knockout mice (Figure 5A-Size: Please explain how the sample size was decided Please provide details of any a prior sample size calculation, if done. objective at a constant exposure time for each channel (30ms for DNA, 200ms for AF488 and 200ms for AF555). Using Image J software for analysis, we set the color balance for each image and kept this threshold constant within each experiment. Images were saved as a Tagged image file format for full resolution quantitative analysis.After acquiring the images, we determined the percentage of total fluorescence positive area and the percentage of fluorescence positive area within and outside the thrombus using Image J software (National Institutes of Health, Bethesda, Maryland). To this end, we subtracted the background at a threshold of 100 pixels and excluded folds and the aortic ring to avoid signal due to autofluorescence and background. Using the polygon tool, we selected the thrombus and the total area (within the thrombus and the vasculature surrounding the aortic ring) containing fluorescence signal. Afterwards, the total area was measured, the fluorescence positive area was determined by setting a threshold for each channel and the percentages of fluorescence positive area were calculated. The thresholds were again kept constant within an experiment. To determine the percentage of fluorescence positive area located outside the thrombus, we subtracted the thrombus area from the total area. These quantification analyses were performed on the section with the highest fluorescence signal and largest thrombus area.In addition to quantifying fluorescence stainings, we also quantified the dense infiltration size and thrombus volume on the Brown-Hopps stained sections using the polygon and area measurement tools. The infiltrate size was calculated on the section with the largest infiltrate and the thrombus volume on each section containing a thrombus on one of the 15 consecutive slides. To calculate the thrombus volume, we added up the areas of all the sections containing a thrombus, multiplied them by the thickness of the section and by the number of consecutive slides. Figure S1 : S1Quantitative analyses of components of the coagulation and immunological defense system in IE or sterile thrombi. A-F, Quantifications of thrombus volume (A) and fibrin inside the thrombus (F) on brightfield images, and neutrophil-specific marker Ly6G located outside the thrombus (B), and von Willebrand factor (VWF, C), platelets CD41 (D) and fibrinogen (E) inside the thrombus on fluorescence images of infected vegetations (red, n=11) compared to sterile thrombi (orange, n=7). For the quantification of fibrin on the MSB stained images, 5 sterile thrombi were included. Median and interquartile range are represented, and statistical significance was evaluated by a Mann-Whitney test. Figure S2 : S2Flow cytometry confirms significant neutrophil depletion. A-F, Flow cytometric analysis of blood samples before (day -1) and after antibody injection (day 1) from isotype control IgG2a-injected (n = 21, 20) (A-C) or neutrophil-depleted (anti-Ly6G, n = 24, 20) (D-F) mice after infection with S. aureus USA300 and the endocarditis surgery. The following markers were identified: CD45+ (leukocytes), Ly6G+/Ly6B+ (neutrophils) and Ly6G-/Ly6B+ (neutrophils and monocytes). Each dot represents a single animal. Mann-Whitney tests were performed. Figure S3 : S3Neutrophil depleted mice contain less evidence of infiltrating neutrophils and released neutrophil extracellular traps (NETs). Histological characterization of infected vegetations, injected with S. aureus USA300, from isotype control IgG2a-treated (n = 4, A) and neutrophil depleted (anti-Ly6G) mice (n = 12, B). A-B, Fluorescence staining of citrullinated histone H3 (H3Cit, green) and myeloperoxidase (MPO, red), bacteria (green) and Ly6G (neutrophils, red), CD41 (platelets, red) and fibrinogen (green), and von Willebrand factor (VWF). DNA is identified by Hoechst 33342 staining, depicted in blue. Scale bars equal 200 μm. C-J, Corresponding quantifications of MPO (C), H3Cit (G) and Ly6G (D), and bacteria (H), CD41 (E), fibrinogen (I) and VWF (F) inside the thrombus of isotype control (black, n = 4)and neutrophil depleted mice (gray, n = 12). J, Thrombus volume calculations. Significance was determined by Man-Whitney test (C-J). Figure S4 :Figure S5 : S4S5PAD4-mediated NET formation does not protect against infective endocarditis in additional models of infective endocarditis. A-F, The impact of NETs impairment was evaluated at day one in mice receiving S. aureus USA300 (A-C) via its tail vein; and in a more severe model whereby S. aureus USA300 was infused via the catheter (D-F). Proportion of mice that developed inflammation-induced endocarditis (A, D) or sterile thrombi (B, E) at day one in neutrophil-specific PAD4 knockout mice compared to control mice. Bacteremia levels at endpoint with corresponding median (interquartile range) in one day tail vein (n = 24, 17, C) and one day catheter delivery model ( n = 15, 10, F). Fisher's Exact (A-B, D-E) and Mann-Whitney test (C, F). Infected vegetations in mice with impaired NET formation do not differ from control vegetations. A-D, The following neutrophil-and coagulation-specific markers were visualized in control (PAD4 fl/fl , n = 11) and neutrophil-selective PAD4 knockout mice (MRP8Cre + x PAD4 fl/fl , n = 8) infected with the clinical S. aureus strain at day three: myeloperoxidase (MPO, red) and citrullinated histone H3 (H3Cit, green) (A); S. aureus (green) and neutrophil-specific marker Ly6G (red) (B); platelet CD41 (green) and fibrinogen (red) (C); and von Willebrand factor (VWF, green) (D). DNA was identified by Hoechst 33342 staining, depicted in blue. Scale bars represent 200 μm. E-K, Quantifications of MPO (E), H3Cit (I), S. aureus (F), Ly6G (J) and VWF (H), platelet CD41 (G) and fibrinogen inside the thrombus (K) in control (black, n = 11) and neutrophil-selective PAD4 knockout (gray, n = 8) mice with endocarditis induced by the clinical strain at day three. L, Quantification of the thrombus volume (n = 11, 8). M-N, Quantification of H3Cit in mice with S. aureus USA300-induced endocarditis in the one-day tail vein delivery (n = 5, 2, M) and in the one day catheter delivery model (n = 5, 4, N). O-P, Quantification of the leukocyte infiltration (O) and the TUNEL+ (P) area in the surrounding aortic wall in control (n = 11) and knockout mice (n = 8) with vegetations injected with the clinical S. aureus strain in the tail vein at day three. Q-T, Gram (Q-R) and TUNEL (S-T) staining of a control (PAD4 fl/fl , n=11) and neutrophil-selective PAD4 knockout mouse (MRP8Cre + x PAD4 fl/fl , n = 8) with endocarditis induced by the clinical S. aureus strain. Median and interquartile range are represented, and statistical significance evaluated by Mann-Whitney test (E-P). Figure S6 : S6Correlation analyses between markers of apoptosis and markers of neutrophils or NETs. A-B, Quantifications of fibrin within the thrombus (A) and the size of the thrombus (B) in C57Bl/6J mice with infective endocarditis induced by WT (black, n = 10) or ΔcoaΔvwb S. aureus (dark gray, n = 9). Medians (and interquartile ranges) are represented in A and B, and statistical significance was determined with a Mann-Whitney test (A-B). C-G, Spearman correlations (r = correlation coefficient) between the TUNEL positive area and the leukocyte infiltration area (C), bacteria (D), MPO (E) and Ly6G (F) positive area located outside the thrombus, and the H3Cit positive area inside the thrombus (G). Graphs C and G contain data from endocarditis vegetations in wild-type mice (C57Bl/6J) infected with WT S.aureus (black, n = 10) and in PAD4-expressing wild-type mice (C57Bl/6J and PAD4 fl/fl ) infected with ΔcoaΔvwb S. aureus (dark gray, n = 10). Graph D-F contain data from endocarditis vegetations in wild-type mice (C57Bl/6J) infected with WT S. aureus (black, n = 10), and PAD4-expressing wild-type (C57Bl/6J and PAD4 fl/fl ) (dark gray, n = 10) and neutrophilselective PAD4 knockout (MRP8Cre + x PAD4 fl/fl ) mice infected with ΔcoaΔvwb S. aureus (light gray, n = 4). Figure S7 : S7Absence of staphylocoagulases does not alter endocarditis outcome or bacteremia in mice with impaired NET formation. Proportions of mice that developed inflammation-induced endocarditis (A) or sterile thrombi (B) at day three, bacteremia levels at endpoint (n = 17, 11, C), and survival rates (n = 18, 11, D) of PAD4 fl/fl and MRP8Cre + xPAD4 fl/fl mice infected with ΔcoaΔvwb S. aureus. Median (interquartile range) is represented in C. Fisher's exact (A-B), Mann-Whitney (C) and log-rank (Mantel-Cox) (D) tests were conducted to determine significance. Clinical S. aureus strain and S. epidermidisSample Size: Please explain how the sample size was decided Please provide details of any a prior sample size calculation, if done.Rat InVivoMAb anti-mouse Ly6G Bio X Cell BE0075-1 8 μg/g 673218M1 / InVivoMAb rat IgG2a isotype control Bio X Cell BE0089 8 μg/g 716718O1 654817M2 / InVivoMAb anti- rat Kappa Immunoglobulin Light Chain Bio X Cell BE0122 4 μg/g / / Anti-mouse CD16/CD32 Biolegend 101302 5 μg/ml / / APC/Cyanine7 anti-mouse CD45 (clone 30-F11) Biolegend 103116 1 µg/mL / / PerCP anti-mouse Ly6G (clone 1A8) Biolegend 127654 1 µg/mL B274299 / Rabbit anti- Staphylococcus aureus antibody Abcam ab20920 5 μg/ml GR3361393-1 / Anti-mouse Ly6G antibody (1A8) Biolegend 127602 2.5 μg/ml B265459 / Human/mouse myeloperoxidase antibody R&D systems AF3667 2.5 μg/ml YBZ0421031 / Rabbit anti-Histone H3 (citrulline R2 + R8 + R17) Abcam ab5103 2.5 μg/ml GR3294584-1 (paraffin) GR3362385-1 (cryo) / Recombinant anti- Histone H3 (citrulline R2 + R8 + R17) Abcam ab281584 2.5 μg/ml GR3389586-11 / Anti-mouse CD41 Biolegend 133902 2.5 μg/ml B285959 / Sheep anti- fibrinogen Bio-Rad laboratories 4440-8004 10 μg/ml 151063 / Rabbit anti-human VWF Dako (Agilent) A008202 8.2 μg/ml 20082415 / Goat anti-rat IgG (H+L) AlexaFluor™555 Invitrogen A21434 1.33 μg/ml 1907302 / Donkey anti-goat IgG (H+L) AlexaFluor™555 Invitrogen A32816 1.33 μg/ml UC282069 / Donkey anti-rabbit AlexaFluor™488 (H+L) Invitrogen A21206 1.33 μg/ml 2256732 / Donkey anti-sheep IgG (H+L) AlexaFluor™488 Invitrogen A11015 1.33 μg/ml 1716970 / 10 nm gold- conjugated goat F(ab')2 anti-rabbit IgG (H/L) antibody Abcam ab39601 1/20 (stock concentration not provided) GR3360046-1 / Other Description Source / Repository Persistent ID / URL S. aureus Newman, USA300, DcoaDvwb, Dnuc Dominique Miasakas (University of Chicago) / University hospitals Leuven / Histamine (200 mM) Sigma-Aldrich (H7125-1G) / Rat intrathecal catheter, 32ga (0.8Fr) PU 18cm, stylet, luer. Fits 27-25ga. Instech (C08PU-RIT1301) / Tryptic Soy Broth Merck Millipore (22092) / CountBright absolute counting beads Invitrogen (C36950) / Percoll® PLUS Sigma-Aldrich (GE17-5445-01) / Ionomycin (4 μM) Invitrogen (I24222) / Hoechst 33342 (2,5 μg/ml) Invitrogen (H1399) / MACSxpress Whole Blood Neutrophil Isolation kit Miltenyi Biotec (130-104-434) / Hanks' Balanced Salt Solution (without calcium and magnesium) Invitrogen (14175095) / RPMI 1640 medium (without phenol red) Gibco (11835030) / Fetal calf serum Gibco (A4736401) / Alul restriction enzyme (4 U) Thermo Fisher Scientific (ER0011) / Citrullinated histone H3 (clone 11 D3) ELISA Cayman Chemical (501620-96) / Epoprostenol(2 μg/ml) Tocris Bioscience (2989) / Apyrase from potatoes (0.02 U/ml) Sigma-Aldrich (A7646-200UN) / Donkey serum (10%) Sigma-Aldrich (D9663-10ML) / Sudan Black B (0.05% or 0.1%) Sigma-Aldrich (380B-1KT) / Antigen retrieval reagent basic solution (1X) R&D systems (CTS013) / Click-iT Plus Tunel Assay labeled with AlexaFluor™647 Invitrogen (C10619) / poly-L-lysine coated coverslips VWR (734-1005) / EM grade PFA (1%) Electron Microscopy Sciences (15710) / Normal goat serum (20%) Vector Laboratories (VEC.S-1000) / EM grade glutaraldehyde (2.5%) Sigma-Aldrich (G5882-50ML) / Osmium tetroxide (1%) Electron Microscopy Sciences (19190) / Sodium cacodylate trihydrate (0.2M) Electron Microscopy Sciences (11650) / ARRIVE GUIDELINES The ARRIVE guidelines (https://arriveguidelines.org/) are a checklist of recommendations to improve the reporting of research involving animals. Key elements of the study design should be included below to better enable readers to scrutinize the research adequately, evaluate its methodological rigor, and reproduce the methods or findings. Study Design Experiment 1: neutrophil depletion in WT mice infected with different bacterial strains (Figure 2) Groups Sex Age Number (prior to experiment) Number (after termination) Littermates (Yes/No) Other descripti on Group 1 (Control: isotopy, S. aureus USA300) male 8 to 14 weeks 26 21 N/A C57Bl/6J Group 2 (anti-Ly6G, S. aureus USA300) male 8 to 14 weeks 28 23 N/A C57Bl/6J Group 1 (Control: isotype, Clinical S. aureus) male 8 to 14 weeks 19 19 N/A C57Bl/6J Group 2 (anti-Ly6G, Clinical S. aureus) male 8 to 14 weeks 20 16 N/A C57Bl/6J Group 1 (Control: isotype, S. epidermidis) male 8 to 14 weeks 12 10 N/A C57Bl/6J Group 2 (anti-Ly6G, S. epidermidis) male 8 to 14 weeks 12 10 N/A C57Bl/6J Sample Size: Please explain how the sample size was decided Please provide details of any a prior sample size calculation, if done.Experiment 3: S. aureus deficient in nuclease (Δnuc) in WT mice (Figure 7) Groups Sex Age Number (prior to experiment) Number (after termination) Littermates (Yes/No) Other description Group 1 (Control: S. aureus USA300) male 8 to 14 weeks 14 12 N/A C57Bl/6J Group 2 (S. aureus Δnuc) male 8 to 14 weeks 14 12 N/A C57Bl/6J Experiment 4: S. aureus deficient in Coa and vWbp (ΔcoaΔvwb) in WT mice (Figure 7)Sample Size: Please explain how the sample size was decided Please provide details of any a prior sample size calculation, if done.Groups Sex Age Number (prior to experiment) Number (after termination) Littermates (Yes/No) Other description Group 1 (Control: S. aureus USA300) male 8 to 14 weeks 29 27 N/A C57Bl/6J Group 2 (S. aureus ΔcoaΔvwb) male 8 to 14 weeks 30 26 N/A C57Bl/6J Sample Size: Please explain how the sample size was decided Please provide details of any a prior sample size calculation, if done.From previous experiments, survival rates of 100% are expected for the S. aureus Δcoa/Δvwb strain and 60% for WT S. aureus at endpoint. A relevant decrease in proportion of mice that survive at endpoint of 40% in PAD4 null mice infected with S. aureus Δcoa/Δvwb is estimated to be relevant.Hence, 36 mice will be needed, based on the Fisher exact test (18 mice in the PAD4 null mice group and 18 mice in PAD4 floxed mice group).Experiment 5: S. aureus deficient in Coa and vWbp (ΔcoaΔvwb) in neutrophil-selective PAD4 knockout mice (Figure S7) Groups Sex Age Number (prior to experiment) Number (after termination) Littermates (Yes/No) Other description Group 1 (Control: PAD4 fl/fl , S. aureus ΔcoaΔvwb) male 8 to 14 weeks 21 18 Yes Group 2 (MRP8Cre + xPAD4 fl/fl , S. aureus ΔcoaΔvwb) male 8 to 14 weeks 11 11 Yes The following parameters were determined a = 0.05 b = 0.20 Brown-Hopps Gram stain. To this end, slides were stained with the following solutions: 1 %crystal violet for 2 min, Gram's iodine (1.2 g potassium iodide and 0.6 g iodine in 200 μl MQ) for 5 min, 2-etoxyethanol for 30 s, 0.5 % basic Fuchsin solution for 5 min (Sigma-Aldrich, St. Louis, USA), Gallego's solution (4 ml. 37 % formaldehyde and 2 ml glacial acetic acid in 194 ml deionized water, VWR, Radnor, USA) for 5 min and Tartrazine solution for 3 s (Sigma- the steps and finally, slides were dehydrated and mounted with DPX (Sigma-Aldrich, St. Louis, USA). To detect fibrin, a Martius Scarlet Blue (MSB) staining was conducted. Slides were first fixated overnight in Bouin's solution (Sigma-Aldrich, St. Louis, USA) and incubated with Celestin blue (1 g Celestine blue B, 10 g Ferric ammonium sulphate, 28 ml glycerin in 200 ml deionized water) for 5 min, Harris hematoxylin (100 ml Harris hematoxylin, 2 ml acetic acid in 100 ml deionized water) for 5 min, acid alcohol (1 % HCl in 70 % ethanol) for 10 s and rinsed with 95 % ethanol (Sigma-Aldrich, St. Louis, USA). In between these steps, washing steps were performed with deionized water. After the rinsing step in 95 % ethanol, slides were incubated with Martius yellow (1 g Martius yellow, 4 g phosphotungstic acid in 200 ml 95 % ethanol) for 2 min, Brilliant Scarlet Blue (2 g Crystal Ponceau 6R, 4 ml acetic acid in 200 ml deionized water) for 10 min, 1 % phosphotungstic acid for 5 min, Methyl blue (1 g Methyl blue, 2 ml acetic acid in 200 ml deionized water) for 2 min, rinsed with 1 % acetic acid, dehydrated and mounted with DPX (Sigma-Aldrich, St. Louis, USA). Table S1 : S1Significance testing of Figure 3BP-values from Kruskal-Wallis test with Dunn's multiple comparison test, conducted inS. aureus USA300 S. aureus Newman Clinical S. aureus S. epidermidis S. aureus Δnuc Vehicle 0.402 0.276 <0.001 >0.999 <0.001 S. aureus USA300 / >0.999 0.421 0.239 0.575 S. aureus Newman >0.999 / 0.600 0.160 0.807 Clinical S. aureus 0.421 0.600 / <0.001 >0.999 S. epidermidis 0.239 0.160 <0.001 / <0.001 Table S2 : S2Significance testing ofFigure 3DP-values from Kruskal-Wallis test with Dunn's multiple comparison test, conducted inVehicle + platelets S. aureus USA300 + platelets S. aureus Newman + platelets Clinical S. aureus + platelets S. epidermidis + platelets Vehicle >0.999 0.076 >0.999 0.085 >0.999 Vehicle + platelets / 0.017 >0.999 0.019 >0.999 S. aureus USA300 + platelets 0.017 / >0.999 >0.999 0.230 S. aureus Newman + platelets >0.999 >0.999 / >0.999 >0.999 Clinical S. aureus + platelets 0.019 >0.999 >0.999 / 0.253

Reviewer acknowledgement Contributing reviewers Bong-Kiun Kaang Seoul National University SeoulRepublic of Korea Min Zhuo minzhuo10@gmail.com University of Toronto TorontoCanada Tim Bliss tim.bliss@crick.ac.uk The Francis Crick Institute LondonUK Marisela Agudelo Yoshiki Arakawa Japan Valeria Avdoshina Nagi Ayad Kasum Azim Tudor C Badea Daehyun Baek Angel Barco Spain Arnab Barik Mark Bear Oren Becher Guoqiang Bi China Haruhiko Bito Japan Vadim Bolshakov Victor Borrell Spain Denis Burdakov Timothy Bussey Marco Canossa Shuwen Cao Francesco Cardona Martine Cattarelli France Sebastiano Cavallaro Italy Sunghoe Chang Brian Chen Canada Gong Chen Xuanmao Chen Youjun Chen Zheyu Chen China Kei Cho Dh Cho Youngshik Choe Soo Young Choi Kimberly Christian Steven Clarke Daniel Cohen France Graham Collingridge Jaqueline Crawley United States of America Mian Cao China L Cao United States of America of America, of America, of America, of America, of America Republic of Korea, of America, of America, of America, of America, United Kingdom, United Kingdom, of America, Italy Republic of Korea, of America, of America, of America, United Kingdom Republic of Korea Republic of Korea Republic of Korea, of America, of America, United Kingdom, of America Reviewer acknowledgement Contributing reviewers 10.1186/s13041-016-0208-4R E V I E W E R A C K N O W L E D G E M E N T Open Access The editors of Molecular Brain would like to thank all our reviewers who have contributed to the journal in Volume 8 (2015). Wim Crusio France Eun-Kyoung Kim Republic of Korea Hyong Kyu Kim Republic of Korea Janghwan Kim Republic of Korea Jaesang Kim Republic of Korea Eunjoon Kim Republic of Korea Sang Jeong Kim Republic of Korea Byung Gon Kim Republic of Korea Jin Kim Republic of Korea Jung-Woong Kim Republic of Korea Hye-Sun Kim Republic of KoreaFernando De Castro Spain Jessica Deslauriers Canada Yuqiang Ding China Tr Doeppner Germany Bo Duan United States of America Sebastian Dworkin Australia Marina Emborg United States of America Jose Antonio Esteban Spain Cinthia Farina Italy André Fischer Germany Patrick Fisher Denmark Masayo Fujita Japan Masaki Fukata Japan Tomoyuki Furuyashiki Japan Peter Gass Germany Anamitra Ghosh United States of America Peter Giese United Kingdom Christian Johannes Gloeckner Germany Kerui Gong United States of America Johannes Graeff Swaziland Kamalesh Guliak India Camilla Gustafsen Denmark Hideo Hagihara Japan Jin-Hee Han Canada Kihoon Han Republic of Korea Young-Goo Han United States of America Daniela Hartl Germany Yasunori Hayashi Japan Yasunori Hayashi United States of America Yiping He United States of America Karl Herrup China Hajime Hirase Japan Xiu-Ti Hu United States of America Andrew Huang Taiwan, Republic of China Eunmi Hur Republic of Korea Jeong-Jin Hwang Republic of Korea Sun Wook Hwang Republic of Korea Su-Kyeong Hwang Republic of Korea Kazutaka Ikeda Japan Masashi Ikeda Japan Masai Ishii Japan Anthony Isles United Kingdom Shigeyoshi Itohara Japan Takuji Iwasato Japan Deok-Jin Jang Republic of Korea Ru-Rong Ji United States of America Zhengping Jia Canada Seonmi Jo Republic of Korea Francois Jouret Belgium Yong-Keun Jung Republic of Korea Bong-Kiun Kaang Republic of Korea Wataru Kakegawa Japan Roger Kamm United States of America Fusao Kato Japan Satoshi Kida Japan Ahmad TariqKaang et al. Molecular Brain (2016) 9:25Page 4 of 5 . Kaang, Molecular Brain. 9Kaang et al. Molecular Brain (2016) 9:25 Page 5 of 5

Professor of Ophthalmology and Otology October, 1893 Alvin A Hubbell M D Professor of Ophthalmology and Otology October, 1893 reports a -case of hypopyon keratitis with enucleation of the ball and sub sequent microscopical examination. He attempts to account for the origin and formation of pus in the anterior chamber-a prob lem that has not been satisfactorily solved. His conclusions are '"that the pus in the hypopyon comes, in the majority of cases, from the uveal tract, the iris and the vessels adjacent to Fontana's spaces, while that in the cornea is derived either from the deep 'ciliary vessels or the anterior border-loop vessels. So much is positive. As to the source of the pus-cells, which appear in the exudate on Descemet's membrane very early in most cases, we are still uncertain. The pus in the cornea may be derived from the conjunctival sac, and may originate from the cornea itself. At least it has not been proven that it does not." CROUPOUS IRITIS. Dr. Adolf Alt, of St. Louis, (Journal of Ophthalmology, Novem ber, 1893,) describes a form of iritis which he terms "croupous." It is that form which Knapp has designated as "spongy iritis," and is scarcely mentioned in the text-books. The literature of the subject began with Schmidt, who reported two cases in 1871. Since then it has been noted by Gunning, Gruening, Kipp, Knapp, Alt, and S. M. Burnett. This is an inflammatory disease of the iris, ushered in by severe pain in and about the eye, edema of the lids and conjunctiva, and circumcorneal injection. This inflam mation leads to a peculiar exudation into the anterior chamber, but which is in no way distinguishable from croupous exudation elsewhere. It forms rapidly, and is first seen as a grayish, grayish-yellow, or grayish-green semi-transparent substance (showing sometimes stripes and dots), which, when the patient is seen, usually fills the anterior chamber to its full extent. After a period varying from a day to a week, and even much more, during which time hemorrhages into the anterior chamber not infrequently take place, the exudation becomes transparent in its periphery, and is liquified and gradually absorbed. This change is visible usually at first in the upper part of the anterior chamber,, where a small strip of iris-tissue becomes uncovered, the exudation sinking by gravitation. It then presents a sharp, well-defined, sometimes perfectly round, sometimes jagged edge upward, and has at this stage an appearance much like that of a cataractous lens, dislo cated into the anterior chamber. This resemblance is the more striking, as the anterior chamber is usually very deep. Gradually the absorbing and melting process goes on, till later only a small piece is seen lying at the bottom of the anterior chamber, and finally after ten to twenty-five days it entirely disappears. The eye then quickly recovers, only one or more posterior synechia remaining to mark the disease. In uncomplicated cases not even a synechia may remain. It seems highly probablb that we have to deal with a special form of infection which is worthy of further study. In treat ment the same measures may be used as in plastic iritis. ERRORS OF REFRACTION AND THEIR CORRECTION IN EPILEPTICS. Mr. Work Dodd read a paper on this subject before the Ophthal-'mological Society of the United Kingdom, (Medical ^Veek, Octo ber 27, 1893,) which was the outcome of a study of 100 cases. The refractions were worked out under mydriatics with every care, under the following conditions : (1) The total refraction under a mydriatic was taken; (2) hypermetropia of 0.75 D was reckoned as emmetropia; (3) astigmatism of 0.25 D was not included. The following are the results of his researches : Emmetropia, 7 ; simple hypermetropia, 42 ; simple myopia, 6 ; total astigma tism, 42, of which there were simple hypermetropic astigmatism, 3 ; compound hypermetropic astigmatism, 24 ; compound myopic astigmatism, 2 ; mixed astigmatism, 6 ; marked anisometropic astigmatism, 7 ; other cases of marked anisometropia, 3. If we compare this table with a classification of percentages of refrac tion, obtained from fifty cases of apparently normal eyes, which were worked out under mydriatics in the same manner, we find emmetropia, 6 ; simple hypermetropia, 70 ; simple myopia, 2 ; total astigmatism, 16. It will be seen that the most marked dif ference is in the amounts of the simple hypermetropia and of simple astigmatism, there being twenty-eight cases per cent, less in the epileptic than in the apparently normal class. Of astigma tism of all kinds there are twenty-six cases per cent, more in the epileptic division than in the normal one, chiefly made up by the large amount of compound hypermetropic astigmatism existing in epileptics. Of the 100 consecutive cases of epilepsy, seventy-five were ordered to wear glasses ; of these there were twenty-three who either did not wear them, or failed to report themselves later and could not be found. Of the remaining fifty-two cases there were : (1) Thirteen who had no fits since using the glasses, during periods varying from four months to one year ; (2) three patients whose condition had not apparently altered; and (3) thirty-six patients whose condition had improved since wearing glasses; in the majority of these the improvement had been marked. In all the cases the ordinary treatment was continued for sometime. Several cases in which patients who had ceased to have fits since wearing the glasses, had suffered from them again through some other form of irritation, but in no case had the fits been as severe as before. Mr. Dodd thinks that we may deduce from the foregoing facts that, given a certain condition of instability of the nervous sys tem, errors of refraction may excite epilepsy, and their correction, with other treatment, may relieve and even cure it. THE TREATMENT OF ULCERS AND ABSCESSES OF THE CORNEA BY CURETTING AND IRRIGATION. DeWecker (Annates <?' Oculistique, July, 1893,) has practised curetting and antiseptic irrigation of corneal ulcers and abscesses for some time with surprising results, of which the following are the most marked : 1. The suppression, sometimes instantaneous, of pain and photophobia. 2. The clearing up of the surrounding parts, followed by a cure infinitely more rapid than is obtained by the employment of various antiseptics and particularly the actual cautery. 3. Reparation by a more transparent tissue is better obtained by this method than by any other mode of treatment. He operates by using sharp curettes of small dimensions, and of various forms, of which one differs but little from that of Critchett, except that it is one-third as wide and its edge is sharp. He endeavors as much as possible to remove from the bottom and edges of the ulcer all the adherent whitish parts in such a way that, under the jet of the irrigator (charged with a four per cent, solution of boric acid), the parts of the cornea not attacked by the curette appear feebly opaline, but uniformly transparent. He does not hesitate to affirm that the curetting (raclage), joined with irrigation, should be used by all clinicians until the progress of our therapeutics furnishes something better. FORMIC ALDEHYDE AS AN OCULAR ANTISEPTIC. Valude, of Paris, (Annates eV Oculistique, July, 1893,) has been led to study the antiseptic properties of formic aldehyde, and believes he has found in this a powerful antiseptic, and, being but little irritating, one that is particularly suited to the eye. He recom mends it in cases to be operated upon, in post-operative infection, in purulent affections of the eye, and in the sterilization of collyria, as it does not precipitate the alkaloids (atropine, eserine, cocaine,) as does bichloride of mercury. He uses it in the eye in solutions of 1 to 100 to 1 to 500, and to preserve collyria 1 to 2000. Finally, as it does not attack metals-steel, silver, or aluminum -it may be used for washing and disinfecting instruments, in solution, for example, of 1 to 500. REMOVAL OF THE STAPES IN CHRONIC NON-SUPPURATIVE DISEASE OF THE MIDDLE EAR. Dr. Clarence J. Blake, of Boston, [Archivesof Otology, 1893,) has recorded his experience in the removal of the stapes for chronic, non-suppurative catarrh of the middle ear. His conclusions are so important that they are here presented in full : " In reviewing the cases reported, . . . it is very evident, so far as conclusions can be drawn from a small number of cases, that the operation of the removal of the stapes does not answer the purpose which might be hoped from it in cases of chronic non suppurative disease of the middle ear. This conclusion is one in which the clinical and operative observations are entirely in accord with the pathology of this class of cases as set forth by numerous observers, and lastly and most clearly by Politzer. For all this class of cases, therefore, I should, as the expression of a personal opinion and as the result of experience, advise an exploratory tympanotomy with local and without general anesthesia, as a pre liminary to, or as the first part of, an operation having in view any form of interference with the middle ear, from simple mobiliza tion of the ossicular chain to the removal of the stapes. " The exploratory tympanotomy, especially where the incision is made, as it should be, close to the periphery of the membrana tympani and of sufficient extent, affords an opportunity for a better determination of the condition of the middle ear in chronic non-suppurative disease than can be obtained in any other way, and after the exploratory incision, if it seems advisable not to operate more extensively, the opening in the membrana tympani can be closed by a simple paper dressing, with the prospect of speedy healing. If, however, the exploratory operation and coin cident tests show that it is advisable to perform an operation in the middle ear, whether synectomy, tenotomy, incudectomy, incudostapedectomy, or stapedectomy, the opening suffices for the purpose. "In the great majority of the cases of stapes fixation, conse quent upon chronic non-suppurative disease of the middle ear, the operation . . . was ineffectual, so far as the removel of the stapes was concerned, the fixation of the base-plate at least being such as to result in fracture of the crura instead of the removal of the ossicle entire. In all the cases of non-suppurative disease in which the stapes was extracted entire, the hearing was definitely and practi cally improved in one only ; and of the two other cases in which definite improvement in hearing resulted from the operation, there was one in which the mobilization of the base-plate, incident to the fracture of the crura, gave an improvement for high tones, and for the voice in ordinary conversation only to the extent of about twenty per cent. " When we take into consideration the secondary changes which may have occurred in the internal ear in the course of a non-sup purative disease of the tympanum, and the injury to the delicate structures in the labyrinth, which might result from the force exerted in the extraction of the stapes, coupled with the inadequate results as set forth in the experience tabulated, it may be justly said that stapedectomy does not afford a promising out look for this class of cases." AURAL REFLEX OF UNUSUAL CHARACTER, DUE TO IMPACTED WAX. Theobold (New Yor/c Medical Record, July 29, 1893,) reports the ease of a female, aged 42, who had suffered for six months with an annoying cough and with spells of inability to swallow food. These symptoms were increased on manipulation of the right ear. Examination showed a piece of wax which had been forced against the drum-membrane by attempts at removal. It was removed by syringing, and the difficulty in swallowing and other symptoms disappeared. The hostess who sends a pitcher of ice water to her guest's room should use as large a lump of ice as will fit the pitcher, and not too much water ; then set the pitcher in the center of a large news paper, gathering the ends up at the top and place a strong rubber band around them, to exclude the air. Treated in this way, the water will remain real ice water all night, and a lump of ice as big as one's fist will not be entirely melted by the next morning. -Buffalo Commercial.

Available online 17 February 2023 Available online 17 February 202310.1016/j.abd.2022.12.001Received 27 October 2022; accepted 21 December 2022;Anais Brasileiros de Dermatologia 2023;98(3):426---427 Anais Brasileiros de Dermatologia CORRESPONDENCE * Corresponding author. (H.A. Miot). On the recurrence rate of cutaneous tumors treated exclusively by micrographic surgery ଝ Dear Editor, We read with interest the article by Dr. Otsuka et al., in which they used a peripheral sampling technique with a parallel incision of the lateral and deep surgical margins of cutaneous carcinomas (basal cell and squamous cell carcinomas). Moreover, they validated the concordance of the identification of compromised margins with subsequent analysis of the paraffin-embedded specimens. 1 We would like to congratulate the authors and make comments regarding the method, its validation, and conclusions about the recurrence rate. We encourage the study of operative techniques in micrographic surgery, as well as in the processing of surgical specimens, which may lead to effective and faster procedures, with lower cost and morbidity rates. Initially, for clarifying purposes, it should be noted that the technique used by the authors is identical to the Tübingen method, one of the most employed modalities in Europe. 2 When meta-analytically analyzed, the results of 10,424 basal cell carcinomas operated on by micrographic surgery, the different micrographic surgery techniques do not show overall differences in recurrence rates between them (1% to 3%), although there are no parallel comparative studies that could explore their peculiarities. Primary tumors have a recurrence rate that ranges from 1% to 3%, and relapses, of 2% to 5%. 3,4 Interestingly, false-negative margins can occur in discontinuous tumors (such as multicentric basal cell carcinoma -BCCs), with thin areas (e.g., tumors with perineural invasion), recurrences under flaps/grafts, and because of technical problems during preparation of the slides. On the other hand, false-positive margins can occur due to confusion with cross-sections of follicular bulbs, due to the inflammatory infiltrate, or in the initial spare sectionning ଝ Study conducted at the Department of Dermatology, Faculty of Medicine, Universidade Estadual Paulista, Botucatu, SP, Brazil. of specimens in the cryostat, necessary for preparing the slides. For this reason, validation of the coincidence of surgical margins in paraffin, as used in the work by Otsuka et al., may not be plausible, as it may overestimate the involvement of the margins, since the outermost sections were previously sampled and analyzed intraoperatively, in thicker sections than the ones in paraffin. When analyzing, separately the recurrence rates described by the authors for primary basal cell carcinomas (0.3%) and the recurrent ones (4.3%), we found that there was supplementation of the surgical treatment with radiotherapy in 97% of the cases, which is unusual, especially for basal cell carcinomas (80% of the sample), given the use of a surgical technique that verifies 100% of the surgical margins aiming at complete cure and recurrence prevention. Although the literature has publications that suggest exclusive radiotherapy as a treatment option for BCCs, with good results and local control of up to 96% of cases, studies on the effectiveness of adjuvant radiotherapy in preventing recurrences in micrographic surgeries are still necessary. 5 In the meantime, the recurrence rate found by the authors cannot be attributed only to the surgical technique, but to the combination between micrographic surgery and complementary radiotherapy. Finally, the completion of micrographic surgeries with only one stage in 72% of cases prompts the discussion of indication criteria, aiming to maximize the cost-benefit to the detriment of conventional oncological surgery, since the latter is more accessible, both technically and financially for the health system. Financial support None declared. Authors' contributions Luiz Eduardo Fabrício de Melo Garbers: Design of the study; writing of the manuscript; review and approval of the final version of the manuscript. Ana Carolina Miola: Design of the study; writing of the manuscript; review and approval of the final version of the manuscript. https://doi.org/10.1016/j.abd.2022.12.001 0365-0596/© 2023 Sociedade Brasileira de Dermatologia. Published by Elsevier España, S.L.U. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Luis Fernando Figueiredo Kopke: Design of the study; writing of the manuscript; review and approval of the final version of the manuscript. Hélio Amante Miot: Design of the study; writing of the manuscript; review and approval of the final version of the manuscript. Conflicts of interest Intraoperative assessment of surgical margins using ''en face'' frozen sections in the management of cutaneous carcinomas. A Otsuka, E Bertolli, M P De Macedo, Cal Pinto, Duprat Neto, J P , An Bras Dermatol. 97Otsuka A, Bertolli E, de Macedo MP, Pinto CAL, Duprat Neto JP. Intraoperative assessment of surgical margins using ''en face'' frozen sections in the management of cutaneous carcinomas. An Bras Dermatol. 2022;97:583---91. Histologic control of excised tissue edges in the operative treatment of basal-cell carcinomas. H Breuninger, J Dermatol Surg Oncol. 10Breuninger H. Histologic control of excised tissue edges in the operative treatment of basal-cell carcinomas. J Dermatol Surg Oncol. 1984;10:724---8. On variations in micrographic surgery and the use of horizontal histological sections in the evaluation of the surgical margin. A C Miola, H A Miot, Lff Kopke, An Bras Dermatol. 95Miola AC, Miot HA, Kopke LFF. On variations in micrographic surgery and the use of horizontal histological sections in the eval- uation of the surgical margin. An Bras Dermatol. 2020;95:545---6. Recurrence rate of basal cell carcinoma among different micrographic surgery techniques: systematic review with metaanalysis. P N Lacerda, E P Lange, N M Luna, H A Miot, Vsn Nogueira, Lpf Abbade, J Eur Acad Dermatol Venereol. 36Lacerda PN, Lange EP, Luna NM, Miot HA, Nogueira VSN, Abbade LPF. Recurrence rate of basal cell carcinoma among different micrographic surgery techniques: systematic review with meta- analysis. J Eur Acad Dermatol Venereol. 2022;36:1178---90. The State of the Art of Radiotherapy for Nonmelanoma Skin Cancer: A Review of the Literature. S Benkhaled, D Van Gestel, Cgs Cauduro, S Palumbo, Del Marmol, V Desmet, A , Front Med (Lausanne). 9913269Benkhaled S, Van Gestel D, Cauduro CGS, Palumbo S, Del Marmol V, Desmet A. The State of the Art of Radiotherapy for Non- melanoma Skin Cancer: A Review of the Literature. Front Med (Lausanne). 2022;9:913269.

Systematic review and meta-analysis of preoperative interventions to support the maturation of arteriovenous fistulae in patients with advanced kidney disease 17 February 2023 Sivaramakrishnan Ramanarayanan s.ramanarayanan@nhs.net 0000-0003-4238-122X Department of Renal Medicine Lister Hospital, East and North Hertfordshire NHS Trust StevenageUK School of Life and Medical Sciences University of Hertfordshire HatfieldHertfordshire, UK Shivani Sharma School of Life and Medical Sciences University of Hertfordshire HatfieldHertfordshire, UK Oscar Swift Department of Renal Medicine Lister Hospital, East and North Hertfordshire NHS Trust StevenageUK Keith R Laws School of Life and Medical Sciences University of Hertfordshire HatfieldHertfordshire, UK Hamza Umar College of Medical and Dental Sciences University of Birmingham BirminghamUK Ken Farrington Department of Renal Medicine Lister Hospital, East and North Hertfordshire NHS Trust StevenageUK School of Life and Medical Sciences University of Hertfordshire HatfieldHertfordshire, UK Systematic review and meta-analysis of preoperative interventions to support the maturation of arteriovenous fistulae in patients with advanced kidney disease 17 February 20234311743089A1FE049667300A8547EC3D10.1093/ndt/gfad040AVF maturationchronic kidney diseasehaemodialysishand exercisepreoperative intervention Background.There is great potential to improve outcomes of arteriovenous fistulas (AVFs) by focusing more on the preoper-ative period of AVF creation.We aim to systematically review the evidence on safety and efficacy of various preoperative interventions that have been tried to improve AVF maturation and success rate. INTRODUCTION Arteriovenous fistulas (AVFs) are the preferred form of vascular access (VA).They have the lowest rate of infection, stenosis and thrombosis among all types of VA [1,2].However, despite the creation of AVFs well before dialysis initiation, a significant proportion fail and patients need to start haemodialysis (HD) through a catheter, putting them at high risk of catheter-related complications.Globally, the rate of primary AVF failure has been estimated at 30-70%, with a 1-year patency rate of 40-70% [3].Variations in the estimates of AVF failure are likely to reflect operational definitions of what constitutes 'failure': e.g.whether the AVF is unsuitable for HD and requires a 'salvage' intervention or failure is defined by other problematic outcomes such as thrombosis alone [4].Bylsma et al. [5], in their systematic review and meta-analysis of AVFs for dialysis, stated that there is ambiguity in how patency is reported across studies, notably that definitions used often do not align to clinical utility. The use of prediction tools to estimate the risk of failure based on a mix of clinical and patient demographic factors [6] as well as preoperative vessel mapping [7] has been suggested to improve AVF maturation, but the rate of primary failure and non-maturation remain at unacceptably high levels.Therefore, interventions aimed at reducing early failure will be advantageous economically, help facilitate patient decision making around VA choices and improve their overall healthrelated quality of life [8]. Most patients with advanced kidney disease are under the care of a nephrologist long before AVF creation is required.This offers a window of opportunity for interventions to improve success rates of AVF maturation.Although there are individual systematic reviews that summarize the outcomes of individual intervention types (hand exercises, pharmacological measures, infrared therapy etc.) on AVF, these have not focused on teasing whether the preoperative period specifically is an important factor in intervention success.For example, Bashar et al. [9] looked at the impact of far infrared therapy in AVF maturation, but the randomized controlled trials (RCTs) included in their review had data from patients who were already receiving HD through an AVF. The aim of this systemic review and meta-analysis is to summarize the types of preoperative interventions that have been implemented to support AVF maturation, to pool data across RCTs specifically to estimate the impact of interventions on outcomes of interest and to undertake subgroup and mediator analyses to understand if the interventions work better for specific patient groups. MATERIALS AND METHODS This review was preregistered in the International Prospective Register of Systematic Reviews (PROSPERO; CRD42020193257). Review process and search strategy The systematic review and meta-analyses were undertaken following the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) [10].The following electronic databases were used to perform searches of published articles without any restrictions and all articles were captured up to 15 May 2022: PubMed, Embase, CINAHL, Cochrane Library and King's Fund Library.The search strategy can be found on https://www.crd.york.ac.uk/PROSPEROFILES/193257_STRATEGY_20200619.pdf. In brief, a combination of terms for advanced kidney disease or end-stage kidney disease (ESKD), haemodialysis, AVF maturation or patency and medical or surgical interventions (including exercises) were combined through the PICO format.Broad terms were used in the search strategy to capture as many studies as possible. The results of searches were then transferred to Endnote citation manager (version X9.3.3;Clarivate, Philadelphia, PA, USA).After removing duplicate publications, titles and abstracts were screened. Study selection criteria S.R. screened the titles and abstracts against the following inclusion criteria: the study involved adult patients with advanced CKD including patients with ESKD; the study explored the efficacy and/or safety of a preoperative intervention aimed at improving the rate of AVF creation or outcome of AVF surgery (using preoperative endpoints such as vein calibre, brachial artery flow etc. or postoperative endpoints such as AVF maturation, primary failure rate); the interventions are confined to the preoperative period of AVF creation; and the study was published in English. We excluded studies that looked at outcomes based on synthetic AV grafts and also studies that were purely investigative, such as aids in vessel selection for AVF creation (e.g.vein mapping) and computational fluid dynamics. The full texts of potentially relevant citations were accessed and assessed using the inclusion/exclusion criteria by two authors (S.R. and O.S.) independently.Any disagreement or discrepancies were discussed and resolved with other members of the study team (K.F. and S.S.). We included RCTs, non-RCTs, case-control studies and cohort studies, including single-arm prospective studies that used baseline values before intervention as a control.We did not include reviews, although they were screened for potentially eligible studies.The primary outcome of interest was maturation of the AVF, assessed by various direct or indirect ways including the proportion of patients achieving the criteria of AVF maturation within a specified time frame in the intervention versus control group; the incidence of primary failure of the AVF in the intervention and control groups; preoperative endpoints such as venous diameter, venous distensibility, arterial diameter or flow and an increase in vessels available for AVF creation, especially in the distal forearm.Additional outcomes of interest included adverse events, dropout rate and adherence. Data extraction The data from eligible studies were extracted by one author (S.R.).The data extracted included author details, country of origin, year published, population studied, eligibility criteria, sample size, study design, baseline demographic and clinical characteristics, intervention characteristics (such as nature, dose and duration, supervised or unsupervised, adherence rate), follow-up time, outcome parameters and adverse events. Where adequate data were not available, attempts were made to contact the principal investigator to obtain the necessary information. Quality and risk of bias assessments The quality and risk of bias assessments were performed by two independent reviewers (S.R. and H.U.).The Cochrane Risk of Bias in Non-randomized Studies of Interventions (ROBINS I) tool [11] was used for risk of bias assessment of non-randomized study designs (Supplemental Table 1) while the Cochrane Risk of Bias 2 (ROB-2) tool [12] was used to determine the quality and risk of bias for randomized study designs (Supplemental Table 2).After independent risk of bias analysis, the two independent reviewers met and discussed their results and discrepancies, if any, were resolved. Statistical analysis The outcome data were grouped according to the type of intervention.For example, studies implementing hand exercise as the intervention were grouped together in order to pool the outcome data.Where it was not possible to pool the data due to significant heterogeneity, the data were summarized narratively.Binary outcomes were expressed as odds ratios (ORs) and 95% confidence intervals (CIs).Only one type of intervention, namely hand exercise, was suitable for meta-analysis, as the relevant studies had common outcome variables, namely vessel diameter.As all hand exercise studies consistently reported cephalic vein diameter in the distal forearm at baseline and end of follow-up (6-8 weeks), this parameter was pooled and meta-analysis was performed using ReviewManager version 5.4 software (https://training.cochrane.org/online-learning/core-software/revman) [13].The effect size was expressed as the weighted mean difference in the final diameter between the intervention and the control arm.A random effects model was applied for meta-analysis of the effect size for hand exercise on vessel diameters.Heterogeneity was assessed using the I 2 statistic, and for interpretation we followed Cochrane guidance [14], where I 2 values of 0-40% identified as might not be important, 30-60% as moderate heterogeneity, 50-90% as substantial heterogeneity and 75-100% as considerable heterogeneity.The presence of any significant publication bias was assessed by examining funnel plots for asymmetry and through the Egger's test. RESULT S Study and participant characteristics The steps of study selection and exclusion are summarized in the PRISMA diagram (Fig. 1).The reason for study exclusion at each step was recorded. Eight studies, conducted between 2003 and 2021, were eligible for inclusion in the systematic review (for study characteristics, see Table 1).The number of participants ranged from 5 patients to a maximum of 138 patients, with total participants across the eight studies being 323. Of the eight included studies, four were RCTs, with three of them involving hand exercise [15][16][17] as the test intervention and one involved a drug, namely cholecalciferol, as the test intervention compared with placebo [18]. Three eligible non-randomized studies tested the efficacy of different regimens of hand exercises on vascular structural and functional parameters.These three studies were single-arm prospective studies in which the intervention was performed on a cohort of patients with advanced CKD or ESKD [19][20][21].However, in the studies by Leaf et al. [20] and Uy et al. [21], the pre-and post-intervention variables were compared between the exercise limb and the non-exercise limb (split-body trial design).This was not possible in the study conducted by Rus et al. [19], as the patients included were HD patients with an AVF in one arm.Yet another non-randomized study involved testing the efficacy of intermittent pneumatic compression of the arm with the Fist Assist device [22]. As hand exercise was the most common type of intervention studied in the preoperative period, these studies are summarized in Table 2. In summary, isometric hand grip exercise was the most common type of exercise advocated and was part of an exercise regime in all the included studies.Additional isotonic exercises were included in studies by Leaf et al. [20] and Barbosa et al. [16].Important additional co-interventions included blood flow restriction using a tensiometer applied to the exercise arm, where the tensiometer was inflated to 50 mmHg of Preoperative interventions to support the maturation of AVFs systolic blood pressure in the study by Barbosa et al. [16], and temperature control during exercise in the study by Leaf et al. [20] to standardize the effect of temperature on venous and arterial calibre. Risk of bias The risk of bias was assessed by the above-mentioned tools and the results are summarized in Table 3.Of the four RCTs, one had a high risk of bias [15], two had some concerns [16,17] and only one had a low risk of bias [18].All four non-RCTs had serious risk of bias due to the small number of participants [20,22] or the split-body trial design [19,21], as the systemic effect of exercise is likely to affect vessel parameters in both upper limbs. Primary and secondary outcomes The outcome measures used for all the exercise studies were predominantly related to a change in vessel calibre or flow at the end of the exercise period.Only the RCT by Aragoncillo Sauco et al. [15] followed patients beyond AVF creation until 12 weeks post-surgery to look at more hard outcomes such as percentage primary failure at 12 weeks as the primary outcome.The cholecalciferol study by Wasse et al. [18] was another RCT that looked at the percentage or proportion of AVF maturation at 6-months after AVF creation using a standard definition of AVF maturation.Neither hand exercise nor cholecaliferol was found to improve the rate of AVF maturation or primary failure. As summarized in Table 2, hand exercise was generally found to have a positive effect on vein calibre.with most of the studies demonstrating a significant increase as compared with no intervention.Effect size was lower in those studies that used the opposite limb of the same patients as a control, possibly because of the systemic effect of exercise affecting the non-exercise arm as well. Meta-analysis of effect in four hand exercise studies was undertaken [15,17,20,21] and the results are summarized in Fig. 2. Two studies were not included due to the lack of a comparative arm or addition of a co-intervention, namely blood flow restriction, to hand exercise.The overall effect size of hand exercise on distal cephalic vein calibre without a tourniquet is ≈0.24 mm (95% CI 0.03-0.45).The heterogeneity is minimal at ≈16%.As can be seen in the forest plot in Fig. 3, the effect size from the RCTs was significantly higher with narrow CIs as compared with non-RCTs, possibly because the non-RCTs used the non-exercise arm of the same patients as a control and hence the systemic influence of exercise on the non-exercise arm underestimated the effect size of hand exercise on the cephalic vein in the exercise arm. The study by Rus et al. [19] also looked at the effect of hand exercise on endothelium-dependent vasodilation, but no significant effect was found. The only single-arm prospective cohort study on intermittent pneumatic compression using a Fist Assist device found a significant improvement in baseline cephalic vein diameter on fixed landmarks of the forearm and upper arm with or Not tested Only one RCT [15] tested this outcome and found no difference between the control and intervention arms (50% lower primary failure in exercise arm, although not statistically significant) One RCT [15] demonstrated significantly high rate of distal AVF creation in the exercise arm versus the control arm: 75% versus 51% (P = .008).In another study [21], potential access sites increased from 12 at baseline to 33 (P < .001) and 23 (P = .047),after 4 and 8 weeks of exercise, respectively Intermittent pneumatic compression (Fist Assist device) Single-arm prospective study [22] Mean increase in forearm cephalic vein diameter by 0.41 mm (CI 0.12-0.without blood pressure cuff application and also increased the proportion of cephalic veins that is suitable for AVF creation [22]. Adverse events of the intervention A common finding seen with hand exercise regimens in all the studies is that, according to logbooks, the adherence rate decreases after 4 weeks, as well as grip strength measurements.In the study by Aragoncillo Sauco et al. [15], 5 of 68 patients dropped out of the elastic band exercise due to shoulder pain and only 60% followed the suggested exercise regimen.Otherwise, overall engagement with exercise was good in all studies, especially in the first 4 weeks.Adherence to the Fist Assist device was also reasonable, with at least 300 recorded sessions in a 3-month period and no adverse events. Publication bias and moderator analyses The funnel plot of the four studies (see Fig. 4) did not indicate any asymmetry; however, the number of studies is small.Similarly, owing to the small number of studies, moderator analysis were not possible. DISCUSSION This systematic review and meta-analysis assessed the efficacy of various forms of preoperative interventions aimed at improving AVF outcomes.The striking finding was the paucity of intervention studies applied to this cohort of patients.Hand exercise was the predominant form of intervention and was used with varying combinations of isometric or isotonic exercise with or without the addition of blood flow restriction.Although the exercise regimens were variable, they were usually applied for ≈6-8 weeks, but peak engagement occurred in the first 4 weeks, after which it appeared to decrease.Most of the exercise regimen seemed to be well tolerated, apart from slow arm contractions using the elastic band, which is associated with shoulder pain, leading to discontinuation of exercise. Although the effect size of hand exercise on cephalic vein size seems to be quite small in terms of an increase in diameter, this might translate to a better outcome, as flow in a vessel is inversely related to the fourth power of its radius.Unfortunately, only one exercise study, by Aragoncillo Sauco et al. [15], followed patients until AVF maturation to assess the effect on primary failure and AVF maturation, which is the hard and desirable outcome measure.Although the study demonstrated a decrease in primary failure in the exercise arm compared with the non-exercise arm, it did not reach statistical significance, due to the overall low rate of primary failure. Two studies employed a split-body trial design, where the non-exercise arm was used as the control.There have been studies [23] that have shown that exercise with one limb leads to systemic vasodilation and changes in vessel calibre in the opposite limb.As typically the dominant arm was the nonexercise arm in these studies (as AVFs are usually created in the dominant arm), exercise with the non-dominant arm may have more influence on the dominant arm.This can lead to underestimation of the effect size of hand exercise. The latest Kidney Disease Outcomes Quality Initiative guidelines [24] suggest not relying on vessel diameter but to test vessel function, as recent studies on AVF maturation have revealed that vessel function parameters are good predictors of AVF maturation [25].Only one study in this analysis tried to study the effect of hand exercise on endothelial-dependent vasodilation [19], but it was undertaken among patients who were already on HD and hence known to have severely impaired endothelial function. There were not enough studies to do a moderator analysis to see which subgroup of patients might benefit more from exercise. The other preoperative intervention that was tested was cholecaciferol supplementation, which was tested in a well-conducted double-blind RCT and was not found to be effective in improving the rate of AVF maturation. Finally, the Fist Assist device [22] demonstrated reasonable safety and efficacy in improving upper arm and forearm cephalic vein calibre and also in increasing the proportion of veins suitable for AVF creation.This is one example where a proven intervention after AVF creation [27] has been tested and proven to be effective in the preoperative period [22]. Several interventions, including infrared radiation [26], that have been employed after AVF creation to assist with the maturation could also be potentially tried in the preoperative period, which is the window of opportunity available to improve AVF outcomes. The limitations of our review are restriction of studies to those in English, inability to do moderator analysis due to the paucity of studies, the paucity of hard outcome data and an inability to account for error in diameter measurements by ultrasound, which is influenced by operator experience, body temperature, environmental temperature, medications etc.However, the strength of the review is identification of gaps in knowledge in the preoperative interventions and the positive effect of exercise on vein calibre that might be enhanced with other co-interventions, thereby improving AVF outcome. C ONCLUSION There is a pressing need to do large-scale RCTs with hard endpoints of preoperative interventions that can improve the structure and function of upper arm vessels prior to AVF creation in order to facilitate favourable remodelling after AVF creation.Multiple co-interventions employing innovative trial designs will help in identifying the synergistic effects of such interventions and filling the current gaps in knowledge. Figure 1 : 1 Figure 1: PRISMA flow diagram. Figure 3 : 3 Figure 3: Forest plot of the effect of hand exercise on end-of-follow-up distal cephalic vein calibre (in mm) without a tourniquet, categorised according to study design. Figure 4 : 4 Figure 4: Funnel plot of eligible studies indicating symmetry and hence absence of publication bias. Table 1 : Summary of included studies with baseline characteristics. 1Outcome measurePrimary: percentage PF by 12 weeks;secondary: mean increase in venouscalibre, mean increase in PSV, %increase in distal AVF creationPrimary outcome: increased CVdiameter (mm) of at least 0.22 mm;secondary: increased CV distensibility(mm), increased RA diameter,increased PSV (cm/s) and meanvelocity (cm/s) in the upper limbs,increased forearm circumference (cm)and increased HGSPrimary: mean change in the distalforearm CV diameter between theintervention and control group atweeks 4 and 8; secondary: HGSPrimary: mean change in venousdiameter between the control and studyarm at weeks 4 and 8; secondary:proportion of subjects that achieved atleast one CV diameter >2.5 mm,proportion of subjects who had asuccessful AVF placementChange/increase in forearmcircumference, venous diameter withand without tourniquet, arterialdiameter and flow, HGS, EDV at8 weeksChange/increase in distal CVcapacitance as measured by CSA withand without a tourniquet, increase inHGSAVF maturation, defined as the abilityto cannulate the AVF with twolarge-bore needles at ≥6 consecutivedialysis sessions and achievement of anAVF blood flow >300 ml/min assessedat 6 months after AVF creationIncrease in upper arm and forearmcephalic vein diameter with andwithout blood pressure cuff, proportionof veins achieving threshold forsuitability for AVFDiabetics(%)IA: 52.8%CA: 55.7%IA: 75%CA: 21.4%IA: 18.8%CA: 16.7%20%35.7%60%IA: 45%CA: 50%59.5%Gender (%Males)IA: 79.2%CA:72.1%IA: 66.7%CA: 71.4%IA:75%CA: 77.8%46%50%100%IA: 75%CA: 62.5%56.8%Mean ageIA: 64.7(21.1) CA:67.9 (55.3)IA:61.33 ± 7.82CA:60.14 ± 10.67IA:48.6 ± 3.4CA:43.4 ± 3.768.7 ± 4.249 ± 1157 ± 9IA:49.9 ± 10.9CA:52.1 ± 14.962.1 ± 12.61Study size167 screened,138randomized, 68 inintervention and 70in control35 screened, 26randomized, 12 inBFR and 14 in CA36 patients, 18intervention, 18control. 2 inexercise arm lost toFU15 patients (28limbs examined, asno detectable distalveins in 2non-exercise limbs)14 patients, 7 maleand 7 female5 patients52 randomized, 25to cholecalciferol,27 to placebo. Finalanalysis: 44: 20cholecalciferol and24 placebo37 patients (46enrolled)Population source4 centres inMadrid, Spain,CKD stage 4/5Nephrologyoutpatients, CKD4/5Nephrologyoutpatients, CKD4/5Nephrologyoutpatients, CKD4/5HD patientsNephrologyoutpatient, CKDwith SCr >1.5Adult ESRDpatients receivingin-centre MHDand planned forAVF creationCKD stage 4 and 5patients anticipatedto start HDMean follow-upAVF surgery after8 weeks of exerciseand follow-up until3 months after AVFcreation8 weeks fromrecruitment8 weeks8 weeks8 weeks6 weeks6 months after AVFcreation84 hours/day for3 monthsStudy designRCT, open labelRCT, double blindRCT, open labelSplit-body trialSingle-armprospective studySplit-body trialRCT, double blindSingle-armprospective studyStudy and year InterventionSauco et al., 2019 IA: isometric[15] exercise, hand gripcontractions; elasticband, arm flexionand extensionCA: no exerciseBarbosa et al., 2017 IA: exercise[16] (isometric andisotonic) BFRtrainingCA: exercisewithout BFRKumar et al., 2020 IA: isometric hand[17] grip exerciseCA: no exerciseUy et al., 2012 [21] IA:isometrichandgrip exercise in onearmCA: opposite limbof same patientRus et al., 2003 [19] Isometrichandgrip exercise innon-AVF armLeaf et al., 2003 IA: isometric hand[20] grip exercise in onearmCA: opposite limbof the same patientWasse et al., 2011 IA: high-dose[18] cholecalciferol200 000 U onceweekly for 3 weeksCA: placeboHammes et al., Intermittent2021 [22] pneumaticcompression of thearm using FistAssist device BFR: blood flow restriction; CA: control arm; CSA: cross-sectional area; CV: cephalic vein; HGS: hand grip strength; IA: intervention arm; MHD: maintenance haemodialysis; PSV: peak systolic velocity; RA: radial artery.2334S. Ramanarayanan et al Table 2 : Summary of exercise regimens and effect on various vessel structure and function parameters across selected studies. 2Artery parametersIncrease in calibre by 0.12 mm(SD 0.31; P = .008) in IADecrease in calibre by 0.02 mm(SD 0.32; P = .55) in CANo significant changes in meanvelocity at all segments acrossboth groups (P = .279, .150and .341 at 2, 10 and 20 cm)Not measuredNot assessedNo significant change inendothelium-dependentvasodilationNot assessedEffect size on vein calibre incontrol armMean decrease of 0.112 mm(SD 0.48; P = .121) at 8 weeksMean increase in CVdiameter of 0.24, 0.39 and0.17 mm for the segments 2,10 and 20 cm (P = .008, .001and .237at these segments)No significant change with orwithout tourniquetMean changes in distal sites:0.81 ± 0.20 at 4 weeks and0.65 ± 0.15 at 8 weeks; inproximal sites:0.71 ± 0.20 mm at 4 weeksand 0.43 ± 0.20 at 8 weeks.No significant difference inproximal or distal sites, asdiameter increased in botharms (inter P = .209, .217,.726 and .826)No control armCephalic vein size increasewas ≈0.4 mm in non-exercisearm (P = non-significant)Effect size on vein calibre inintervention armMean increase of 0.71 mm(SD 0.49; P < .001) at 8 weeksMean increase of 0.20, 0.16and 0.15 mm for thesegments 2, 10 and 20 cm inCV (P = .16, .20 and .33 atthese segments)Mean change 0.31 ± 0.04 at4 weeks (P < .05) withouttourniquet and 0.40 ± 0.06with tourniquet (P < .05)Mean changes in distal sites:0.48 ± 0.16 at 4 weeks and0.32 ± 0.20 at 8 weeks; inproximal sites:0.59 ± 0.25 mm at 4 weeksand 0.52 ± 0.32 at 8 weeksAverage vein diameterwithout tourniquet remainedunchanged for 4 weeks butsignificantly increased after8 weeks (P = .015). Averagevein diameter after tourniquetsignificantly greater after4 weeks (P = .007) and even.001). greater at 8 weeks (P <However, distensibilityremained unchanged2-fold increase in CVdiameter noted in mostpatients (P < .05)Grip strengthSignificant increase of 4.33 kg(SD 4.5) in IA (P < .001);non-significant increase of3.4 kg (SD 2.5) in the CA(P = .46)Significant increase indynamometry: in CA, 2.36 kgincrease (P = .003); in IA,2.25 kg increase (P = .06). Nosignificant difference inincrease in strength betweenarms, inter P = .302Increased by median of 4 kgon exercise arm; nosignificant increase in controlarmIncrease in the exercised armfrom 0 to 8 weeks:24.50 ± 2.05 kg to27.04 ± 2.20 kg (P = .025).No change in control arm:26.68 ± 2.60 kg to26.82 ± 2.40 kg P = .929Maximal hand grip strengthmeasured with dynamometerincreased significantly from24.1 ± 2.95 mm to26.2 ± 3.06 mm after 4 weeksand to 28.5 ± 3.17 mm after8 weeks (P < .001)Paradoxically, the increase invenous size wasunaccompanied by anincrease in hand grip strengthIntervention, co-interventions and controlsTwo sets of 30 ISM hand grip contractionstwice a day. At noon: slow contractionsession using elastic band with arm in flexionand extension. Comparator: no exercise.Duration 8 weeksISM + IST with tensiometer for BFR inintervention arm. Exercise only without BFRin control. Duration 8 weeksISM handgrip exercise: 20 repetitions/min,30 min/day. Comparator: no exercise.Duration 8 weeksISM forearm strengthening exercises inpreferred access arm: 10 sets of 20repetitions/min. Comparator: non-exercisearm. Duration 8 weeksISM hand grip exercise using a rubber ring:4.5 cm inner and 7.5 cm outer diameter,maximal compression force 50 N. Frequencyand intensity: trained and supervised bydialysis staff on HD days6 weeks of ISM exercise at 30-40% MVC for80-360 sec with increased frequency overtime and repetitive isotonic squeezing of asquash ball or racquet ball. Non-exercise armwas comparator. Duration and frequency: 4times per week for 6 weeks. Controlledheating employedAuthor andyearSauco et al.,2021 [15]Barbosa et al.,2018 [16]Kumar et al.,2020 [17]Uy et al., 2012[21]Rus et al., 2003[19]Leaf et al.,2003 [20] CA: control arm; CV: cephalic vein; IA: intervention arm; ISM: isometric; IST: isotonic; MVC: maximal voluntary contraction; RA: radial artery. Table 3 : Comparison of effect size of various preoperative interventions on AVF outcomes and some salient outcome measures tested by these interventions. 3Proportion of veins achieving thethreshold for AVF creationNot testedEffect on primary failure/thrombosis/re-interventions/re-anastomosis/plastiesNot testedEffect on AVF maturationAVF maturation at 6 months:45% in cholecalciferol groupand 54% in the placebo group(P = .8)Effect size on forearm cephalic veindiameterNot testedMean increase in vein diameter of0.24 mm (CI 0.03-0.45). If only RCTsare included, the mean increase is0.29 mm (CI 0.11-0.47)Source of data1 RCT [17]Meta-analysis of 4 trialsconducted by us (2 RCT and2 non-RCT) [15, 17, 20, 21]Intervention typeCholecalciferolsupplementationHand exercise Forest plot of the effect of hand exercise on end-of-follow-up distal cephalic vein calibre (in mm) without a torniquet. ExerciseControlMean differenceMean differenceStudy or subgroup Aragoncillo et al. Kumar Leaf Uy Total (95% CI) Heterogeneity: τ 2 = 0.01, χ 2 = 3.59, df = 3 (P = 0.31), I 2 = 16% Mean (mm) 3.52 2.15 SD (mm) 0.93 0.4 Total 68 16 Mean (mm) 3.21 1.87 SD (mm) 0.98 0.21 2.47 1.98 1.42 0.73 5 15 1.51 2.2 1.18 0.79 Test for overall effect: Z = 2.25 (P = 0.02) 104 Exercise Control Figure 2: Study or subgroup Mean SD Total Mean SDTotal 70 16 5 13 104 TotalWeight 32.5% 53.5% 1.6% 12.3% 100.0% WeightIV, Random, 95% CI 0.31 [-0.01, 0.63] 0.28 [0.06, 0.50] 0.96 [-0.66, 2.58] -0.22 [-0.79, 0.35] 0.24 [0.03, 0.45] Mean difference IV, Random, 95% CI-430% of forearm and 43% of upper Increase in vein calibre arm veins crossed the threshold IV, Random, 95% CI needed for AVF creation from being 0 Decrease in vein calibre -2 2 Mean difference IV, Random, 95% CI4 under the threshold(mm)(mm)(mm)(mm)2.1.1 RCTAragoncillo et al.3.520.93683.210.987032.5%0.31 [-0.01, 0.63]Kumar2.150.4161.870.211653.5%0.28 [0.06, 0.50]Subtotal (95% CI)848686.0%0.29 [0.11, 0.47]Heterogeneity: τ 2 = 0.00, χ 2 = 0.02, df = 1 (P = 0.88), I 2 = 0% Test for overall effect: Z = 3.12 (P = 0.002) 2.1.2 Non-RCTNot testedLeaf2.471.4251.511.1851.6%0.96 [-0.66, 2.58]Uy1.980.73152.20.791312.3%-0.22 [-0.79, 0.35]Subtotal (95% CI)Heterogeneity:Not tested.006)7; P =2336S. Ramanarayanan et al Preoperative interventions to support the maturation of AVFs S. Ramanarayanan et al SUPPLEMENTARY DATASupplementary data are available at ndt online.FUNDINGNone.AU THORS' C ONTRIBU TIONS S.R. was responsible for the design, analysis and interpretation of data and drafting of the manuscript.O.S. was responsible for analysis of data and study selection.S.S. was responsible for the design, analysis and interpretation of data and critical review of the manuscript.K.L. was responsible for statistical analysis, interpretation of data and critical review of the manuscript.H.U. was responsible for analysis and interpretation of data and the risk of bias analysis.K.F. was responsible for the concept, design, analysis and interpretation of data, critical review of the manuscript and final approval of the manuscript submitted.DATA AVAIL ABILIT Y STATEMENTThe data underlying this article will be shared upon reasonable request to the corresponding author.C ONFLICT OF INTEREST STATEMENTNone declared.The results presented in this article have not been published previously in whole or part. 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SUPPLEMENTARY DATA for 5-methyl-cytosine stabilizes DNA but hinders DNA hybridization revealed by magnetic tweezers and simulations Xiao-Cong Zhao Institute for Advanced Studies College of Life Sciences State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis Wuhan University 430072WuhanChina Hai-Long Dong Department of Physics School of Physics and Technology Key Laboratory of Artificial Micro & Nano-structures of Ministry of Education Wuhan University 430072WuhanChina Xiao-Lu Li Institute for Advanced Studies College of Life Sciences State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis Wuhan University 430072WuhanChina Hong-Yu Yang Institute for Advanced Studies College of Life Sciences State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis Wuhan University 430072WuhanChina Xue-Feng Chen Institute for Advanced Studies College of Life Sciences State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis Wuhan University 430072WuhanChina Liang Dai Department of Physics City University of Hong Kong 999077Hong KongChina Wen-Qiang Wu School of Life Sciences State Key Laboratory of Crop Stress Adaptation and Improvement, Key Laboratory of Plant Stress Biology Henan University 475001KaifengChina Zhi-Jie Tan zjtan@whu.edu.cn Department of Physics School of Physics and Technology Key Laboratory of Artificial Micro & Nano-structures of Ministry of Education Wuhan University 430072WuhanChina Xing-Hua Zhang Institute for Advanced Studies College of Life Sciences State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis Wuhan University 430072WuhanChina SUPPLEMENTARY DATA for 5-methyl-cytosine stabilizes DNA but hinders DNA hybridization revealed by magnetic tweezers and simulations S1 # These authors contributed equally to this work. *Corresponding authors S2 Section1. Preparation of DNA for MT experiments. As shown in Figure 1 in the main text, we prepared the DNA hairpin sample by following steps. (i) We amplified the 209-bp dsDNA (5206-5415 bp of λ-DNA) and the 211-bp dsDNA (11075-11286 bp of λ-DNA) as hairpin handles through PCR using hairpinF1-Thiol/hairpinR1_BsaI and hairpinF4_BsaI/hairpinR4 as primers, respectively. For the DNA sample used in Figures 1B-C, 2-3 and Figures S2-S8, S12A, we amplified the head-to-head two 502-bp dsDNA segments (3315-3817 bp of λ-DNA) as the hairpin stem through PCR using hairpinF2-BsaI/hairpinR2_BsaI and hairpinF3_BsaI/hairpinR3_BsaI as primers, respectively. We used Taq DNA polymerase (Thermo Scientific) to generate dsDNA. We used 5-methyl-dCTP to replace dCTP (New England Biolabs) in PCR to add methyl groups to cytosine of the dsDNA. (ii) We digested the four dsDNA segments to generate the 4-nt sticky ends using BsaI-HF (New England Biolabs). (iii) We ligated the four dsDNA segments to generate a 1424-bp combined dsDNA using T4 DNA ligase (New England Biolabs). (iv) We added biotin groups to 3' ends of the dsDNA using Terminal transferase (New England Biolabs) and biotin-11-dUTP (Roche). (v) We anchored the thiol-labelled end of dsDNA to the sulfo-SMCC (Thermo Fisher) coated glass surface. Then, we tethered a streptavidin-coated superparamagnetic microbead Dynabeads) to the other end of dsDNA through the biotin-streptavidin bond. We exerted a stretching force of ~40 pN on the dsDNA using magnetic tweezers. (vi) We peeled off the untethered ssDNA strand in the dsDNA at 100 mM NaOH and left the tethered ssDNA stretched at ~40 pN. (vii) Because the two hairpin stems contained complementary sequences, the long DNA hairpin zipped at 8 pN when we changed the buffer into 1 mM Tris-HCl pH7.5 and 150 mM NaCl. For the other DNA sample used in Figure S9, S10 and S12B, we amplified the head-to-head two 502-bp dsDNA segments (40701-41202 bp of λ-DNA) as the new hairpin stem through PCR using 2_hairpinF2-BsaI/2_hairpinR2_BsaI and 2_hairpinF3_BsaI/2_hairpinR3_BsaI as primers, respectively. We prepared this DNA sample by above steps except changed the sequence of the long hairpin. We synthesized all the oligos with the following sequences (Sangon Biotech). Figure S1. Home-built MT and flow cell. We built the MT using an inverted microscope equipped with an oil objective, on the top of which a flow cell was placed. We controlled the temperature with an objective heater. To assemble one flow cell, we stuck together two cover slides with doubles-side taps and draw the inlet and outlet with silicone rubber. Figure S13. The effects of 5mC on the unzipping and rezipping forces obtained at 150 mM NaCl using MD simulations. We stretched the 2-bp DNA hairpin through a force-cycle containing a force-increasing scan through a set of constant forces where the hairpin unzipped, followed by a force-decreasing scan through the same set of constant forces where the hairpin rezipped. At each constant force, we held the DNA for 150 ns during which we calculated the average in the extension of DNA in the last 50 ns. The force-cycle yielded an unzipping force-extension curve (filled symbols) and a rezipping force-extension curve (empty squares symbols). We determined the unzipping force to be the lowest force where an abrupt increase in extension occurred in the unzipping force-extension curve (upward arrow). Similarly, we determined the rezipping force to be the highest force where an abrupt decrease in extension occurred in the rezipping force-extension curve (downward arrow). Figure S2 . S5 Figure S3 .Figure S4 . S2S5S3S4The effects of 5mC on the unzipping-rezipping cycle of long DNA hairpin at various NaCl concentrations (A-D), MgCl2 concentrations (E-H) and temperatures (I-L) using a constant loading rate of 0.2 pN/s. (A-B), (E-F) and (I-J) Representative unzipping (filled squares) and rezipping (empty squares) forceextension curves. The determined unzipping and rezipping forces were marked as upward and downward arrows, respectively. (C), (G) and (K) The effects of 5mC on unzipping (filled squares) and rezipping (empty S4 squares) forces. The error bars were the standard deviations obtained from four molecules. We fitted the forces as linear functions of log([NaCl]/1 M), log([MgCl2]/1 M or temperature, respectively. We obtained the slopes donated them in the figures. (D), (H) and (L) The effects of 5mC on hysteresis in the unzipping-rezipping cycle. The error bars were the standard deviations obtained from four molecules. We fitted the hysteresis as linear functions of log([NaCl]/1 M), log([MgCl2]/1 M or temperature, respectively. We obtained the slopes donated them in the figures. The obtained slopes were: Non-Me DNA, unzipping force versus log([NaCl]/1 M), 4.7 pN (Figure S2C) 5mC DNA, unzipping force versus log([NaCl]/1 M), 4.7 pN (Figure S2C) Non-Me DNA, rezipping force versus log([NaCl]/1 M), 4.7 pN (Figure S2C) 5mC DNA, rezipping force versus log([NaCl]/1 M), 4.8 pN (Figure S2C) Non-Me DNA, unzipping force versus log([MgCl2]/1 M), 1.3 pN (Figure S2G) 5mC DNA, unzipping force versus log([MgCl2]/1 M), 1.2 pN (Figure S2G) Non-Me DNA, rezipping force versus log([MgCl2]/1 M), 1.3 pN (Figure S2G) 5mC DNA, rezipping force versus log([MgCl2]/1 M), 1.6 pN (Figure S2G) Non-Me DNA, unzipping force versus temperature, -0.22 pN/ o C (Figure S2K) 5mC DNA, unzipping force versus temperature, -0.21 pN/ o C (Figure S2K) Non-Me DNA, rezipping force versus temperature, -0.20 pN/ o C (Figure S2K) 5mC DNA, rezipping force versus temperature, -0.04 pN/ o C (Figure S2K) Non-Me DNA, hysteresis versus log([NaCl]/1 M), -57.8 pN· nm (Figure S2D) 5mC DNA, hysteresis versus log([NaCl]/1 M), -114.6 pN· nm (Figure S2D) Non-Me DNA, hysteresis versus log([MgCl2]/1 M), -43.3 pN· nm (Figure S2H) 5mC DNA, hysteresis versus log([MgCl2]/1 M), -305.8 pN· nm (Figure S2H) Non-Me DNA, hysteresis versus temperature, -7.8 pN· nm/ o C (Figure S2L) 5mC DNA, hysteresis versus temperature, -64.3 pN· nm/ o C (Figure S2L) The effects of 5mC on the unzipping-rezipping cycle of long DNA hairpin at various NaCl concentrations (A-D), MgCl2 concentrations (E-H) and temperatures (I-L) using a constant loading rate of 1 pN/s. (A-B), (E-F) and (I-J) Representative unzipping (filled squares) and rezipping (empty squares) forceextension curves. The determined unzipping and rezipping forces were marked as upward and downward arrows, respectively. (C), (G) and (K) The effects of 5mC on unzipping (filled squares) and rezipping (empty squares) forces. The error bars were the standard deviations obtained from four molecules. We fitted the forces as linear functions of log([NaCl]/1 M), log([MgCl2]/1 M or temperature, respectively. We obtained the slopes donated them in the figures. (D), (H) and (L) The effects of 5mC on hysteresis in the unzipping-rezipping cycle. The error bars were the standard deviations obtained from four molecules. We fitted the hysteresis as linear functions of log([NaCl]/1 M), log([MgCl2]/1 M or temperature, respectively. We obtained the slopes donated them in the figures. The obtained slopes were: Non-Me DNA, unzipping force versus log([NaCl]/1 M), 4.5 pN (Figure S3C) 5mC DNA, unzipping force versus log([NaCl]/1 M), 4.6 pN (Figure S3C) Non-Me DNA, rezipping force versus log([NaCl]/1 M), 4.8 pN (Figure S3C) 5mC DNA, rezipping force versus log([NaCl]/1 M), 4.9 pN (Figure S3C) Non-Me DNA, unzipping force versus log([MgCl2]/1 M), 1.4 pN (Figure S3G) 5mC DNA, unzipping force versus log([MgCl2]/1 M), 1.2 pN (Figure S3G) Non-Me DNA, rezipping force versus log([MgCl2]/1 M), 1.4 pN (Figure S3G) 5mC DNA, rezipping force versus log([MgCl2]/1 M), 1.7 pN (Figure S3G) S6 Non-Me DNA, unzipping force versus temperature, Comparison of the extension histograms with free energy landscape at 150 mM NaCl 22 o C under a constant force of 18.0 pN. (A) The free energy landscape as a function of the location of the unzipping fork (proportional to extension) was calculated using the 10 nearest-neighbor base-pair energies obtained in forceinduced DNA unzipping experiments by optical tweezers. (B) The extension histograms obtained from four molecules. Figure S5 .Figure S6 . S5S6The repetitive constant-force DNA unzipping experiments at 150 mM NaCl and 22 o C. The extensions of fully zipped and unzipped DNA are marked as dashed horizontal lines. The beginning and end of unzipping are enlarged for clarity. (A) Unzipping of non-Me DNA using other three independent DNA molecules. (B) Unzipping of 5mC DNA using other three independent DNA molecules. The repetitive constant-force DNA rezipping experiments at 150 mM NaCl and 22 o C. The extensions of fully zipped and unzipped DNA are marked as dashed horizontal lines. The beginning and end of DNA rezipping are enlarged for clarity. (A) Rezipping of non-Me DNA using other three independent DNA molecules.(B) Rezipping of 5mC DNA using other three independent DNA molecules. Figure S7 .Figure S8 . S7S8The effects of 5mC on the unzipping-rezipping cycle of long DNA hairpin at 150 mM NaCl, 100 mM MgCl2 and 36 o C. The unzipping (filled squares) and rezipping (empty squares) force-extension curves of 5mC DNA (magenta) and non-Me DNA (olive) in one unzipping-rezipping cycle are plotted. For each molecule at each salt and temperature condition, we repeated the force-cycles three times and used at least four molecules to obtain the averages and standard deviations of the unzipping force, rezipping force and hysteresis. The effects of 5mC on the time-courses of DNA unzipping and rezipping at constant force at 100 mM MgCl2, 150 mM NaCl and 36 o C. The beginning and end of unzipping or rezipping are enlarged for clarity. (A) Unzipping of non-Me DNA. (B) Unzipping 5mC DNA. (C) Rezipping of non-Me DNA. (D) Rezipping 5mC DNA. (E) The probability of rezipping for 5mC DNA as a function of applied force obtained by 25 repetitive measurements at each constant force for each molecule. The error bars were the standard deviations obtained from four molecules. Figure S9 . S10 Figure S10 . S9S10S10The effects of 5mC on the time-courses of DNA unzipping and rezipping at constant force using the other 502-bp DNA sequence at 150 mM NaCl and 22 o C. The beginning and end of unzipping or rezipping are enlarged for clarity. (A) Unzipping of non-Me DNA. (B) Unzipping 5mC DNA. (C) Rezipping of non-Me DNA. (D) Rezipping 5mC DNA. (E) The probability of rezipping for 5mC DNA as a function of applied force obtained by 25 repetitive measurements at each constant force. The error bars were the standard deviations obtained from four molecules. The effects of 5mC on the time-courses of DNA unzipping and rezipping at constant force using another 502-bp DNA sequence at 150 mM NaCl, 100 mM MgCl2 and 36 o C. The beginning and end of unzipping or rezipping are enlarged for clarity. (A) Unzipping of non-Me DNA. (B) Unzipping 5mC DNA. (C) Rezipping of non-Me DNA. (D) Rezipping 5mC DNA. (E) The probability of rezipping for 5mC DNA as a function of applied force obtained by 25 repetitive measurements at each constant force for each molecule. The error bars were the standard deviations obtained from four molecules. Figure S11 . S11 Figure S12 . S11S11S12The 5mC slowed down DNA hybridization at 150 mM NaCl and 100 mM MgCl2 revealed by MD simulations. (A) The average H-bond distance as a function of the MD simulation time from three independent MD trajectories. The dash lines donated the H-bond distance of the folded state. The average and standard deviation of the time to fold () obtained from three independent MD trajectories were donated. (B) The hybridization time for non-Me DNA, and 5mC DNA at different salt conditions. The error bars denoted the standard deviations obtained from different MD trajectories. The beginning of non-Me DNA rezipping at 150 mM NaCl and 22 o C using the 502-bp DNA hairpin. (A) The DNA hairpin used in Figures 1B-C, 2-3 and Figures S2-S8. (B) The DNA hairpin used in Figures S9-S10. hairpinF1 - hairpinF1Thiol: thiol/TACCGAGGCTGCAGTGTAChairpinR1_BsaI: CGATCGGTCTCGCTGGCACCACGTC hairpinF2-BsaI: CGATCGGTCTCACCAGTTCGTCGCGGCTTTTCCG hairpinR2_BsaI: CGATCGGTCTCAAAAACGCCTCCCAGCCGGACCGG hairpinF3_BsaI: CGATCGGTCTCTTTTTCGCCTCCCAGCCGGACCGG hairpinR3_BsaI: CGATCGGTCTCATCAGTTCGTCGCGGCTTTTCCG hairpinF4_BsaI: CGATCGGTCTCGCTGACGTTTAACCAGACCAGCG hairpinR4: ACACGTTATGGAACTGGCGAGCCATC 2_hairpinF2-BsaI: CGATCGGTCTCACCAGTGTTCACAACCTGTATCCA 2_hairpinR2_BsaI: CGATCGGTCTCAAAAAGCAGTACAGCAAATCCTT 2_hairpinF3_BsaI: CGATCGGTCTCTTTTTGCAGTACAGCAAATCCTT 2_hairpinR3_BsaI: CGATCGGTCTCATCAGTGTTCACAACCTGTATCCA S3

Virtual fashion experiences in virtual reality fashion show spaces 17 November 2023 SeJin Kim sejinkim@changwon.ac.kr Osvaldo Gervasi Annamaria Recupero Se Jin Kim Department of Clothing and Textiles Changwon National University ChangwonRepublic of Korea University of Perugia Italy University of Siena Italy Junwei Cao Yangzhou University China Virtual fashion experiences in virtual reality fashion show spaces 17 November 2023F819B4FEBB3D71057C6C95ACBE10435710.3389/fpsyg.2023.1276856RECEIVED 13 August 2023 ACCEPTED 07 November 2023virtual fashion experiencefashion showvirtual fashion spacecognitive presencesensible immersionemotional immersionaesthetic interaction Introduction: Virtual reality (VR) provides a new fashion space and fashion experience.This study focuses on immersive VR and fashion shows to empirically explore the VR fashion space and fashion experience.Insights specific to fashion have not been presented in as much depth in the literature; thus, the current findings are particularly valuable and insightful.Methods: This study employed three immersive VR (IVR) fashion show stimuli and in-depth interviews according to a semi-structured questionnaire.Collected data were analyzed based on the concept of VR space and VR experience derived through literature research.Results: The VR fashion space was divided into three types and VR experiences of cognitive presence, sensible immersion, emotional immersion, and aesthetic interaction were derived accordingly.First, the physical representation of a fashion show induced a cognitive and emotional sense of presence, in which users felt as though they had moved to the same time and place as those at the fashion show.Second, participants experienced cognitive confusion owing to the differences with a priori experiences in the fashion show space (i.e., reality and imagination coexist).Third, participants transcended the limitations of physical reality while in the fashion show space of pataphysics (which was realized with human imagination), and they moved beyond the stage of confusion that is experienced while facing realistic objects to connect to creative inspiration.Discussion: The difference in the properties of VR space may be associated with distinct VR fashion experiences.The findings suggest that (1) a priori elements such as sociocultural contexts and personal experiences differ in the experiential dimension of virtual space, (2) the VR fashion show space induces a psychological experience between brand and consumer, and (3) creative inspiration and exploratory play can be greatly induced in a user if the immersive fashion space is further from the original source. Introduction Humans accumulate experience when interacting with space, which is a medium through which the world is perceived (Tuan, 2011).Space is signified through dynamic interrelationships between itself and the various elements that constitute it (Lefebvre, 1991).As space is not just a static, physical background, attention needs to be paid to its active aspects. Virtual reality (VR) spaces provide humans with virtual spaces, with social functions that are similar to space in reality (Barreda-Ángeles and Hartmann, 2022).For example, VR can facilitate social interactions between people who are geographically distant, enabling collaboration.Humans accumulate novel experiences in this new space.A VR space can provide an environment that is either similar to reality or one that is completely new.The space allows users to feel an immediate sense of immersion, even more so than the sense of presence in the real world and physical reality, and this virtual human experience is real (Pelet et al., 2017).Therefore, the experiential impact that the new digital environment has on humans needs to be determined. VR affects fashion shows, which are a site for the presentation of fashion and serve as a means of communication between fashion brands and audiences.Space has not been given the same consideration as clothes vis-à-vis fashion shows.Yet, fashion shows cannot be held without space.It is an important component in developing and conveying the concept and image of a fashion collection (Mendes, 2019).Various places have been used effectively to convey the fashion show's image and concept (Strömberg, 2019).VR technology allows users to access fashion shows at any time and from any place.Advances in communication infrastructure such as the Internet of Things and 5G technology support this, and a Metaverse world that can be accessed from anywhere in the world is opening up.VR spaces based on the properties of digital media share the characteristics of imagined and physical images (Wideström, 2019).They transcend regional, cultural, and temporal boundaries and have unique spatial characteristics that cannot be experienced in a physical space.Thus, VR fashion show spaces can affect the fashion experiences of users. Fashion studies have focused on topics such as the use of virtual environment technology in the development of fashion stores and designs and the effect they have on user experience.Park et al. (2018) showed that VR fashion stores have a positive impact on users' shopping behavior vis-à-vis enjoyment and purchase intention.Sina and Wu (2023) revealed that the appropriate use of design elements in VR fashion stores leads to high results regarding consumers' emotions, perceptions, and satisfaction levels.Lighting color and color temperature in a 360-degree VR space are also related to consumers' shopping motivation.Jung et al. (2021) noted that expanding the experience of a luxury fashion show (which had been allowed to a small, limited audience previously) to the public would popularize luxury items. These findings are significant as they suggest that human perception and experience of the fashion industry can be affected by the relevant VR technology.However, a fashion show differs from a fashion store with regards to purpose, content, and experience.Fashion shows provide fashion images, communication, and entertainment.VR fashion show spaces allow various interactions between brands, designers, and users to create new meaning for fashion shows and novel fashion experiences.Therefore, investigating such content is meaningful as it will expand on previous studies to present a new perspective. This study focused on immersive VR environments and students majoring in fashion.Immersive VR (IVR) uses wearable devices such as head-mounted displays (HMDs;Shahrbanian et al., 2012).Ricci et al. (2023) noted that IVR was associated with higher hedonic and utilitarian values, and better user experience when compared to general VR.IVR provides higher levels of sense of presence, immersion, and emotion than does general VR (Kerrebroeck et al., 2017).The experiential dimension could differ based on user characteristics; users with higher levels of openness to experience (Costa and McCrae, 1997; which is a prominent characteristic of artists) experience a deeper aesthetic experience through the sense of presence in VR (Starkey et al., 2021).Therefore, fashion students are required to have emotional and sensory experiences that will enhance their IVR experiences compared to general students. This study investigated users' fashion experiences in IVR fashion show spaces, in which VR and an HMD are used.VR fashion show spaces refer to the virtual realm in which fashion shows are conducted using IVR technology.This study explored the following research questions: First, how are VR spaces and experiences defined?Second, what kind of fashion experiences do users have in VR fashion shows?A framework of analysis was prepared through a literature review for research question 1.For research question 2, in-depth interviews were conducted with undergraduate fashion students with higher levels of immersion to analyze users' virtual fashion experiences through VR fashion shows. 2 Literature review 2.1 The VR space as an experience-creating medium Perspectives and characteristics of space Space is the realm or world in which certain substances and objects can exist or events can occur and is simultaneously universal and abstract.It is a medium that defines human thoughts and forms individuals' unique experiences (Buttimer and Seamon, 2015).Space is formed in the relationship between an object and the human who perceives and recognizes it (Ashihara, 1981). Experiences are formed during a human's interactions with a given space.This sociological approach to space explains the interactions between people, space, and human behavior rather than focusing on the meaning of physical backgrounds.This approach is useful in establishing a conceptual framework to unpack human experiences in VR space. There are many discussions on space from a sociological perspective.The first is the space of physical interactivity.Space is not an abstract entity that is distinct from the objects existing within it; it can only be understood through concrete events or objects (Choi, 2016).Löw and Weidenhaus (2017) defined space as a relational arrangement of social goods and living beings existing in places.Space does not exist independently.It is formed by exchanges that take place among the sociocultural background, and the actors and products within it.Therefore, space is created by social institutions and actors and cannot exist independently from these elements.Simmel (2005) paid attention to the kinds of spatial experiences that people undergo in a city, and the cognitive functions that take place, and found that the meaning of space is created, and interactions are amplified through human engagement.Space is nothing in itself but becomes full through interactions and gains meaning vis-à-vis individuals and social groups (Schroer, 2010). The second is the space of fluidity and dynamism.Modern society is flexible and fluid because of its fast pace and advancements in information technology.Bauman (2005) distinguished this from the solidity of the modern era and described the spatial characteristics of modern society as being liquid.He considered a city an intensely liquid space that is not fixed in one space and is consistently moving owing to the loose solidarity between the individualized elements constituting the liquid.Advancements in information technology cause changes in social patterns and create new spatial logics called the Space of Flow (space-time created by the fluidity of changes among social institutions, cultures, and members; Castells, 2010).Therefore, space is not fixed and eternal, but rather expands, integrates, and fractures in its relationship with changing social institutions, cultural contexts, and members. The third is the space of symbolism.Foucault presented the concept of heterotopia; for example, everyday spaces such as public baths, prisons, and colonies embody either deviant ideals within the institutions of modern society or incompatible utopian concepts (Foucault, 1967).This interpretation disproves the idea that space has a symbolic meaning.Lefebvre (1991) considered space a social product and stated that it can control or influence human consciousness and behavior.The framework of social space was based on trialectics, which includes representations of space as perceived through human activities, cognitive space such as lifestyles, and space of representations that is experienced with a combination of symbols and images (Lefebvre, 1991).It is a view of space as a social product and its representation differs from the second perspective in terms of the formative identity of space.Soja (1989) stated that space is formed as material spatial practices that reproduce the actual form and concrete patterns of lifestyles, which are transformed into an imaginary conceived space that becomes conceptual and is expressed symbolically, changing into a lived space that is structured by the experiences of individuals or groups.These discussions suggest that space is created by humans, the agents of action, and can be symbolically realized through interactions with social institutions.Thus, space is a result of human interaction and a social construct. Accordingly, this study approaches the spatial characteristics of VR space by considering the properties of digital space in the next chapter based on the three perspectives regarding the concept of space. Approach to fashion shows and VR spaces By reviewing previous studies on digital media spaces (Choi and Park, 2019;Suh, 2020) to determine their characteristics, this study identified the following: ambiguity and extensibility of space-time boundaries; complexity and flexibility of information; virtuality of reality; interactions that enable two-way communication between information providers and users; and ease of access to information.These characteristics are classified as follows by comprehensively considering the properties of space examined earlier (Table 1). First, the space of physical representation is the space in which the real world's physical appearance is projected and reconstructed.Although VR space is artificial, it is reproduced by reflecting reality (Lombard et al., 2006;Deng et al., 2019).New media interacts with old media (Bolter and Grusin, 2000).Second, the mixed space of reality and imagination is the space in which reality and virtuality coexist, which is characterized by the flexibility and dynamism of information, and in which space and time are not clearly separated.Modern space has a complex and fluid character and breaks away from the existing fixed concept of space and time owing to the rapid exchange of information and advances in the media (Bauman, 2005).Digital media spaces are characterized by a property of variability that differs from physical space and old media (Manovich, 2001).Therefore, the VR space has a mixed character that encompasses all these properties, in which reality and virtuality coexist owing to the variability and fluidity of digital images.For example, while components of the traditional fashion show reproduce the space-time of reality in the VR space, they construct a new fashion show space by creating an ideal object or by transforming the existing appearance.Third, the space of pataphysics is based on symbolic creation.The term pataphysics was proposed by Alfred Jarry and refers to an area of study that extends beyond metaphysics, and to the virtual nature of things based on imagination (Bok, 2002).Space in pataphysics cannot exist in reality but is realistically created by human imagination.Modern society is a world of simulacra in which reality has been replaced by symbols (Baudrillard, 1994).This VR space is the space of symbolic ideals in which symbolic objects are realized without material substance.The spatial characteristics of pataphysics are significant in the image-oriented fashion industry as they can express images that designers want to convey without limitations and communicate with users. VR experience Humans accumulate new experiences by interacting with various elements surrounding digital media.For example, digital images have a color range different with nature, which means it gives humans supernormal stimulation.VR technology also provides opportunities to travel to outer space and other places.As an example of VR fashion show, the 21FW Balenciaga collection-set in universe and in the form of a 3D virtual video game-was presented and audiences could easily access it anytime, anywhere. The VR resulting from digital media is a 3D environment that is reproduced to enable physical experiences as in the real world.In the VR space, users can navigate the space as they do in real life, interact with objects, and experience one or more of their five senses being stimulated in real time (Burdea and Coiffet, 2003).While more recent studies (Freeman et al., 2019;Rathinam et al., 2019) have focused on the possibility of VR technology to help human psychological treatment and physical rehabilitation, there is a lack of research that identifies emotional and aesthetic experiences, especially in fashion Thus, this study explored the virtual fashion experience through the concept of space and virtual experience.For this, a framework was built through a literature review about the concept of space as its focus, previous studies on the relationship between brands and users, and relevant studies on VR experiences, summarizing the results (Table 2). Cognitive presence The sense of presence refers to the psychological experience of feeling as though you are in a specific space in the virtual world or perceiving created objects, people, and events as being real (Lombard et al., 2006).The sense of presence can be divided according to the degree of interaction between the user and virtual space and media.Among the subdivisions, the cognitive sense of presence refers to the experience of perceptual illusion, in which the user mistakenly feels as though it is a real physical environment or object owing to the physical stimulations provided by digital media.This is a cognitive experience in which the user feels they are physically present in a specific space and perceives various information through sensory stimulations (Biocca and Levy, 2000); the cognitive sense of presence causes a user to experience place illusion, in which they feel as though they are in a specific place by experiencing the virtual space even though they are not actually present (Slater, 2009). As a sense of presence is based on sensory information, it increases when the media provides more detailed and elaborate information as if in real life (Lombard and Ditton, 1997).It is classified as the characteristics of media systems that generate a sense of presence concerning vividness and interactivity (Steuer, 1992).If high and intense levels of sensory information are acquired in the VR space, it causes high levels of vividness (Li et al., 2002).Interactivity refers to the degree to which a user can affect the content and form of a mediated environment in real time, and it is determined by the speed at which user inputs are reflected, as well as their range, and how natural the mapping is (Steuer, 1992).For example, naturalness in a VR environment is related to high fidelity and it enhances the sense of presence (McMahan et al., 2012).Therefore, interactivity can be increased by high levels of fidelity in the VR space, in which elaborate, realistic, and sensory expressions are possible. Although not much research on the effect of interaction in the VR space exists, there have been some studies on VR related to sports (Sigrist et al., 2015;Vogt et al., 2015).According to them, the sense of presence is related to the electroencephalogram and was highest in the interactive VR condition, showing that interactive VR produces physical performance in a highly competitive spirit.However, the impact of the high fidelity of VR fashion presentation and user experience has not been proven yet. Sensible immersion Immersion refers to the state in which the user's perceptual and psychological awareness are almost completely cut off from the outside world while participating in VR, and the pleasurable experience of being transported to an elaborately simulated place, alongside the sensory, emotional experience of being surrounded by a completely different reality that takes over their attention and sense organs (Murray, 1997).It is the flow that is experienced owing to external stimuli, and it is primarily used to describe the experience of using VR devices (Cheng et al., 2017).This study examined the characteristics of immersion by distinguishing between sensory and emotional immersion.Sensory immersion refers to the range of sensory channels involved in virtual simulation (Kim and Biocca, 2018).Using purely sensory receptors such as one's sight and hearing to acquire information also increases the immersion effect when compared to using one type of sensory channel.It can further focus on the sensory properties of the garment as an object.Thus, this study aimed to determine whether users obtain tactile effects in non-contact situations of VR. Emotional immersion Emotion is a personal response to internal or external stimuli that is caused by a somewhat specific cause or event; it provides information on a specific object or situation (Payne and Cooper, 2001).Sensory organs sense external stimuli and the sensed information is transmitted to the brain, which undergoes mental processes called emotions and reactions.The implicit memories of a person, psychological experiences, feelings that are expressed as emotions through external physical stimuli, and the resulting highlevel emotional responses are sensibilities (Han and Choi, 2020).A user can experience human senses and emotions through the VR space, without directly induced stimuli and events, which is the plausibility illusion (Slater, 2009).Although sensibilities and emotions can be discussed separately, this study uses the term emotion to collectively define psychological responses that are distinct from perception.Users of the VR space experience emotional immersion through objects and emotions such as joy and sadness (Han and Choi, 2020). Emotional immersion is closely related to sensory experience.The sense of space and distance are related to creating emotional experiences in themselves (Diemer et al., 2015;Peperkorn et al., 2015).Facial expressions are effective in inducing emotions (Han, 2018), and the hyper-realistic expression of digital humans in virtual space induces interest and pleasure; through this satisfaction, the level of immersion increases owing to intrinsic rewards and the sense of selfachievement, even if there are no special goals or extrinsic rewards (Kim and Seo, 2017).Thus, visual expressions bring about emotional immersion.In fashion presentations, emotional communication such as fashion images is important.This study revealed how users gain virtual fashion experiences through the emotional immersion provided by the IVR environment. Aesthetic interaction The aesthetic experience refers to an attitude, perception, experience, or interest related to art appreciation (Wanzer et al., 2020)."Aesthetic" refers to a sensory experience that is related to the visual form, texture, harmony, order, and beauty of an artifact (Venkatesh and Meamber, 2008), and is an ideal experience characterized by an aesthetic quality that is distinct from everyday experiences (Marković, 2012).This experience arises from the dynamic interactions between the perceptions of objects and processing of perceived information and is formed with the addition of judgment and evaluation (Venkatesh et al., 2010).It is influenced by culture, reference groups, and personal taste (McCracken, 2005). Aesthetic experience is not only limited to exhibitions and works of art; it is also found in various areas of activity such as in sports, games, and exploration (Csikszentmihalyi and Robinson, 1990).It refers to the element of pleasure that is based on the understanding of the object.Norman (2002) stated that the aesthetic experience in the goods and services system includes the immediate sense of the object and the joy and pleasure that are experienced in the process of comparing the characteristics to the information saved in one's memory. An aesthetic interaction refers to the dynamic exchange of information, such as product characteristics and sensory and cognitive experiences that include user behavior between the person and artifact contexts (Locher et al., 2010).Instead of being seen as a unilateral experience, an aesthetic interaction should be considered an active and agentic concept in which information is shared between humans and objects.Kang (2023) analyzed the aesthetic experience of the VR exhibition and showed that the IVR exhibition was highly evaluated in the emotional and intellectual factors than the VR exhibition.Ma et al. (2023) showed that education using VR technology can foster children's aesthetic creativity.These provide important grounds suggesting that IVR can affect aesthetic experience and creativity.Nevertheless, the aesthetic experience of fashion presentation in an IVR environment has not been considered so far. The experience of aesthetic interactions is appropriate to define this complex experience as a separate characteristic for examining the user's experience of VR fashion show spaces.This is because each experiential element in the VR space can appear in a complex manner unlike in real spaces, and because the nature of fashion, unlike other fields, has aesthetics that reflect the internal experience of external objects.This study examined the experience of aesthetic interactions in the VR space separately concerning experiences such as the sensory and emotional pleasure regarding objects, and the psychological state of satisfaction with fashion collections and brands as consumer goods. 3 Materials and methods Participants Interviews were conducted in a controlled laboratory on campus.Denzin and Lincoln (2003) argued that content was more important than the number of cases and suggested no more than 10 cases.Consequently, this study selected 21 participants, with 7 per stimulus (Table 3).Participants were recruited through purposive and snowball sampling.Recruitment notices were distributed through social media and in-depth interviews were conducted with respondents.According to Lee and Park (2018), those who prefer communicating with new communication technologies are in their 20s and 30s.Thus, this study selected male and female participants in South Korea who were in their 20s, majoring in fashion/clothing, and had experience watching fashion shows or videos, but had no experience with VR.Those who satisfied the necessary conditions and expressed their intention to participate were selected (Table 3).This study received ethics approval from an institutional review board (no.7001066-202301-HR-003). Stimuli and devices Of the case selection methods suggested by Jason and John (2008), a typical method was used to select the most representative cases, which were 360-degree VR fashion shows that had the three types of characteristics vis-à-vis virtual space.The following method was used to select the stimuli: In January 2023, keywords combining "360, " "VR, " and "fashion show" were entered on YouTube VR to search for fashion shows of different brands that had the highest number of likes and that clearly showed each of the three characteristics of virtual space.Accordingly, three videos were selected.Fashion show spaces comprise models, runway clothes, music, lighting, stage, venue, and the audience (Kim and Ahn, 2016;Kim, 2018).Accordingly, stimuli were selected by considering whether the elements of the traditional fashion show were reproduced, transformed, and maintained; these stimuli were created according to the characteristics of a VR space when the above material elements of the fashion show were converted to digital images.Stimulus 1, Dior's 2017 Spring/Summer Haute Couture Show, was produced by recording the live fashion show so that participants could watch the show as a 360-degree video from an audience perspective.The concept of this show was a labyrinth, and the space was constructed using grass and trees inside a tent that was set up in the Musée Rodin Garden (Mower, 2017).It was a fashion show space that reproduced the appearance of physical reality.Stimulus 2, Prada's 2021 Spring/Summer Womenswear Show, was held without an audience and comprised a stage that was blocked off on all sides by digital screens and curtains.As with Stimulus 1, this space had a variable and flexible appearance as the viewer's perspective was not fixed in place and the background moved at different stages.Digital screens were set up on the walls of the space to create another fashion show scene.It was a mixed space comprising a blend of reality and virtuality.Stimulus 3, TTSWTRS's Technological Singularity Show, was produced in 2020.This fashion show displayed the transformation of objects, animals, and human figures into virtual objects and backgrounds that were made using digital images.A space of manufactured objects is one of pataphysics, which is expressed beyond the limitations of reality through interactions between the human imagination and VR technologies. Although there are several types of IVR display devices, Oculus Quest 2 was used from among the wireless HMD devices that allow a wearer to move freely.This device was selected because it had a reduced weight and more comfortable, ergonomic design when compared to previous devices that could be attached to the wearer's head.Released in 2020 by Meta, the Oculus Quest 2 comprises a VR headset and controller, and the screen can be moved while wearing the VR headset, by using the controller and neck movements. Detailed interview questions For feelings such as the sense of presence that users experience in VR, many consider that subjective statements are the standard method of measurement (Ijsselsteijn et al., 2000).Therefore, this study conducted in-depth interviews and used the participants' statements for data analysis.Semi-structured questionnaires were used to facilitate detailed conversations in an open environment without any constraints (Karanika and Hogg, 2010).The questionnaire comprised items that could determine the characteristics of the VR experience.Items were referenced from previous studies on experience (Pinker, 1997;Shedroff, 2001;Mascarenhas et al., 2006), VR experience and space (Ji et al., 2022), and the sense of presence and immersion in VR environments (Steuer, 1992;Narciso et al., 2019;Kim and Choo, 2023) (Table 4). In-depth interview and methods of data collection and analysis In a qualitative study, it is important to create a favorable atmosphere by building rapport with participants so that they can express themselves freely (Boyce and Neale, 2006).Two moderators who were students majoring in clothing and textiles helped build rapport with participants to create a relaxed atmosphere and encourage them to speak so that they would voluntarily share and interact with each other.In-depth interviews were conducted in the following manner: (1) Before watching the stimuli, participants were asked simple questions about themselves; (2) Participants who were going to experience VR were given an explanation on using the HMD, and a simple simulation was conducted to help them understand the device and learn how to operate it; and (3) After watching the stimuli (for approximately 3 to 4 min), participants were interviewed for 30 min using the prepared questionnaire.Their responses were recorded and transcribed using Naver CLOVA NOTE, an automatic recording program.The responses were moved to Microsoft Word for refinement and preparation of the data.Data were analyzed using qualitative methods.Participants' responses were recorded in writing and compared with the key concepts and characteristics that were derived through the literature review.Data analysis was conducted in line with Giorgi (2004), where: (1) the content of the descriptions was understood in full, (2) the content of participants' descriptions were organized into meaningful units, (3) the organized words were transformed into academic expressions and the themes and central meanings were determined, and (4) the central meanings were identified and categorized into groups. Results Virtual fashion experience representing space-time Cognitive sense of the place In Stimulus 1, which comprised a 360-degree video of a real-life show, participants described experiencing place illusion, in which they felt as though they were with the audience in a specific place in which the fashion show was being held. "The background was green and felt like a field.It felt like the spectators were sitting on a path in the field, and it was so realistic that I felt I was there" (Participant 7). "If I turned my head just a little, I could see people right next to me talking to and looking at each other, so it felt like I was with the audience…It felt like I was in Paris so it somehow felt like I was dreaming!"(Participant 4). These responses demonstrate that with the reproduction of the stage, background, and audience in the VR fashion show space, participants were fully aware that it was a fashion show.Rather than focusing on the details of elements such as the runway outfits and models, they were fascinated by the atmosphere in the space, and experienced the cognitive sense of presence, wherein they felt as though they were in the venue, which was actually in another region. Assimilation into the fashion show In the space of Stimulus 1, participants were reminded of brands or images they had seen on other visual media, and fashion images were formed in addition to the atmosphere of the fashion show that was provided by the VR space. "The location of the fashion show felt like a spring garden, and the floral atmosphere felt a lot like old-fashioned dresses" (Participant 6)."There were a lot of dresses, and I thought of the image of a spring picnic" (Participant 4). "The brand generally had a luxurious and elegant image" (Participant 7). Participant 7 linked the fashion image to that of the brand.The VR fashion show space is a container in which fashion images are created through the interactions of each component and the user's overall a priori experience, perception, and emotional response.As the space of a reproduced fashion show captures the appearance of a specific reality, participants had the emotional experience of forming fashion images based on previous experiences and then relating them to the brand identity.Participants said that they felt a sense of belonging to the brand as it felt as though they had attended a fashion show that was only open to a few people. "I looked around a lot because it felt like I was experiencing a fascinating fashion show in the forest.I was surprised to see that famous actors were there, too!" (Participant 3). "It felt novel and exclusive.I felt like I was watching the fashion show after getting a VIP seat invitation from the director" (Participant 1).Experiencing a fashion show in which a specific time and place was reproduced created a sense of closeness among all the elements that shared the same space and time.This is a state of brand attachment □ What had you known about the brand and design previously? □ How does it compare to watching a fashion show on social media? Sense □ Did the models and objects in the video feel three-dimensional?Why do you think so? □ How was the visual texture of the runway outfits, models, audience, etc.? □ How was the music in the fashion show space? □ How were the form and visual texture of the stage, background, and props?Please describe them in detail. Emotion □ How did you feel after watching the fashion show? □ Did any images come to mind?Why is that? □ What was the most memorable thing after watching the video and how did it make you feel?Why is that? Behavior □ How was the process of putting on the head-mounted display? that was formed in the fashion show space, wherein one created an emotional bond in the way they would with someone close to them (Thomson et al., 2005).It represented an emotional experience. □ Contextual understanding of runway outfits During the fashion show, participants could see three-dimensional runway outfits and perceive their form, color, and texture.However, instead of remembering the outfits in detail, they appeared to try to understand the outfits in the context of the fashion show space. "I saw a female model, and I was impressed because the stage and outfit that the model was wearing matched so well!" (Participant 7). "It was nice to be able to see environmentally friendly outfits that fit the atmosphere of the fashion show" (Participant 3). "It was different from the style of the brands I know, but it was good in that it showed various sides and reflected the changes by the trend" (Participant 1). These responses contrasted the purpose of watching a fashion show, as participants had articulated in the beginning of the interview.Participants who were majoring in clothing and textiles stated that they occasionally watched fashion shows to gain information on outfits.However, in the reproduced VR space, participants were more immersed in the space and atmosphere than in the clothes and demonstrated the aesthetic experience of trying to understand the outfits in the context of the space. The VR fashion show space, which is a physical representation of the original in real life, allows users to experience a representation of reality.Through this representational space, the user shows that they are forming an emotional bond by observing the fashion show in the context of brand identity and the sociocultural context that they perceive in reality and being immersed rather than focusing on fashion information. Virtual fashion experience of mixed reality and virtuality Cognitive confusion Participants experienced cognitive confusion, in which all the objects constituting this mixed space were considered artificially created virtual objects. "I was mistaken about whether seeing real three-dimensional models was virtual reality or reality.I looked into the model's eyes, and even though they were a fake person, I got goosebumps because they looked real" (Participant 10). In Stimulus 2, some objects that comprised the digital screens and stage were created virtually.However, the models, runway outfits, and stage were real.Participants could not tell the difference.These responses suggest that although an enclosed virtual space that is blocked from the outside world can increase the psychological experience of immersion for users, it can cause them to experience cognitive confusion (where reality is considered virtual) if users are not given sufficient clues that relate to the physical reality. Sensory and emotional immersion of closeness As with perception, the process of judging a situation begins with the senses (Lokesh et al., 2022).In the VR fashion show space, the movements of models and perspectives caused sensory experiences such as the three-dimensional effect and sense of distance.The elements of a fashion show, such as a model's walk, differ from other mediums of fashion presentation as dynamism is emphasized.In the case of VR fashion shows, the dynamism provides a three-dimensional effect and a sense of space. "I felt a strong sense of three-dimensionality.When the model came back, I felt like I could touch them if I reached out, and I felt like I was in the fashion show space with them" (Participant 9)."The models made eye contact with me as they walked by, and it seemed like they were really looking at me and that the model was aware of me" (Participant 10). Visual elements that are presented can have an effect on inducing emotions (Diemer et al., 2015;Peperkorn et al., 2015).An approach that reflects the sense of space and distance concerning visual perception acts to induce emotions; facial expressions also induce emotions (Han, 2018).As they felt that the models were walking by them, participants said that they experienced a sense of threedimensional space and presence.Some said that they felt scared or a sense of familiarity because of the models' eyes or expressions. "The models' eyes and expressions seemed a bit threedimensional, and they felt real as they came closer.I was overwhelmed when I saw the models' eyes and the fashion together" (Participant 10). "When they approached me, I could feel it so vividly, like they were going to reach me right away, and it felt like having a friend right in front of me!" (Participant 8). Regarding the eyes of digital humans, the addition of mutual interactivity, emotional expressions, visual processing technology, and biological characteristics can increase levels of immersion in visual immersion, self-purposefulness, concentration, and external insensitivity (Yun et al., 2020).Participants' responses demonstrate that when the model's eyes faced them and they came closer, this induced emotional experiences such as a sense of fear or familiarity.Although Stimuli 1 and 3 had dynamism, which is a component of fashion shows, when there is not enough information on the space (that is, when it is difficult to understand the space in the sociocultural context of reality), this suggests that there could be higher levels of emotional experience concerning the fashion objects. Authenticity of a fashion image The new information and emotional experiences that are acquired in the VR fashion show space have an impact on the perceived image of the fashion brand.Participants recalled the fashion image of the brand they had known and explained that experiencing a VR fashion show that mixed reality and imagination either renewed their brand image or strengthened the existing one. "I think the feeling of models walking up and making eye contact with me made me focus on the show more, and made me feel good because it felt like they were holding the show just for me" (Participant 8). "It did not have that feeling that was characteristic to the brand.It had a very bright feeling…The show rather felt friendlier and a little different" (Participant 10). Participants did not stop with these emotional experiences.They went on to connect them with their evaluation of the fashion brand.This is connected to fidelity, which is a measure of how well virtual environments represent the real world (Meyer et al., 2012), and is also strongly related to realism (Bowman and McMahan, 2007).In the VR fashion show space, in which reality and virtuality are mixed, participants said that low levels of fidelity increased virtuality and created doubts regarding brand originality. "The overall quality of the fashion show I watched on YouTube was very high…but this somehow did not feel like Prada" (Participant 14). It appears that participants felt more confused about the brand identity when they were not sufficiently provided with information that is generally well-known. "As there wasn't a logo or anything to symbolize the brand, I did not think anyone would know that they were that brand's clothing" (Participant 10)."I do not think I ever felt like it was a real stage…so I really did not think it was that brand" (Participant 11). The VR fashion show space (in which there is a mix of reality and imagination) creates a new space-time that has fluidity and dynamism, and is a mix of the real and virtual.This fashion show space makes it difficult to understand objects based on the user's past experiences, which is the sociocultural context of reality.Therefore, participants experienced immersion that was dependent on sensory elements, and as a result, the technology of graphics such as fidelity affected the authenticity of objects.This way, a fluid space can become an obstacle for users who are trying to immerse themselves in a brand (that is, trying to maintain the relationship between a brand and consumer; Gundlach et al., 1995), which is because the solidarity with the history, tradition, and identity that the brand built in reality weakens in this space.However, it is meaningful as it creates aesthetic experiences in which fashion images (that are strengthened or decreased) can be perceived or experienced through individual subjective insight and new spaces. Virtual fashion experience of pataphysics 4.3.1 Connection to creative inspiration Although participants experienced a sense of presence as though they were in the same fashion show space as others, they considered the objects they observed unrealistic because they had neither seen nor touched them and could not touch them outside of the show.As a result of this "unreality, " participants said that they became curious about how the elements of an ideal fashion show space are created, and said that they felt inspired. "As the video moved with me when I turned my head and moved my gaze, it really felt like I was in a fashion show…First, I felt it was a little unrealistic because the video's composition and flow were unrealistic, and I had never experienced it and could not touch it" (Participant 21). "I wonder how the graphics were implemented so well…It makes me wonder how long it must have taken to make this great video with the designs…I thought that it could be expressed in a more fun way if it was done this way" (Participant 16). Before watching, participants majoring in the field of fashion stated that they ordinarily acquired new ideas and information through fashion shows.These responses demonstrate that unfamiliar images can provide new visual stimulation and cognitive experiences to a greater degree than when compared to other stimuli; this shows that they could provide material for creative inspiration concerning academic and work-related activities. Aesthetic exploratory play Exploratory behavior obtains information on stimuli.Exploration is divided into specific exploration, in which a new object is manipulated and tested using one's senses, and diverse exploration, which is a long-term and continuous exploration that takes place internally after an object's characteristics have been identified (Hutt, 1971).Unlike exploratory behaviors, which are driven by external stimuli, playing is a multifaceted behavior that is driven by intrinsic motivation.It takes place after the information has been acquired and feelings of pleasure are induced in the process, and not in the results.With exploratory play, a combination of exploration and play takes place.It refers to the behavioral pattern that appears after a new object is observed based on an a priori experience related to the said object.The unfamiliar emotions that participants experienced in Stimulus 3 resulted in the behavior of collecting information by moving the field of view to various angles.Through this experience, participants explained that they became more curious about the brand's concept and level of completeness of the content.This is similar to the exploratory play that young children engage in while exploring a new world."It was the first time I had seen a model or clothes threedimensionally, so it felt like I was on a new amusement park ride" (Participant 21).Participants' physical activity in the VR fashion show space was limited to their eye movements.However, they were seen to acquire audiovisual information with virtual objects regardless of material achievements and physical behaviors.Even if participants could not use their will to control the situation directly, they used the information they acquired by guessing the connectivity between the fashion show's components to see the fashion show as a work of art and understand its concept, which was an aesthetic exploratory experience. The fashion show space of pataphysics, which was realized with human imagination, is an imaginary space that comprises a combination of images that are somewhat far from the experiential symbolism of lifestyles and social groups.This space (where the relationship between the social signifiers and the signified are disconnected) provides aesthetic experiences in which users engage in new exploratory play and dynamic interactions take place between fashion images and the users' aesthetic exploration. Conclusion Summary and discussion This study approached the VR space with three characteristics based on the perspective of social space and proposed that differences in spatial character led to differences in the fashion experiences of users. First, the fashion show space, which is a physical representation, induced a cognitive and emotional sense of presence, in which users felt as though they had moved to the same time and place as those of the fashion show.Through this fashion show space, users experienced aesthetic interactions in the cultural context, in which runway outfits were interpreted by connecting them to the atmosphere of the representational space.Jung and Ko (2023) explored the experiences of luxury fashion brands and determined that the components of the virtual fashion space are related to virtual experiences such as immersion, presence, and interaction.A priori elements such as sociocultural contexts and personal experiences differ in the experiential dimension of virtual space.Stimulus 1 was the reproduction of a real luxury brand fashion show, and it was determined that the user became immersed by connecting the fashion images that were accumulated in reality to the virtual space.This immersion played a large role in forming the emotional bond that is referred to as a sense of belonging. Second, in the mixed fashion show space (in which reality and imagination coexist), participants experienced cognitive confusion owing to the differences with a priori experiences.Participants' sensory experience was connected to the formation of the brand's image and emotional experiences.The three-dimensionality and a sense of space that occurs in enclosed spaces provided by VR technology induce a psychological experience in which the user feels as though they have a special and intimate relationship with the brand.Given that the stimuli are fashion shows of luxury brands, it is unusual that an emotional experience of intimacy is induced concerning a private relationship, which is unlike the sense of belonging in a representational space.Jung et al. (2021) argued that VR technology reflects a utopia in which traditional luxury fashion shows (that had been characterized by privilege and status in the past) can be experienced equally.This could lead to different consequences based on the properties of the space. Third, participants transcend the limitations of physical reality while in the fashion show space of pataphysics (which was realized with human imagination) and that they move beyond the stage of confusion that is experienced while facing realistic objects to connect to creative inspiration.Unlike other spaces, the fashion show space of pataphysics is far from social symbolic meanings and is a space that results from the creator's imagination.In virtual space, which is difficult to understand in the sociocultural context of reality, the user engaged in active exploratory behavior to acquire new information and underwent aesthetic experiences concerning aesthetic exploration.This confirmed the results of Kim and Choo (2023), who found that experiences of shopping in VR increased levels of perceptual curiosity and creativity in consumers.Research findings suggest that creative inspiration and exploratory play can be induced in a user to a greater degree if the immersive fashion space is further from the original source and if the relationship between the signifiers and the signified is further disconnected. Implications and limitations Studies in the field of fashion that have dealt with fashion experiences through IVR technology have primarily focused on fashion stores or consumer experiences.This study presented the characteristics of new virtual spaces created by media technology, and empirically determined the differences in fashion experiences in each virtual space.It followed a more detailed approach to the virtual experience and subdivided it.This study provides a basic framework for research on fashion content production and interactions with users in the Metaverse, which has been used increasingly in recent years.Therefore, this study makes an academic contribution by expanding the field of digital fashion design, which is still in its early stages, by narrowing the research gap. The VR fashion show spaces of pataphysics provide creative motivation and aesthetic experiences of exploratory play.Unlike previous studies that focused on the environment of fashion stores (whose primary purpose is sales), and consumption behavior and intention, this study focused on fashion show environments.Current fashion shows are significant as they are communication channels that convey brand concepts and designers' creativity.Experience is an important mechanism for improving professionalism (Dreyfus and Dreyfus, 1986), and the VR space can create a variety of experiences.Therefore, the current findings suggest that IVR fashion shows, and the spatial experience of pataphysics, could have an educational effect and help enhance the professionalism of students majoring in fashion, who are required to have abundant creative sensibilities.Future research should focus on its educational effects. IVR technology changes the way that traditional fashion shows communicate.This study demonstrated that VR fashion shows play a positive role in fashion media by interacting with users, building a sense of closeness, and creating new fashion images.This is unlike previous fashion shows, which used a few unilateral methods of delivery.Unlike in the real world, in which new designers can be placed at a disadvantage when compared to luxury fashion brands that have a long history and tradition, VR fashion show spaces can give them the opportunity to effectively expand into new markets.Although physical fashion shows only last for about 20 min, there are concerns about the negative environmental effects they have, such as the vast amount of energy that is used and their heavy carbon footprint (Webb, 2022).Holding VR fashion shows can minimize their impact on the physical environment, and contribute to the development of a sustainable fashion industry by enhancing diversity in the industry, which is centered on mainstream fashion companies.This study has some limitations.First, it did not adequately examine how technical problems in the devices used to experience VR fashion shows can affect a user's fashion experience.Pallavicini et al. (2020) found that the images, sounds, and interactions of VR experience devices induced positive or negative reactions.Marsh et al. (2001) explained that the effect of sense of presence decreased and emotions such as discomfort and fear could be induced if interactions with the user were not smooth and the images on the device or screen were difficult to discern owing to issues such as slow buffering speeds.Some experienced problems with image quality owing to the Wi-Fi connection during the experiment, whereas some complained of discomfort owing to the weight of the device.Therefore, future research should exclude the technical limitations of the experience devices.Second, there could be differences in perception and cognition by sex (Rosa et al., 2014).Future research can evaluate differences based on sex and individual perceptions.Third, the experiment was conducted with fashion students; thus, there could be individual differences in students' personalities and the results may not be generalizable to students in other fields.According to Dewey (2007), experience is formed by the interaction between the reciprocity of external conditions and subjective internal conditions.Thus, different dimensions of VR experience could be derived if other populations were targeted (e.g., consumers, other fashion professionals, or tourists). TABLE 1 1 The characteristics of VR space. Social spaceDigital media spaceVR spacePhysical interaction-InteractionSpace of physical representation-Ease of accessFluidity and dynamism-Complexity and flexibility of informationMixed space of reality and idea-Ambiguity and extensibility of space-time boundariesSymbolismVirtualitySpace of pataphysics TABLE 2 2 The dimension of VR experience in VR space. Brand experienceVR experienceVR experience in VR spacePerceptionPresenceAuthenticityCognitive presenceSenseSensible immersionEmotionImmersionEmotional immersionBehaviorEmotion SimulationInteractionAesthetic interaction TABLE 3 3 Participants. No.Age (years)SexStimuli126MS1221MS1324MS1426MS1521FS1627FS1723FS1822FS2923FS21022FS21125FS21225FS21325MS21424FS21523FS31624FS31722FS31827MS31922FS32023FS32123FS3 All were undergraduates majoring in clothing and textiles except for no.6 (postgraduate in fashion design).No one had any VR experience. TABLE 4 4 Detailed interview questions.Age, occupation, education level, residence, use of social and digital media, and experience watching fashion shows □ Reason for watching fashion shows and what is expected of them Item AcknowledgmentsI thank all participants and Donghyun Lee, Woogyun Kim, and Minseo Gwon for collecting survey data.Data availability statementThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.Ethics statementThe studies involving humans were approved by the Institutional Review Board in Changwon National University(no. 7001066-202301-HR-003).The studies were conducted in accordance with the local legislation and institutional requirements.The participants provided their written informed consent to participate in this study.Author contributionsConflict of interestThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Publisher's noteAll claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. 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Detailed Seed Cone Morpho-Anatomy Provides New Insights into Seed Cone Origin and Evolution of Podocarpaceae; Podocarpoid and Dacrydioid Clades 19 November 2023 Raees Khan raees.khan@adelaide.edu.au School of Biological Sciences The University of Adelaide 5005AdelaideSAAustralia CAS Key Laboratory for Plant Diversity and Biogeography of East Asia Kunming Institute of Botany Chinese Academy of Sciences 650201KunmingChina State Herbarium of South Australia 5005AdelaideSAAustralia Robert S Hill 0000-0003-4564-4339 School of Biological Sciences The University of Adelaide 5005AdelaideSAAustralia Veit M Dörken veit.doerken@uni-konstanz.de 0000-0003-4564-4339 Department of Biology University of Konstanz 78457KonstanzGermany Ed Biffin edward.biffin@adelaide.edu.au State Herbarium of South Australia 5005AdelaideSAAustralia Detailed Seed Cone Morpho-Anatomy Provides New Insights into Seed Cone Origin and Evolution of Podocarpaceae; Podocarpoid and Dacrydioid Clades 19 November 2023924347CBE1D11452069D3389CA44A45910.3390/plants12223903Received: 29 July 2023 Revised: 13 September 2023 Accepted: 25 October 2023conifersdevelopmental biologydispersalevolutionfossilsleaf dimorphismpaleobotany The study of reproductive morphology and trait evolution provides a vital insight to understand the evolutionary history of plants.The conifer family Podocarpaceae has a remarkable diversity of seed cones, with distinct morphology among the genera and with conifers in general.However, we lack a good understanding of the seed cone morpho-anatomy and trait evolution of Podocarpaceae.We investigated detailed seed cone morpho-anatomy using staining and sectioning techniques to clarify the anatomical, morphological diversity and evolution of functional traits.The presence of a fleshy receptaculum is a characteristic feature of both clades.However, species of Retrophyllum, Afrocarpus and some species of Nageia and Podocarpus form a fleshy sarcotesta-like seed coat, lacking a fleshy receptaculum.The ancestral state reconstructions show a shift between and sometimes within the genus.Although both clades demonstrate fleshiness as an ancestral trait, the shift in fleshy structures provides evidence for complex multiple evolutions of fleshy morphologies.These seed cone traits (e.g., fleshiness and size), along with the broad, flattened and well-adapted (leaf dimorphism) foliage in both clades, are largely congruent with efficient light harvesting and bird dispersal.These traits make these two clades well adapted to their environment, when growing in communities including tall and broad-leaved angiosperms (closed-canopy angiosperm forests), compared to other podocarps, making them more successful in achieving a wider distribution and species richness. Introduction Podocarps are unique among conifers in their ability to successfully compete with angiosperms, especially in tropical forests [1].Podocarps achieved this capacity by evolving some functionally significant traits, e.g., flattened broad leaves, fleshy seed cones and large seed sizes [2][3][4][5].Podocarps have an amazing diversity of fleshy seed cones and have evolved several complex functional structures in different genera [6][7][8][9][10].Recent phylogenetic analyses show three major clades in the Podocarpaceae, i.e., Prumnopityoid, Podocarpoid and Dacrydioid, along with some separate and distinct lineages [4,11].The Podocarpoid and Dacrydioid clades have evolved an amazing diversity in both leaf and seed traits [4,[12][13][14].The Podocarpoid clade has a suggested crown age of approximately 75 Ma (54-85 Ma) and is the largest genus within Podocarpaceae (consisting of more than 130 species), distributed in Southeast Asia and through much of the Southern Hemisphere [13,15].The Dacrydioid clade is the second most species-rich clade and includes three genera (Dacrydium, Dacrycarpus and Falcatifolium) with a suggested crown age of approximately 75 Ma and is predominantly distributed in the Southern Hemisphere [4,13].Some studies have evaluated seed cone evolution in conifers based on morpho-anatomy, and some have used model-based ancestral reconstruction [6,8,14,[16][17][18][19].However, detailed and comprehensive studies of the seed cone morpho-anatomy are still lacking.Klaus and Matzke [6], in concluding that the ancestral state for the Podocarpaceae seed cone is fleshy, considered the Podocarpoid clade genera Afrocarpus and Retrophyllum, and some species of Nageia and Podocarpus as non-fleshy.The study by Chen et al. [19] considers the epimatium of Dacrydium, Falcatifolium, Microcachrys and Lepidothamnus as non-fleshy and reported a complete absence of fleshy receptacles in Nageia in their ancestral reconstructions.Although some studies have been undertaken to evaluate different aspects of the reproductive cycles of Acmopyle, Podocarpus, Dacrydium and Dacrycarpus [14,[16][17][18], no comprehensive studies are available on detailed seed cone morpho-anatomy and the evolution of functional traits and structures for both the Podocarpoid and Dacrydioid clades.Similarly, because seed cone morphology is complex in podocarps and there has been a lack of detailed studies on the seed cone morpho-anatomy, it is difficult to correlate fossil seed cones with extant taxa [4].The recent studies on the detailed seed cone morphoanatomy of three paleoendemic genera [9] and the Prumnopityoid clade [10] provide vital insights into the origin and evolution of seed cone types in Podocarpaceae [20].The goal of this study is to understand and describe the detailed morpho-anatomical structures of the two largest species rich clades, i.e., Podocarpoid and Dacrydioid clades' seed cones, and discuss the complex evolutionary history of fleshiness in Podocarpaceae.The morphoanatomical similarities and differences among the genera are evaluated and compared with the available related fossil data.The origin, evolution, and potential drivers of fleshy seed cone evolution among the genera of these clades are also discussed. Material and Methods Seed Cone Collection Seed cones at different developmental stages were collected from the Australian National Botanic Gardens, Canberra, ACT, Australia; Mount Lofty Botanical Garden, South Australia; and The Tasmanian Arboretum, Devonport, Tasmania.Seed cones of Nageia (AN-395) and Retrophyllum (RB-23) were collected from preserved specimens in the State Herbarium of South Australia and the Department of Ecology and Evolution, University of Adelaide.The spirit collection of the seed cones, with voucher numbers from UOA-10 to UOA-60, are stored at the Department of Ecology and Evolution, University of Adelaide, Australia.We included Acmopyle in this study as an outgroup because it has a fleshy receptacle, a papery epimatium fused with the outer testa and a woody sclerotesta (endotesta).Data on seed dispersal were collected from the available literature. Morphology and Distribution of the Investigated Taxa Members of the Podocarpoid clade are usually large trees or sometimes shrubs, with broad and flattened leaves and fleshy seed cones.Representative species from each genus that cover the variation in the seed cone morphology were included here.In this study, we examined: 1. The South African Afrocarpus falcatus (Thunb.)C.N. Page, which forms large trees [21]. 2. Two species of Nageia, N. nagi (Thunb.)Kuntze, which is an evergreen tall tree, distributed in China (introduced), Japan, Taiwan (introduced) and Vietnam [21,22] and N. wallichiana (Presl.)Kuntze, which is a tall tree distributed in Brunei Darussalam, Cambodia, China, India, Indonesia, Laos, Malaysia, Myanmar, Papua New Guinea, the Philippines, Thailand and Vietnam [21,22]). 3. Three species of Podocarpus subgenus Podocarpus (P.henkelii Stapf., P. elongatus (Aiton) L'Herit.Ex Persoon and P. oleifolius D. Don).Podocarpus henkelii and P. elongatus occur in southern Africa, while P. oleifolius is found in Central America [23]. 4. Two species of Podocarpus subgenus Foliolatus (P.spinulosus (Smith) R. Br.Ex Mirbel and P. elatus R. Br. ex Endlicher).Podocarpus spinulosus and P. elatus are dioecious species from northeastern Australia [24]. 5. Retrophyllum comptonii (Buchholz) C.N. Page, which is a tree endemic to New Caledonia [25]. Additionally, we examined the following members from the Dacrydioid clade: 1. Dacrycarpus dacrydioides (A.Richard.) de Laub., a tall tree endemic to New Zealand. 2. Dacrydium cupressinum Solander ex G. Forst., a tall tree endemic to New Zealand. 3. Falcatifolium papuanum de Laub., a tall tree endemic to Papua New Guinea [26]. Finally, we included Acmopyle pancheri (Brongniart et Grisebach) Pilger, which is endemic to New Caledonia [21,22,27] as an outgroup to the two main clades. Taxon Processing and Sectioning Specimens were fixed in 200 mL of FAA (100 mL 95% ethanol + 80 mL dH 2 O + 20 mL 37% formaldehyde solution) immediately after collection.Whole seed cones, plus longitudinal and cross sections of the seed cones were photographed with a Nikon-SMZ25 stereomicroscope.For histology, seed cones were fixed for 48-72 h.The seed cones were processed for a 48 h cycle on a Sakura Tissue-Tek VIP6 Vacuum Infiltration Tissue Processor (Torrance, CA, USA).The seed cones were embedded in paraffin wax (Sakura Tissue Tek embedding centre) and then longitudinal and cross sections of 8-10 µm thickness, were produced using a Leica RM 2235 Rotary Microtome, (Wetzlar, Germany) and stained with H & E (DAKO Cover Stainer (Agilent, Santa Clara, CA, USA)) and Toluidine blue stains.The sections were brought to water and to slide.Each slide was individually stained for 10 s in Toluidine blue stain by pipetting a few drops of Toluidine blue stain into each slide.The slides were observed under light microscopy and photographed at several magnifications. Measurements and Trait Reconstructions Different morpho-anatomical and embryological characters were recorded (Table 1).The anatomical layers were measured from mature seed cones (ten seed cone replicates for each species).The measurements were taken using ImageJ 1.8.0_112 software.Seed cone morpho-anatomical trait data were collected mainly from living and Herbarium material.Quantitative seed cone data were mainly based on Herbarium observations and information in Farjon [21].For ancestral reconstruction, we used the dated phylogeny of Khan et al. [13], based on three chloroplasts (rbcL, matK and trnL-trnF) and three nuclear genes (NEEDLY, PHYP and ITS2).The characters were mapped for their evolution using RASP 4.2-BayesTraits [28], Mesquite 3.6 [29] with maximum likelihood (ML) and the Markov chain Monte Carlo (MCMC) reconstruction method. Results Afrocarpus Seed Cone Morpho-Anatomy Afrocarpus falcatus seed cones are yellow, obovoid, and are produced abundantly annually (Figure 1A).It is wind-pollinated, and the reproductive cycle completes in one year from initiation to the formation of the mature cone.The terminal bud initiates in the form of two primordia and develops into an ovule-epimatium complex.This structure differentiates into a seed cone apex and later into an ovule with an integument and epimatium.The megaspore mother cell differentiates from other cells surrounded by the nucellus.The seed cone (12-20 × 6-14 mm) is surrounded by 1-2 bracts and the ovule is inverted.The seed cone cross sections show six major zones: (i).Epimatium: 8-14 layers of small irregular isodiametric cells.Scattered sclereids cells are also present in this zone.(ii).Exotesta: 10-18 layers of round and elongated cells.Multiple vascular traces (four ascending and several descending), as well as resin canals, were observed in this zone.Scattered sclereids cells are also present.The epimatium is connate with the exotesta forming a fleshy sarcotesta-like seed coat (Section 3.6, a-c in the figure).(iii).Mesotesta: 3-5 layers of elongated cells.The vascular bundles from the exotesta enter this zone.These layers are highly resinous.(iv).Endotesta: 20-32 compact layers of small dense cells.The endotesta is hard and stony when mature, forming a sclerified sclerotesta-like structure.(v).Nucellus: The protective cover of the embryo consisting of 4-10 layers of dense and smaller cells and (vi).Gametophyte: 10-22 layers of cells with a mature embryo which is about 0.8-1.2× 0.3-0.5 mm. Nageia Seed Cone Morpho-Anatomy Nageia nagi produces brown globose (12-18 × 10-16 mm) seed cones that are windpollinated.In its native range, the reproductive cycle initiates in February and completes in November to December.Pollination occurs in April-May.The ovule is inverted.The seed cone cross sections show five major zones (Supplementary Figure S1): (i).Epimatium: 4-8 layers of small and large irregular isodiametric cells.(ii).Exotesta: 12-20 layers of round and elongated cells.Several vascular bundles and resin ducts are present in this layer.The epimatium is connate with the exotesta forming a fleshy sarcotesta-like seed coat.(iii).Endotesta: 10-18 compact layers of small dense cells.The sclerotesta is hard and stony when the seed cone is mature, forming a sclerified sclerotesta-like structure.(iv).Nucellus: 6-12 layers of dense and smaller cells.(v).Gametophyte: 12-20 layers of cells with a mature embryo which is about 0.8-1.5 × 0.3-0.4mm.The N. nagi seed cone has no fleshy receptacle, similar to N. fleuryi, N. formosensis and N. maxima, but distinct from N. wallichiana, where the seed cones have a fleshy receptaculum (Table 1). Podocarpus Seed Cone Morpho-Anatomy Podocarpus henkelii Podocarpus henkelii produces obovoid seed cones with one inverted ovule and completes its reproductive cycle in one year.The seeds are yellow-green and are positioned on a non-fleshy receptacle (Figure 1D).The seed cones are 15-20 mm long and 8-12 mm wide.The seed cone cross section shows six zones: (i).Epimatium: 12-16 layers of small, irregular isodiametric cells.Sclereids are present in this zone.The epimatium is fleshy and fused with the exotesta surrounding the whole seed, forming a fleshy sarcotesta-like seed coat.(ii).Exotesta: 12-18 layers of round and elongated cells.Several vascular traces, small resin ducts and large resin canals and sclereids are present in this zone (Figure 2c,d).(iii).Mesotesta: 16-22 layers of dense round and rectangular sclerenchymatous cells.(iv).Endotesta: 12-20 compact layers of small isodiametric cells.The mature endotesta is woody and stony, forming a sclerified sclerotesta-like structure.(v).Nucellus: 6-12 layers of isodiametric cells, forming the protective cover of the embryo.(vi).Gametophyte: 14-24 layers with a straight embryo (about 0.65-1.2× 0.2-0.4mm in size). Podocarpus elongatus Podocarpus elongatus has an oblong seed cone with one inverted ovule positioned on the bright red receptaculum.The seed cone reproductive cycle completes in one year.The seed cone cross section shows seven major zones: (i).Epimatium: 6-10 layers of small irregular isodiametric cells.Sclereids are present in this zone.The epimatium is papery and fused with the outer testa surrounding the whole seed.(ii).Exotesta: 8-13 layers of round and elongated cells.Several enlarged vascular traces and normal-sized resin canals and sclereids are present in this zone.The epimatium is papery and fused with the exotesta surrounding the whole seed.(iii).Mesotesta: 3-5 layers of dense, round, and rectangular sclereid cells.(iv).Endotesta: 6-12 layers of large and small irregular and isodiametric cells (Figure 2e,f).The cells are sclerified when the seed cone is mature, but they are not woody.(v).Nucellus: 8-14 layers of large and small irregular and isodiametric cells, forming the protective cover of the embryo.(vi).Gametophyte: 14-18 layers of cells with a straight embryo (about 0.5-0.9× 0.2-0.3mm in size).(vii).Receptacle: 12-25 layers of small and large rounded cells.The longitudinal section shows that three vascular bundles enter the receptaculum.The central vascular bundle enters the seed. Podocarpus oleifolius Podocarpus oleifolius produces elongated-oblong fleshy seed cones (12-18 × 4-7 mm) and completes its reproductive cycle in one year (Figure 1J,K).One or two inverted ovules are placed on the yellowish red fleshy receptacle with two small bracts.The seed cone cross section shows seven zones: (i).Epimatium: 8-12 layers of small and large isodiametric cells.Sclereids are present in this zone.The epimatium is papery and fused with the exotesta surrounding the whole seed (Figure 2a,b).(ii).Exotesta: 10-14 layers of small and large rounded cells.(iii).Mesotesta: 10-16 layers of small and large cells.Several vascular traces, resin ducts and resin canals are present.(iv).Endotesta: 10-16 compact layers of small dense cells that are sclerified when the seed cone is mature.However, the cone is not woody.(v).Nucellus: 7-10 layers of large and small rounded cells that form the protective cover of the embryo.(vi).Gametophyte: 12-22 layers of large and small isodiametric cells with a straight embryo (about 0.6-0.8× 0.2-0.3mm in size).(vii).Receptacle: 10-15 layers of small and large rounded cells with four vascular traces in the centre of the receptaculum (Figure 3B). Podocarpus spinulosus Podocarpus spinulosus completes its reproductive cycle in one year and produces elongated oblong fleshy seed cones (11-22 × 6-7 mm).The inverted ovule is positioned on the blueish black fleshy receptacle.Six major zones can be observed in the cross section of the seed cone: (i).Epimatium:14-18 layers of small and large rounded and elongated cells.Sclereids are present in this zone.(ii).Exotesta: 12-16 layers of round and elongated cells.Several vascular traces, resin ducts, resin canals and sclereids are present in this zone.The epimatium is papery and fused with the exotesta surrounding the whole seed.(iii).Endotesta: 5-8 compact layers of small and large dense cells that become highly sclerified when the seed cone matures.However, the endotesta is not woody.(iv).Nucellus: 6-8 layers of dense cells form the protective cover of the embryo.(v).Gametophyte: 12-22 layers of large and small isodiametric cells with a straight embryo (about 0.6-0.9× 0.2-0.4mm in size).(vi).Receptacle: 9-18 layers of small and large rounded cells.The longitudinal section shows that three vascular bundles enter the receptaculum.The central vascular bundle enters the seed (Figure 4c,d). Podocarpus elatus Podocarpus elatus completes its reproductive cycle in about one year, initiated from the last week of August in the new vegetative buds.It produces abundant elongated oblong fleshy seed cones, with one or sometimes two inverted ovules positioned on the bright red fleshy receptacle, which is formed by two bracts.Two weeks after seed cone initiation, a bract ovule complex appears.The peduncle extends the seed cone away from the shoot.This bract ovule complex differentiates into the nucellus and integument.Pollination and fertilization occur in November-December.Six major zones can be observed in the cross section of the seed cone: (i).Epimatium: 6-10 layers of small and large isodiametric cells.Sclereids are present in this zone.(ii).Exotesta: 18-26 layers of round and elongated cells.Several vascular traces, small and large resin canals (resin ducts at the end of the exotesta) and sclereids are present in this zone.The epimatium is papery and fused with the exotesta surrounding the whole seed (Figure 2g,q,h).(iii).Endotesta: 4-6 compact layers of small dense cells that become sclerified when the seed cone matures.However, the endotesta is not woody.(iv).Nucellus: 2-4 layers of small, dense cells form the protective cover of the embryo.(v).Gametophyte: 14-20 layers of large isodiametric cells with a straight embryo (about 0.7-1.2× 0.3-0.5 mm in size).(vi).Receptacle: 14-18 layers of small and large rounded cells and six elongated vascular bundles arranged spirally, with several resin ducts. Retrophyllum Seed Cone Morpho-Anatomy Retrophyllum comptonii produces solitary reddish oblong-subglobose seed cones (10-20 × 7-15 mm).The seed cone is surrounded by 3-5 bracts.The ovule is inverted.Six major zones can be observed in the cross section of the seed cone: (i).Epimatium: 6-12 layers of large isodiametric cells.Sclereid cells are dominant in this zone.(iii).Exotesta: 16-20 layers of dense round cells.Several vascular traces, resin canals and sclereids are present in this zone.The epimatium is fleshy and fused with the exotesta surrounding the whole seed, forming a fleshy sarcotesta-like seed coat.(iii).Mesotesta: 6-8 layers of isodiametric cells, containing several vascular traces and resin ducts.(iv).Endotesta: 12-18 compact layers of small dense sclerified cells.This zone is hard and stony when the seed matures, forming a sclerotesta-like structure.(v).Nucellus: 4-8 layers of dense and smaller cells forming the protective cover of the embryo.(vi).Gametophyte: 8-12 layers of dense isodiametric cells with a straight embryo (about 0.4-0.9× 0.2-0.4mm in size). Acmopyle Seed Cone Morpho-Anatomy Acmopyle pancheri seed cones initiate in September, pollination and fertilization occur in December and the cones mature in May-June.The elongated oblong (18-34 × 10-18 mm), reddish brown seed cones are produced on scale-leaved branches of the second order.The receptacle is verrucose, papillate and fleshy (Figure 5G).The peduncle and scaly leaves transform into a fleshy structure, forming the receptacle.The seed cone has 6-10 bracts, with one or sometimes two erect ovules.Seven major zones can be observed in the cross section of the seed cone: (i).Epimatium: 5-8 layers of small and large isodiametric cells, with sclereids dominant.(ii).Exotesta: 14-20 layers of dense round cells, including several vascular traces, resin canals and sclereids.The epimatium is papery and fused with the exotesta surrounding the whole seed.(iii).Mesotesta: 6-12 layers of small dense cells.(iv).Endotesta: 12-18 compact layers of small dense sclerified cells, forming a thick, woody and stony zone.(v).Nucellus: 4-8 layers of dense and smaller cells forming the protective cover of the embryo.(vi).Gametophyte: 8-12 layers of dense isodiametric cells with a straight embryo (about 0.3-0.6 × 0.1-0.2mm in size).(vii).Receptacle: A fleshy elongate receptaculum is present, consisting of 12-18 layers of small and large rounded cells with four vascular bundles and resin ducts at the centre (Figure 4g,h). Dacrycarpus Seed Cone Morpho-Anatomy Dacrycarpus dacrydioides seed cones initiate in March and mature in January-February.The seed cones are elongated oblong (10-18 × 4-8 mm) and fleshy, with an orangered colour.The peduncle and keel-shaped leaves transform into a fleshy receptacle.The seed cone usually has 2-3 bracts and one inverted ovule.Six major zones can be observed in the cross section of the seed cone: (i).Epimatium: 3-6 layers of elongated cells.(ii).Exotesta: 10-14 layers of dense small round and elongated cells, including several vascular traces and resin canals, which are elongated resin ducts.The epimatium is papery and fused with the exotesta surrounding the whole seed (Figure 6d-f).(iii).Endotesta: 4-6 compact layers of small dense sclerified cells.However, the endotesta is not woody.(iv).Nucellus: 3-6 layers of elongated irregular cells form the protective cover of the embryo.(v).Gametophyte: 10-15 layers of dense large isodiametric cells with a straight embryo (about 0.6-1.0 × 0.2-0.5 mm in size).(vi).Receptacle: A fleshy receptaculum is present, consisting of 12-18 layers of small and large rounded cells, with three vascular bundles and resin ducts. Dacrycarpus Seed Cone Morpho-Anatomy Dacrycarpus dacrydioides seed cones initiate in March and mature in January-February.The seed cones are elongated oblong (10-18 × 4-8 mm) and fleshy, with an orange -red colour.The peduncle and keel-shaped leaves transform into a fleshy receptacle.The seed cone usually has 2-3 bracts and one inverted ovule.Six major zones can be observed in the cross section of the seed cone: (i).Epimatium: 3-6 layers of elongated cells.(ii).Exotesta: 10-14 layers of dense small round and elongated cells, including several vascular traces and resin canals, which are elongated resin ducts.The epimatium is papery and fused with the exotesta surrounding the whole seed (Figure 6d-f).(iii).Endotesta: 4-6 compact layers of small dense sclerified cells.However, the endotesta is not woody.(iv).Nucellus: 3-6 layers of elongated irregular cells form the protective cover of the embryo.(v).Gametophyte: 10-15 layers of dense large isodiametric cells with a straight embryo (about 0.6-1.0 × 0.2-0.5 mm in size).(vi).Receptacle: A fleshy receptaculum is present, consisting of 12-18 layers of small and large rounded cells, with three vascular bundles and resin ducts.Dacrycarpus dacrydioides longitudinal section (d), cross section (e,f); and Dacrydium cupressinum longitudinal section (g), cross section (h,i).Shown are the fleshy sarcotesta-like seed coat (Slsc), mesotesta (Mes), endotesta (End), epimatium (Em), megagametophyte (Mg), micropyle (Mi), embryo (Emb), nucellus (Nu), resin canal (Rc), resin duct (Rd) and receptaculum (Rp). Dacrydium Seed Cone Morpho-Anatomy Dacrydium cupressinum seed cones initiate in August-September and mature in March (~18 months later).The seed cones are elongated oblong (6-10 × 2-4 mm), fleshy and bright red.The fertile bracts transform into a swollen and fleshy bright-coloured structure at maturity.The seed cone has 8-14 bracts and one inverted ovule.An asymmetrical cuplike fleshy epimatium is also present (Figures 5D,E and 6g-i).Six major zones can be observed in the cross section of the seed cone: (i).Epimatium: 3-8 layers of small irregular cells.The epimatium is a fleshy, asymmetrical, cup-like structure covering half of the seed.(ii).Exotesta: 1-2 layers of elongated cells.No vascular traces or resin canals are present.(iii).Endotesta: 8-18 compact layers of small dense sclerified cells.(iv).Nucellus: 6-10 layers of dense and smaller rounded cells, forming the protective cover of the embryo.(v).Gametophyte: 8-14 layers of dense isodiametric cells with a straight embryo (about 0.25-0.6 × 0.1-0.2mm in size).(vi).Receptacle: 12-18 layers of small and large rounded cells, including three vascular bundles and several resin ducts (Figure 4e,f). Falcatifolium Seed Cone Morpho-Anatomy Falcatifolium papuanum seed cones are solitary with a fleshy asymmetrical cup-like epimatium and receptaculum.The seed cones are obovoid and fleshy (10-16 × 4-6 mm).Dacrycarpus dacrydioides longitudinal section (d), cross section (e,f); and Dacrydium cupressinum longitudinal section (g), cross section (h,i).Shown are the fleshy sarcotesta-like seed coat (Slsc), mesotesta (Mes), endotesta (End), epimatium (Em), megagametophyte (Mg), micropyle (Mi), embryo (Emb), nucellus (Nu), resin canal (Rc), resin duct (Rd) and receptaculum (Rp). Dacrydium Seed Cone Morpho-Anatomy Dacrydium cupressinum seed cones initiate in August-September and mature in March (~18 months later).The seed cones are elongated oblong (6-10 × 2-4 mm), fleshy and bright red.The fertile bracts transform into a swollen and fleshy bright-coloured structure at maturity.The seed cone has 8-14 bracts and one inverted ovule.An asymmetrical cup-like fleshy epimatium is also present (Figures 5D,E and 6g-i).Six major zones can be observed in the cross section of the seed cone: (i).Epimatium: 3-8 layers of small irregular cells.The epimatium is a fleshy, asymmetrical, cup-like structure covering half of the seed.(ii).Exotesta: 1-2 layers of elongated cells.No vascular traces or resin canals are present.(iii).Endotesta: 8-18 compact layers of small dense sclerified cells.(iv).Nucellus: 6-10 layers of dense and smaller rounded cells, forming the protective cover of the embryo.(v).Gametophyte: 8-14 layers of dense isodiametric cells with a straight embryo (about 0.25-0.6 × 0.1-0.2mm in size).(vi).Receptacle: 12-18 layers of small and large rounded cells, including three vascular bundles and several resin ducts (Figure 4e,f). Falcatifolium Seed Cone Morpho-Anatomy Falcatifolium papuanum seed cones are solitary with a fleshy asymmetrical cup-like epimatium and receptaculum.The seed cones are obovoid and fleshy (10-16 × 4-6 mm).The peduncle and keel-shaped leaves transform into a fleshy red receptacle.The seed cone usually has 6-12 bracts.The ovule orientation is inverted, but this gradually changes to nearly erect when the seed cones are mature.Six major zones can be observed in a cross section of the seed cone: (i).Epimatium: 4-8 layers of small, elongated and irregular-shaped cells.(ii).Exotesta: 3-6 layers of small rounded and elongated cells, including vascular traces or resin canals.(iii).Endotesta: 6-14 compact layers of small dense sclerified cells.(iv).Nucellus: 4-8 layers of dense and small rounded cells forming the protective cover of the embryo.(v).Gametophyte: 8-16 layers of dense isodiametric cells with a straight embryo (about 0.3-0.6 × 0.15-0.25 mm in size).(vi).Receptacle: 10-18 layers of small and large isodiametric cells, with two vascular bundles and resin ducts. Comparison of Seed Cone Morpho-Anatomical Characters The seed cone shape of the species studied in both clades varies from obovoid (Afrocarpus falcatus, Podocarpus henkelii, Retrophyllum comptonii, Dacrydium cupressinum and Falcatifolium papuanum), to globose (Nageia nagi), to oblong (N.wallichiana, Podocarpus elongatus, P. oleifolius, P. spinulosus, P. elatus, Dacrycarpus dacrydioides and Acmopyle pancheri).The ovule orientation is inverted in all species except A. pancheri.The number of ovules per seed cone is usually one and sometimes two (Table 1).The testa is observed to be differentiated into three zones (exotesta, mesotesta and endotesta) or two zones (exotesta and endotesta).In the Podocarpoid clade, three zones are present in Afrocarpus falcatus, Podocarpus henkelii, P. elongatus, P. oleifolius and Retrophyllum comptonii and two zones in Nageia nagi, N. wallichiana, Podocarpus spinulosus and P. elatus.In the Dacrydioid clade, the testa is divided into two zones (exotesta and endotesta) in the species examined (Dacrydium cupressinum, Dacrycarpus dacrydioides and Falcatifolium papuanum).An epimatium is present in all species (Figures 1-6).The epimatium and outer testa in all species, except Dacrydium cupressinum and Falcatifolium papuanum, are connate, forming a papery/coriaceous or fleshy sarcotestalike seed coat.This sarcotesta-like seed coat is papery/coriaceous in all species studied except Afrocarpus falcatus, Nageia nagi, Podocarpus henkelii and Retrophyllum comptonii, which possess a fleshy sarcotesta-like seed coat, but lack a fleshy receptacle (Figures 1 and 2).The mesotesta is woody and stony in Acmopyle pancheri, while the endotesta is woody and stony in Afrocarpus falcatus, Podocarpus henkelii and Retrophyllum comptonii.Species with a fleshy sarcotesta-like seed coat have a hard woody mesotesta or endotesta, except for Acmopyle pancheri, which has a papery sarcotesta-like seed coat at maturity.An asymmetrical cup-like fleshy epimatium is present in Dacrydium cupressinum and Falcatifolium papuanum.Nageia wallichiana and N. motleyi are the only species in the genus with a fleshy, bright red receptaculum.The receptacle surfaces of Acmopyle pancheri, A. sahniana and Dacrycarpus dacrydioides have a papillate appearance (Figures 3 and 5).Whitish wax is present on both the receptacle and seed surface of most species, and in Acmopyle pancheri and Afrocarpus falcatus, the entire seed cones are covered by stomata containing white wax.Vascular bundles, resin canals and resin ducts are present in the testa of all species and are especially prominent in Acmopyle sahniana, Dacrycarpus dacrydioides, both Nageia species, Retrophyllum comptonii, all Podocarpus species examined and Afrocarpus falcatus.A system of several small vascular traces and resin ducts is present in the exotesta of Afrocarpus falcatus, Podocarpus elongatus, P. henkelii, P. spinulosus, P. elatus, Nageia nagi, N. wallichiana and Retrophyllum comptonii, and in the mesotesta of Podocarpus oleifolius.A similar complex system of vascular traces and resin canals is present in Dacrycarpus dacrydioides in the inner layers of the exotesta.A resinous layer or a system of enlarged resin ducts is present in the mesotesta of Afrocarpus falcatus, Podocarpus elongatus, P. henkelii and Retrophyllum comptonii; enlarged resin canals are present in the mesotesta of Podocarpus henkelii and the endotesta of Podocarpus spinulosus and P. elatus.A similar complex system of enlarged resin ducts is present in Dacrycarpus dacrydioides in the inner layers of the mesotesta.No such system was observed in Dacrydium cupressinum or Falcatifolium papuanum. Seed Cone Morpho-Anatomical Traits and Structures Both clades have evolved diverse structures and traits in their seed cones. Fleshy Seed Cones The species of both clades produce fleshy seed cones, although the fleshy structures (receptaculum, epimatium or fleshy sarcotesta-like seed coat) differ among the genera.The ancestral state reconstruction of fleshiness and non-fleshiness in podocarps is not very meaningful in an evolutionary sense, as the fleshy structures differ across the clades, and sometimes within genera.All four genera of the Podocarpoid clade (Afrocarpus, Podocarpus, Nageia and Retrophyllum) and three genera of the Dacrydioid clade (Dacrydium, Dacrycarpus and Falcatifolium) produce fleshy seed cones, but the fleshy structures differ among the genera.A fleshy receptaculum and sarcotesta-like seed coat are the main fleshy structures. Epimatium Morphology An epimatium is present in all species in both clades.The ancestral state reconstructions infer that the presence of an epimatium is an ancestral state in the Podocarpoid and Dacrydioid clades.However, the epimatium morphology differs among genera, from free to fused and from fleshy to papery.Ancestral state reconstructions indicate that the papery fused epimatium is an ancestral trait in the Podocarpoid clade and the fleshy fused epimatium evolved several times (Figure 7).An epimatium fused with the outer testa is present in all species of the Podocarpoid clade and evolved independently in Dacrycarpus (Dacrydioid clade).Dacrydium and Falcatifolium have a free asymmetrical cup-like epimatium that usually becomes fleshy when the seed cone is mature.Dacrycarpus and Podocarpus species with a fused epimatium have a papery morphology, except in P. smithii, P. henkelii, P. madagascariensis and P. capuronii and in four of the six species of Nageia, where the epimatium is fused to the outer testa and develops a fleshy sarcotesta-like seed coat, akin to that in Afrocarpus and Retrophyllum (Figure 1). Receptaculum Morphology The presence of a fleshy receptacle is a characteristic feature in both clades.In most species of Podocarpus, the fleshy structure is a receptaculum (the exceptions are P. smithii, P. henkelii, P. madagascariensis and P. capuronii).A receptaculum is also present in two species of Nageia (N.wallichiana and N. motleyi) and all species of Dacrydium, Dacrycarpus, Falcatifolium and Acmopyle (Figure 4).The ancestral state reconstructions elucidate that the presence of a fleshy receptaculum is most likely an ancestral trait in both clades examined here (Supplementary Figure S2).The fleshy receptaculum is absent in some Podocarpus species (P.smithii, P. henkelii, P. madagascariensis and P. capuronii), which develop a fleshy fused epimatium with the outer testa, with a woody and stony endotesta.The receptaculum is usually brightly coloured.The seed cone bracts or scale leaves around the seed and the asymmetrical cup-like epimatium transform into a fleshy structure at seed cone maturity in Dacrydium and Falcatifolium.The receptacle in Acmopyle has a papillate surface (similar to Dacrycarpus) with white (waxy) stomata. Testa Morpho-Anatomy In Dacrydium, Dacrycarpus, Falcatifolium, Nageia (N.wallichiana and N. motleyi), Podocarpus (except P. smithii, P. henkelii, P. madagascariensis and P. capuronii) and Acmopyle, the testa is non-fleshy and has a papery structure.The endotesta is also non-woody in all these species, except Acmopyle, where the endotesta is woody and stony.The testa is fused with the epimatium, forming a fleshy sarcotesta-like seed coat, in Retrophyllum, Afrocarpus, Nageia and in some species of Podocarpus (e.g., P. smithii, P. henkelii, P. madagascariensis and P. capuronii).These species also have a hard woody mesotesta or endotesta.This strategy possibly evolved in these species to encourage endozoochory but to prevent digestion of the seed.The ancestral state reconstructions of the fleshy sarcotesta-like structure show that it evolved several times in the Podocarpoid clade (Figure 8).Retrophyllum has no fleshy receptaculum and is comparable to Afrocarpus in developing a fleshy sarcotesta-like seed coat.The main morpho-anatomical difference between these genera is the presence of a hard stony sclerotesta in Afrocarpus and Retrophyllum and a non-woody sclerotesta in most species of Podocarpus and two species of Nageia.Several vascular bundles and resin canals were present in the exotesta of all the Podocarpus species examined. that it evolved several times in the Podocarpoid clade (Figure 8).Retrophyllum has no fleshy receptaculum and is comparable to Afrocarpus in developing a fleshy sarcotesta-like seed coat.The main morpho-anatomical difference between these genera is the presence of a hard stony sclerotesta in Afrocarpus and Retrophyllum and a non-woody sclerotesta in most species of Podocarpus and two species of Nageia.Several vascular bundles and resin canals were present in the exotesta of all the Podocarpus species examined. Ovule and Embryo Traits Prior to pollination, ovules are inverted and the micropyle points towards the cone axis/base in all species of both clades.However, the micropyle of the mature seed cones of Dacrydium, Falcatifolium and Acmopyle points towards the apex, i.e., the ovule orientation changes post-pollination.The number of prosuspensor cells varies widely in the Podocarpaceae (Table 2), and it ranges from seven to twenty-three in the Podocarpoid clade and from five to fourteen in the Dacrydioid clade.The number is lower in other reported Podocarps (3)(4)(5)(6), and there is variable reporting of the number of binucleate cells and elongating cells present.The reproductive cycle varies from one to two years.Afrocarpus falcatus, Nageia and Dacrycarpus dacrydioides complete their reproductive cycle in one year from seed cone initiation to the production of mature seed cones, while Dacrydium cupressinum and Acmopyle pancheri complete their reproductive cycle in two years.The reproductive cycle also varies among species of the same genus, e.g., Podocarpus species complete their reproductive cycle in one to two years, depending on the species.Most of the living podocarps (about 18 genera) produce fleshy seed cones.Fleshy seed cones are present in all genera of both clades.However, the fleshy structures are different (e.g., fleshy sarcotesta-like seed coat, bracts, receptacle and epimatium) and evolved multiple times.Podocarps, in general, evolved fleshy seed cones using different functional structures, e.g., receptaculum, epimatium, fleshy sarcotesta-like seed coat, aril or fleshy bracts [9,10,13,20]. Klaus and Matzke [6] reported that seven genera of the Podocarpaceae (including some Podocarpus species) have non-fleshy seed cones, while Herting et al. [8] reported that three genera of the Podocarpaceae (Manoao, Saxegothaea and Pherosphaera) have non-fleshy seed cones.Khan and Hill [9] suggest that 18 podocarp genera show fleshiness (the exceptions being Saxegothaea and Pherosphaera).This study confirms the presence of fleshy epimatium in Dacrydium, Falcatifolium, Microcachrys and Lepidothamnus, contrary to the study by Chen et al., [19].However, the current study demonstrates that fleshy seed cones have evolved several times, using different functional structures.Klaus and Matzke [6] suggested that a reversion from fleshy to non-fleshy seed cones took place by the Early Eocene in the Retrophyllum + Nageia + Afrocarpus clade.The current study suggests no reversion to non-fleshy seed cones, because these genera have fleshy seed cones.Herting et al. [8] also considered the seed cones of Retrophyllum + Nageia + Afrocarpus clade as fleshy in their ancestral state reconstructions.Klaus and Matzke [6] considered a seed cone fleshy in the Podocarpoid clade if the taxa had a fleshy receptacle and were likely bird-dispersed.The fossil record demonstrates the presence of fleshiness or fleshy structures in the female cone of the Podocarpaceae by the Middle Cretaceous [32]. The fossil record also provides some evidence for the presence of some of these traits.Fossil seed cones of the extinct species Podocarpus witherdenensis, from the Eocene of Vegetable Creek, NSW, Australia, show the presence of a fleshy receptaculum and a nonfleshy fused epimatium [33].The paleo data also suggest that the presence of fleshy cones, similar to crown podocarps, may have existed by the Early Cretaceous, in the Rajmahal flora in India [34].Seed cones with a free epimatium that are regarded as belonging to an extinct species of the genus Lepidothamnus were recorded from the Middle Cretaceous of Winton, Queensland [32].Similarly, some extinct podocarps (e.g., Mataia, Nipaniostrobus, Harrisiocarpus gucikii and H. cracoviensis) also show the presence of an epimatium-like structure [35][36][37][38][39].The fossil cone records also show the presence of a fleshy receptaculum in Dacrycarpus in the Miocene-Eocene [33].Seed cones with a fleshy receptaculum are recorded in fossil Dacrycarpus guipingensis from the Miocene of Guangxi, South China [40], D. puertae from the Eocene of Patagonia [41] and D. mucronatus from the Miocene of Balcombe Bay, Victoria, Australia [42]. Shifts in the Fleshy Structures In Retrophyllum, Afrocarpus, Nageia (except N. wallichiana and N. motleyi) and in some species of Podocarpus (e.g., P. smithii, P. henkelii, P. madagascariensis and P. capuronii), the seed coat (epimatium plus exotesta) is fleshy.The Podocarpoid clade exhibits a shift from a fleshy receptaculum to a fleshy sarcotesta-like seed coat on two separate occasions.A fleshy receptacle of several bright colours has been recorded in different species of Podocarpus, Dacrydium, Dacrycarpus, Falcatifolium, Acmopyle and two species of Nageia.The presence of a fleshy receptaculum is reconstructed as ancestral in both the Podocarpoid and Dacrydioid clades.However, in the Podocarpoid clade, the fleshy receptaculum is lost several times and reappears once in Nageia.This is also contrary to the study by Chen et al. [19], which suggested the absence of a fleshy receptacle.The only other genus with a fleshy receptaculum is Lepidothamnus [43].The Podocarpoid clade shows a transition from a fleshy receptaculum to a fleshy fused epimatium and from a fleshy fused epimatium to a fleshy receptaculum.This transition is more evident in the Podocarpoid clade and was not limited to the ancestor of the Afrocarpus-Nageia-Retrophyllum clade but has also occurred within Podocarpus and Nageia.In the case of Podocarpus, this transition has occurred several times.A similar transition has occurred in the Prumnopityoid clade, suggesting that the evolutionary history of fleshiness in the Podocarpaceae is complex, because of the presence or absence of fleshiness and the functional structures that contribute to fleshiness among the seed cones (i.e., receptaculum, bracts and/or epimatium).The presence of a fleshy receptaculum in Nageia was previously reported by Mill, 2001 [44].Klaus and Matzke [6] also reported that Podocarpus latifolius, P. milanjianus and P. elongatus have no fleshy receptacle, but our observations show that a fleshy receptaculum is present in P. latifolius and P. elongatus. In contrast, the Dacrydioid clade is different because Dacrycarpus produces a fleshy receptaculum and a fused papery epimatium, while Dacrydium and Falcatifolium produce a free asymmetrical fleshy epimatium, that surrounds the seed and the subtending multiple bracts, or a fleshy receptaculum at maturity [45,46].The epimatium is considered homologous to the ovuliferous scale of other conifers, and this conclusion is supported by Herting and Stützel, 2020 [47]. Significance of the Evolutionary Reconstruction of Fleshiness Both clades produce brightly coloured fleshy seed cones.The ancestral reconstruction of the conifer seed cones implies that the ancestral seed cones of extant conifers were sclerified [8,45].However, the fleshiness of the Podocarpaceae seed cones is an ancestral trait [8].Klaus and Matzke [6] reported that fleshy seed cone structures appeared seven times in the Podocarpaceae independently and that the ancestral trait was non-fleshy.However, a reconstruction of fleshiness and non-fleshiness is not very meaningful in an evolutionary sense for podocarps, because the fleshy structures are quite different across clades, sometimes even within genera, and ancestral state reconstructions show that these fleshy structures have evolved multiple times in the Podocarpaceae. Broader Perspective with other Podocarps A broader podocarp perspective suggests that the type of morpho-anatomical adaptations and evolution of traits and structures seen in the Dacrydioid and Podocarpoid clades are typical of podocarps.A similar evolutionary pattern is present in the Prumnopityoid clade and Acmopyle [22].Different functional structures of podocarps seed cones (sterile bracts, fertile bracts, epimatia and occasionally arils) produce fleshiness either singly or in combination.This shows that there is strong evolutionary pressure among the living podocarps, especially in the Podocarpoid clade, to produce fleshy seed cones.This supports the theory that species of the Dacrydioid and Podocarpoid clades evolved fleshy seed cones and broad flattened foliage as an adaptive strategy in the canopy-forming Cenozoic Forest evolution [2,48].Fleshy fruits in podocarps are generally associated today with tropical forests [49].A parallel shift in the seed cone size is also noticeable with Cenozoic Forest evolution [50].The Podocarpoid clade species have relatively large seed cones (Supplementary Figure S4).Furthermore, Leslie et al. [50] reported that seed sizes are generally larger in animal dispersed species [5].The number of seeds per cone varies in the Podocarpoid clade from one to two, which have, for example, been reported in Podocarpus [51].Most genera in the Prumnopityoid clade (except Parasitaxus) possess one to two or else multiple seeds per cone, as does Acmopyle and the relict genera Microcachrys; Saxegothaea and Pherosphaera have multiovulate (seeded) cones [52].Species with one or two seeds per cone generally have larger seed cones than the multiovulate cone.All species of both the Dacrydioid and Podocarpoid clades have inverted ovules.The Prumnopityoid clade also has inverted ovules.Pherosphaera, Phyllocladus and Acmopyle are the only three genera with mature erect ovules.In Acmopyle, ovules are usually horizontally inclined to the fertile bract initially and become erect after pollination [12]. Evolution of Morphological Structures and Animal Dispersal The Dacrydioid and Podocarpoid clades show a remarkable tendency towards the evolution of fleshiness through a complex mechanism.Fleshiness has probably evolved through several different ways, in order to attract animals to disperse the seeds.Around 40% of living conifer species show fleshy morphologies [22].Eighteen podocarp genera show fleshy structures that support the zoochorous dispersal of seed cones.The seed cone of podocarps is generally dispersed by birds and small animals, which are attracted by the brightly coloured fleshy structures [9,10,13].Eleven podocarp genera show features consistent with avian endozoochory [6,53].For example, masting is well known in many temperate podocarps, which may favour zoochory [54].Both clades considered here show zoochory as the main seed dispersal strategy.For example, in the Podocarpoid clade, Podocarpus is the most dominant and widely distributed genus in Podocarpaceae [13].Most Podocarpus species produce seed cones with a colourful, fleshy receptacle and are mainly dispersed through endozoochory, with frugivory being dominant (Table 1).Afrocarpus falcatus is reported to be dispersed by birds and several other vertebrates, (Table 1) because its fleshy epimatium and testa complex serve as a food source [43,55].Retrophyllum, Nageia and Acmopyle produce colourful fleshy seed cones that also support zoochory [45,52,56].Klaus and Matzke [6] reported that the morphological characteristics of the Retrophyllum seed cone do not support avian endozoochory, although they regarded the presence of a fleshy receptaculum as the key evidence.Retrophyllum minus is a rheophytic species at low elevations in New Caledonia, for which hydrochory has been reported [56,57].The seed cone morphology of the Dacrydioid clade also supports zoochory, as all species produce fleshy seed cones (Table 1).The fleshy receptaculum of Dacrycarpus dacrydioides helps in the bird dispersal of seed cones [58,59].The fossil record shows the unique geographic distribution of Dacrycarpus and is a good example of probable long-distance dispersal [40,41].The fossil seed cone morphology also supports zoochory and bird dispersal.For example, the fossil seed cone records from Patagonia (Eocene) and South China (Miocene) show the presence of a fleshy receptacle [40,41].The seed cones of Dacrydium cupressinum are dispersed by frugivory (Table 1).These cones are small and favour local dispersal (10-40 m) by wind [60], but they are well adapted for frugivory when the fleshy receptacle is fully developed [61][62][63][64][65][66].Falcatifolium is also reported to be dispersed through zoochory (Table 1).The living species of the Dacrydioid and Podocarpoid clades have evolved fleshy and coloured seed cone structures and demonstrate a shift in the fleshy parts, and adaptation to endozoochory as the main seed dispersal strategy [6]. Conclusions The Podocarpoid and Dacrydioid clades produce fleshy seed cones and have zoochory as their main dispersal strategy.Ancestral state reconstruction shows that although fleshiness in both clades is an ancestral trait, this fleshiness is achieved through diverse and complex mechanisms of similar and different functional structures, e.g., the receptaculum, epimatium and fleshy sarcotesta-like seed coat (fleshy fused epimatium with testa).The Podocarpoid clade follows a similar evolutionary pathway in achieving fleshiness (e.g., a shift in fleshiness from the receptaculum to the epimatium fused with the testa and vice versa).Similarly, the Dacrydioid clade shows both receptaculate seed cones with a papery, fused epimatium and receptaculate seed cones with a fleshy, free epimatium.This shows that in podocarps, fleshy seed cones have evolved via different functional structures, suggesting that the evolutionary pressure to evolve fleshy cones is high in these podocarps and they have the genetic and morphological flexibility to respond to this pressure in several different ways.This provides an exciting opportunity for investigating the evolutionary history of fleshy cones in conifer and connections between plants and animals in the early Cenozoic. Figure 1 . 1 Figure 1.Seed cone of Afrocarpus falcatus (A), longitudinal section (B), cross section (C); Podocarpus henkelii (D), longitudinal section (E), cross section (F); Podocarpus spinulosus (G), longitudinal section (H), cross section (I); and Podocarpus oleifolius (J), longitudinal section (K), cross section (L).Shown are the epimatium (Em), bract (Br), fleshy sarcotesta-like seed coat (Slsc), mesotesta (Mes), endotesta (End), megagametophyte (Mg), micropyle (Mi), embryo (Emb), resin canal (Rc), resin duct (Rd), peduncle (Pd), receptaculum (Rp) and resin layer (Rl). Figure 1 . 1 Figure 1.Seed cone of Afrocarpus falcatus (A), longitudinal section (B), cross section (C); Podocarpus henkelii (D), longitudinal section (E), cross section (F); Podocarpus spinulosus (G), longitudinal section (H), cross section (I); and Podocarpus oleifolius (J), longitudinal section (K), cross section (L).Shown are the epimatium (Em), bract (Br), fleshy sarcotesta-like seed coat (Slsc), mesotesta (Mes), endotesta (End), megagametophyte (Mg), micropyle (Mi), embryo (Emb), resin canal (Rc), resin duct (Rd), peduncle (Pd), receptaculum (Rp) and resin layer (Rl). Figure 2 . 2 Figure 2. Seed anatomy of Podocarpus oleifolius longitudinal section (a), cross section (b); P. henke longitudinal section (c), cross section (d); P. elongatus longitudinal section (e), cross section (f); an P. elatus longitudinal section (g), cross section (h).Shown are the epimatium (Em), bract (Br), fles sarcotesta-like seed coat (Slsc), mesotesta (Mes), endotesta (End), megagametophyte (Mg), embr (Emb), resin canal (Rc), resin duct (Rd) and vascular bundles (Vb). Figure 2 . 2 Figure 2. Seed anatomy of Podocarpus oleifolius longitudinal section (a), cross section (b); P. henkelii longitudinal section (c), cross section (d); P. elongatus longitudinal section (e), cross section (f); and P. elatus longitudinal section (g), cross section (h).Shown are the epimatium (Em), bract (Br), fleshy sarcotesta-like seed coat (Slsc), mesotesta (Mes), endotesta (End), megagametophyte (Mg), embryo (Emb), resin canal (Rc), resin duct (Rd) and vascular bundles (Vb). Figure 3 . 3 Figure 3. Cross sections of the receptaculum of Podocarpus elongatus (A); Podocarpus oleifolius (B); Podocarpus spinulosus (C); Dacrycarpus dacrydioides (D); Dacrydium cupressinum (E) and Acmopyle pancheri (F), showing vascular bundles (Vb) and resin ducts (Rd). Figure 3 . 3 Figure 3. Cross sections of the receptaculum of Podocarpus elongatus (A); Podocarpus oleifolius (B); Podocarpus spinulosus (C); Dacrycarpus dacrydioides (D); Dacrydium cupressinum (E) and Acmopyle pancheri (F), showing vascular bundles (Vb) and resin ducts (Rd). Figure 4 . 4 Figure 4. Receptacle anatomy of Podocarpus oleifolius cross section (a,b); Podocarpus spinulosus cross section (c,d); Dacrydium cupressinum cross section (e,f); and Acmopyle pancheri cross section (g,h).Shown are the vascular bundles (Vb) and resin canals (Rc). Figure 4 . 4 Figure 4. Receptacle anatomy of Podocarpus oleifolius cross section (a,b); Podocarpus spinulosus cross section (c,d); Dacrydium cupressinum cross section (e,f); and Acmopyle pancheri cross section (g,h).Shown are the vascular bundles (Vb) and resin canals (Rc). Figure 5 . 5 Figure 5. Seed cone of Dacrycarpus dacrydioides (A) longitudinal section (B) cross section (C); Dacrydium cupressinum (D), longitudinal section (E), cross section (F); and Acmopyle pancheri (G), longitudinal section (H), seed (I).Shown are the epimatium (Em), bract (Br), fleshy sarcotesta-like seed coat (Slsc), mesotesta (Mes), endotesta (End), megagametophyte (Mg), micropyle (Mi), embryo (Emb), resin canal (Rc), resin duct (Rd) and receptaculum (Rp). Figure 5 . 5 Figure 5. Seed cone of Dacrycarpus dacrydioides (A) longitudinal section (B) cross section (C); Dacrydium cupressinum (D), longitudinal section (E), cross section (F); and Acmopyle pancheri (G), longitudinal section (H), seed (I).Shown are the epimatium (Em), bract (Br), fleshy sarcotesta-like seed coat (Slsc), mesotesta (Mes), endotesta (End), megagametophyte (Mg), micropyle (Mi), embryo (Emb), resin canal (Rc), resin duct (Rd) and receptaculum (Rp). Figure 6 . 6 Figure 6.Anatomy of seed cones of Afrocarpus falcatus longitudinal section (a), cross section (b,c); Dacrycarpus dacrydioides longitudinal section (d), cross section (e,f); and Dacrydium cupressinum longitudinal section (g), cross section (h,i).Shown are the fleshy sarcotesta-like seed coat (Slsc), mesotesta (Mes), endotesta (End), epimatium (Em), megagametophyte (Mg), micropyle (Mi), embryo (Emb), nucellus (Nu), resin canal (Rc), resin duct (Rd) and receptaculum (Rp). Figure 6 . 6 Figure 6.Anatomy of seed cones of Afrocarpus falcatus longitudinal section (a), cross section (b,c); Dacrycarpus dacrydioides longitudinal section (d), cross section (e,f); and Dacrydium cupressinum longitudinal section (g), cross section (h,i).Shown are the fleshy sarcotesta-like seed coat (Slsc), mesotesta (Mes), endotesta (End), epimatium (Em), megagametophyte (Mg), micropyle (Mi), embryo (Emb), nucellus (Nu), resin canal (Rc), resin duct (Rd) and receptaculum (Rp). Figure 7 . 7 Figure 7. Character mapping of fused epimatium fleshiness and non-fleshiness in different genera of Podocarpaceae-maximum likelihood.Figure 7. Character mapping of fused epimatium fleshiness and non-fleshiness in different genera of Podocarpaceae-maximum likelihood. Figure 7 . 7 Figure 7. Character mapping of fused epimatium fleshiness and non-fleshiness in different genera of Podocarpaceae-maximum likelihood.Figure 7. Character mapping of fused epimatium fleshiness and non-fleshiness in different genera of Podocarpaceae-maximum likelihood. Figure 8 . 8 Figure 8. Character mapping of the fleshy sarcotesta-like structure in different genera of Podocarpaceae-maximum likelihood.Figure 8. Character mapping of the fleshy sarcotesta-like structure in different genera of Podocarpaceae-maximum likelihood. Figure 8 . 8 Figure 8. Character mapping of the fleshy sarcotesta-like structure in different genera of Podocarpaceae-maximum likelihood.Figure 8. Character mapping of the fleshy sarcotesta-like structure in different genera of Podocarpaceae-maximum likelihood. Table 1 . 1 Seed cone morphological and anatomical, and qualitative and quantitative characteristics for the Podocarpoid and Dacrydioid clades. CharactersAfrocarpus falcatusNageia wallichianaNageia nagiPodocarpus henkelii (subgenus Podocarpus)Podocarpus elongatus (subgenus Podocarpus)Podocarpus oleifolius (subgenus Podocarpus)Podocarpus spinulosus (subgenus Foliolatus)Podocarpus elatus (subgenus Foliolatus)Retrophyllum comptoniiDacrydium cupressinumDacrycarpus dacrydioidesFalcatifolium papuanumAcmopyle pancheriReproductive cycle1 year1 year1 year1 year1 year1 year1 year1 year1 year2 years1 year1 year1-2 yearsCone shapeobovoidovoidgloboseobovoidovoidovoidovoidovoidovoid-subgloboseobovoidovoidobovoidovoidCone size (mm)12-20 × 8-1618-38 × 6-1012-18 × 10-1615-20 × 8-1213-20 × 4-812-22 × 4-711-24 × 6-725-45 × 10-2010-20 × 7-156-10 × 2-410-18 × 4-810-16 × 4-618-34 × 10-18Colouryellowishreddishbrownyellowishreddishreddishdark purpledark purplereddishreddishreddishreddishreddish-brownNumber of seeds per cone11-21111-211-21-211-211-2Seed size (mm)8-18 × 6-1414-20 × 10-1610-15 × 8-1212-17 × 4-66-12 × 3-55-8 × 3-68-10 × 5-715-20 × 12-157-17 × 5-122.5-6 × 2-36-10 × 4-86-7 × 4-56-8 × 5-7Seed surfacerugoserugoserugoserugosesmoothsmoothrugoserugoserugosesmoothsmoothsmoothrugoseSeed colourbrownbrownbrownlight brownbrownbrownbrownpurplish blackbrownbrownish blackpurplish blackdark brownbrownOvule orientationinvertedinvertedinvertedinvertedinvertedinvertedinvertedinvertedinvertedinvertedinvertedinvertederectCuticlethickthickthickthickthinthinthinthinthickthickthinthinthickEpidermal layers11-21-21111-2111111Epidermal cell shaperectangularround-triangularround-triangularround-triangularround-triangularrectangularround-triangularround-triangularisodiametricdome-shapeddome-shapeddome-shapedtriangularExotestafleshycoriaceouscoriaceousfleshycoriaceouscoriaceouscoriaceouscoriaceousfleshycoriaceouscoriaceouscoriaceouscoriaceousExotesta layers of cells10-1814-2212-2012-188-1310-1412-1618-2616-201-210-143-614-20Mesotesta layers of cells3-5--16-223-510-16--6-8---6-12Endotesta layers of cells20-3212-2010-1812-206-1210-165-84-612-188-104-66-1412-18Nucellus layers4-105-106-126-128-147-106-82-44-86-103-64-84-8Embryo (megaga-metophyte)straightstraightstraightstraightstraightstraightstraightstraightstraightstraightstraightstraightstraightshapeEmbryo size (mm)0.8-1.2 × 0.3-0.50.8-1.7 × 0.3-0.50.8-1.5 × 0.3-0.40.65-1.2 × 0.2-0.40.5-0.9 × 0.2-0.30.6-0.8 × 0.2-0.30.5-1.3 × 0.3-0.50.7-1.2 × 0.3-0.50.4-0.9 × 0.2-0.40.25-0.6 × 0.1-0.20.6-1.0 × 0.2-0.50.3-0.6 × 0.15-0.250.3-6 × 0.1-0.2 Table 1 . 1 Cont. Characters Afrocarpus falcatus Nageia wallichiana Nageia nagi Podocarpus henkelii (subgenus Podocarpus) Podocarpus elongatus (subgenus Podocarpus) Podocarpus oleifolius (subgenus Podocarpus) Podocarpus spinulosus (subgenus Foliolatus) Podocarpus elatus (subgenus Foliolatus) Retrophyllum comptonii Dacrydium cupressinum Dacrycarpus dacrydioides Falcatifolium papuanum Acmopyle pancheri Plants 2023, 12, 39036 of 25Bracts1-24-74-61-2222-423-58-142-36-124-10Stomata on bractspresentpresentpresentpresentpresentpresentpresentpresentpresentpresentpresentpresentpresentEpimatiumpresentpresentpresentpresentpresentpresentpresentpresentpresentpresentpresentpresentpresentEpimatium shapefleshy and fused with outer testa surrounding the whole seedpapery and fused with outer testa surrounding the whole seedfleshy and fused with outer testa surrounding the whole seedfleshy and fused with outer testa surrounding the whole seedpapery and fused with outer testa surrounding the whole seedpapery and fused with outer testa surrounding the whole seedpapery and fused with outer testa surrounding the whole seedpapery and fused with outer testa surrounding the whole seedfleshy and fused with outer testa surrounding the whole seedfleshy asymmetrical cup-likepapery and fused with outer testa surrounding the whole seedfleshy asymmetrical cup-likepapery and fused with outer testa surrounding the whole seedEpimatium structurefleshyfleshyfleshyfleshypaperypaperypaperypaperyfleshyfleshypaperyfleshypaperyEpimatium colouryellowishpurplepurpleyellowishpurpleolive greendark purplepurplish blackreddishbrownish blackpurplish blackreddish brownbrownReceptaculumabsentpresentabsentabsentpresentpresentpresentpresentabsentpresentpresentpresentpresentReceptaculum colour-reddish-absentbright redyellowish redblueish blackblueish black-reddishorange-redreddishreddish brownSclereidspresentpresentpresentpresentpresentpresentpresentpresentpresentpresentpresentpresentpresentResin canalspresentpresentpresentpresentpresentpresentpresentpresentpresentpresentpresentpresentpresentzoochory [NewDispersalzoochory [birds, bats (Rousettus aegyptiacus), monkeys (Chlorocebus pygerythrus), woodland dormouse (Graphiurus murinus) and Verreaux's mousezoochoryzoochoryzoochory (birds)zoochory (birds)zoochory (birds)zoochory (birds)zoochory (birds)zoochoryzoochory [New Zealand bellbird (Anthornis melanura) and T ūī habroptilus)] Kākāp ō (Strigops novaeseelandiae), (ProsthemaderaZealand pigeon (Hemiphaga novaeseelandiae), T ūī (Prosthemadera novaeseelandiae) melanura, Anthornis melanura), (Anthornis and New Zealand bellbirdzoochory (birds)zoochory (birds)(MyomyscusHemiphagaverreauxii)]novaeseelandiae,Prosthemaderanovaeseelandiae] Table 2 . 2 Number of prosuspensor and binucleate embryonic cells in Podocarpaceae. TaxonNumber of Prosuspensor CellsNumber of Binucleate Embryonic CellsReferencePodocarpoid CladeAfrocarpus18-239-11Buchholz, 1941 [30]Nageia18-237, 9-11#Podocarpus glomeratus11-132-3#P. chinensis14-151-2#P. lawrencei7-101-2#P. nivalis7-101-2#P. totara7-101-2#P. laetus7-101-2#P. urbanii11-132-3#Dacrydioid CladeDacrycarpus dacrydioides11-144 or 5Buchholz, 1941 [30]Dacrydium7-115-9#Other PodocarpaceaePhyllocladus4-69-12Doyle and Looby, 1939 [31]Saxegothaea3-410-12#Pectinopitys ferruginea7-97-9Buchholz, 1941 [30]Prumnopitys taxifolia6-99-12#4. Discussion4.1. 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Evolution Mechanism of Sputtered Film Uniformity with the Erosion Groove Size: Integrated Simulation and Experiment 18 November 2023 Guo Zhu 0000-0002-9996-201X School of Mechanical & Electrical Engineering Hunan City University 413000YiyangChina Yutong Yang yangyutong896@163.com 0000-0002-9996-201X School of Mechanical & Electrical Engineering Hunan City University 413000YiyangChina Baijun Xiao School of Mechanical & Electrical Engineering Hunan City University 413000YiyangChina Zhiyin Gan zhiyingan@126.com School of Mechanical Science & Engineering Huazhong University of Science & Technology 430074WuhanChina Evolution Mechanism of Sputtered Film Uniformity with the Erosion Groove Size: Integrated Simulation and Experiment 18 November 202310C2E0EFED0356BA40F3A24A2553980210.3390/molecules28227660Received: 28 September 2023 Revised: 13 November 2023 Accepted: 16 November 2023Zhu, G.Yang, Y.Xiao, B.Gan, Z. Evolution Mechanism of Monte Carlo methodmolecular dynamicsmagnetron sputteringsputtered particle transportfilm thickness uniformity In this work, Cu thin films were experimentally fabricated at different target-substrate distances by 2-inch and 4-inch circular planar magnetron targets.Meanwhile, the sputtering deposition of Cu thin films was investigated via an integrated multiscale simulation, where the magnetron sputtering discharge was modeled using the Monte Carlo (MC) method, and the sputtered particle transport was simulated using a coupled Monte Carlo (MC) and molecular dynamics (MD) method.Experimental results indicated that, as the target-substrate distance increased from 30 to 120 mm, the film thickness distribution of the 2-inch target sputtering changed from a bell-shaped curve to a line-shaped curve, while that of the 4-inch target sputtering varied from a saddle-shaped curve to a line-shaped curve.The simulation results were accordant with the experimental results.The simulation results revealed that, at a target-substrate distance of 30 mm, the sputtering particle flow from the 2-inch target overlapped strongly near the substrate center, leading to a bell-shaped film thickness distribution, while the increased diameter of the erosion groove on the 4-inch target reduced the superposition effect of the sputtering particle flow near the substrate center, resulting in a saddle-shaped film thickness distribution.In addition, when the target-substrate distance ranged from 30 to 120 mm, the film thickness uniformity of 4-inch target sputtering was superior to that of 2-inch target sputtering, and the underlying mechanism was discussed in detail. Introduction Due to the convenient process tuning and low coating temperature, magnetron sputtering is widely employed in manufacturing semiconductor, photo-electronic, and thermoelectric thin film devices [1][2][3].The film thickness uniformity, as a critical index, significantly determines the performance consistency of film devices [4].The thickness uniformity of the sputtered film is influenced by sputtering conditions, including the target-substrate geometrical configuration, relative motions between the target and substrate, and the target surface erosion.Investigating the interplay between sputtering conditions and film thickness uniformity is of great significance to improving the performance of thin film devices. Currently, real-time monitoring for the sputtered film deposition cannot be realized through experimental means.Improvements in the thickness uniformity of sputtered films solely via empirical inference and posterior experiments are difficult, time-consuming, and expensive [5].Consequently, the analytical models for the deposition uniformity of the circular planar single-target [6][7][8][9][10] and rectangular planar single-target [11,12] sputtering systems were successively developed to reinforce the understanding of experimental results and reveal the underlying mechanism.Then, extended analytical models were further proposed for the circular planar dual-target [13] and circular planar triple-target [14] sputtering systems.During magnetron sputtering deposition, fast-moving particles undergo no collision before they reach the substrate surface, while slow-moving particles are transported to the substrate surface by diffusion due to scattering collisions.However, the diffusion transport of slow-moving particles is neglected in the analytical model [14], although it significantly influences the film thickness distribution. On the other hand, the sputtered particle transport can also be simulated by the MC method, in which the free flight and scattering of sputtered particles are both taken into consideration.Thus, a more precise distribution of deposited particles on the substrate can be obtained through MC simulation.In most MC simulations, the post-collision flight angles of colliding particles are approximately calculated based on the assumption that the argon atom is at rest before the collision [15][16][17][18][19][20][21][22], since the accurate calculation of the scattering angle requires complex coordinate transformations [23].In this context, a coupled MC-MD method was developed to simulate the transport processes of sputtered particles [24], in which the velocities of colliding particles after collisions were calculated using the MD method.Furthermore, the initial emission positions of sputtered particles are determined by the ionization density distribution near the target surface during magnetron sputtering discharge [25].However, due to the disconnect between the magnetron sputtering discharge research and sputtered particle transport research, the initial emission positions of sputtered particles in existing MC simulations are approximately selected based on the uniform distribution [16], Gauss distribution [17,26], or the measured surface profile of the etched target [27].Accordingly, a combined numerical investigation of the magnetron sputtering discharge and sputtered particle transport is needed to precisely characterize the non-uniform erosion of the target surface, which is critical to understanding the control mechanism of sputtered film uniformity. In the present work, a combined numerical investigation of the magnetron sputtering discharge and sputtered particle transport was conducted.In this combined numerical simulation, the magnetron sputtering discharge was modeled using the MC method, in which the sputtering possibility distribution on the copper target surface was evaluated based on the calculated ionization density distribution near the target surface.Then, the transport processes of sputtered Cu atoms were further simulated by the coupled MC-MD method, in which the initial emission positions of sputtered Cu atoms were selected from the calculated sputtering possibility distribution.In particular, the thickness distributions of sputtered films deposited from 2-inch and 4-inch targets were investigated through simulation and experiments, respectively.The evolution mechanism of sputtered film uniformity with the erosion groove size was skillfully explored by investigating the deposition behavior of sputtered atoms ejected from fan-shaped sputtering sources.This work attempted to bridge the gap between magnetron sputtering discharge research and sputtered atom transport research in the DC magnetron sputtering field, such that the state data of sputtered atoms arriving at the substrate surface can be calculated more realistically. Results The Radial Distributions of the Sputtering Possibility on the 2-Inch and 4-Inch Target Surface Figure 1 shows distribution nephograms of ionization density for the 2-inch target sputtering and 4-inch target sputtering, respectively.In Figure 1a,b, all the ionization density values were normalized by the maximum ionization density value in the simulation domain, and all the points in the simulation domain were colored based on the normalized ionization density values.Figure 1 displays the typical ionization density distribution nephograms of balanced magnetron sputtering discharge, which resembles those reported in ref. [28].As shown in Figure 1a,b, ionization points are concentrated in the region approaching the target surface, since almost all the energetic electrons are trapped in this region with the magnetic field almost parallel to the target surface [29].As the target diameter increases from 2 inches to 4 inches, the width and central diameter of the annular ionization regime both increase due to the increase in the width of the magnetic field on the target surface. this region with the magnetic field almost parallel to the target surface [29].As the target diameter increases from 2 inches to 4 inches, the width and central diameter of the annular ionization regime both increase due to the increase in the width of the magnetic field on the target surface.Argon ions, generated by the collision between energetic electrons and argon atoms, are accelerated by the electrostatic field between ionization points and the target surface.They almost impact perpendicularly onto the target surface, since the effect of the magnetic field on argon ion trajectory is negligible.The bombardment of argon ions results in the sputtering of near-surface target atoms.The sputtering yield is dependent on the bombarding energy.Herein, the sputtering density is introduced and defined as the number of sputtered atoms per unit area, which can be expressed by [30] S j = ∑ Y E i ) n 0 i=1 2πr j ∆r(1) where Y is the energy-dependent sputtering yield, Ei is the bombarding energy of the i-th argon ion, n0 is the total number of argon ions impinging the annular region with a radial coordinate of rj on the target surface, ∆r is the radial width of the annular region, and Sj is the sputtering density within the annular region.Based on Equation (1), the radial distributions of the sputtering density for the 2-inch and 4-inch targets can be calculated, respectively.Then, the sputtering possibility of a sputtered atom ejected from the j-th annular region can be calculated by P j = S j ∑ S k n 1 1 (2) where n1 is the total number of the annular regions divided on the target surface.The radial distributions of the sputtering possibility on the target surface can be evaluated according to Equation (2). Figure 2 displays the radial distribution of the sputtering possibility for the 2-inch and 4-inch targets, respectively.As shown in Figure 2, the peaks of the sputtering possibility distribution are situated at the radial coordinates of ±16 mm for the 2-inch target sputtering, while the peaks of the sputtering possibility distribution are located at the radial coordinates of ±30 mm for the 4-inch target sputtering.These radial coordinates correspond to the points on the target surface where the vertical component of the magnetic field is zero [31].Therefore, the initial emission position (rj) of a sputtered atom can be sampled from the sputtering possibility distribution via the acceptance-rejection method.Then, the x and y coordinates of the sputtered atom on the target surface can be determined by the random number ϕ distributed uniformly in [0, 2π]. x = rjcos(ϕ) (3) Argon ions, generated by the collision between energetic electrons and argon atoms, are accelerated by the electrostatic field between ionization points and the target surface.They almost impact perpendicularly onto the target surface, since the effect of the magnetic field on argon ion trajectory is negligible.The bombardment of argon ions results in the sputtering of near-surface target atoms.The sputtering yield is dependent on the bombarding energy.Herein, the sputtering density is introduced and defined as the number of sputtered atoms per unit area, which can be expressed by [30] S j = ∑ n 0 i=1 Y(E i ) 2πr j ∆r(1) where Y is the energy-dependent sputtering yield, E i is the bombarding energy of the i-th argon ion, n 0 is the total number of argon ions impinging the annular region with a radial coordinate of r j on the target surface, ∆r is the radial width of the annular region, and S j is the sputtering density within the annular region.Based on Equation (1), the radial distributions of the sputtering density for the 2-inch and 4-inch targets can be calculated, respectively.Then, the sputtering possibility of a sputtered atom ejected from the j-th annular region can be calculated by P j = S j ∑ n 1 1 S k(2) where n 1 is the total number of the annular regions divided on the target surface.The radial distributions of the sputtering possibility on the target surface can be evaluated according to Equation (2). Figure 2 displays the radial distribution of the sputtering possibility for the 2-inch and 4-inch targets, respectively.As shown in Figure 2, the peaks of the sputtering possibility distribution are situated at the radial coordinates of ±16 mm for the 2-inch target sputtering, while the peaks of the sputtering possibility distribution are located at the radial coordinates of ±30 mm for the 4-inch target sputtering.These radial coordinates correspond to the points on the target surface where the vertical component of the magnetic field is zero [31].Therefore, the initial emission position (r j ) of a sputtered atom can be sampled from the sputtering possibility distribution via the acceptance-rejection method.Then, the x and y coordinates of the sputtered atom on the target surface can be determined by the random number φ distributed uniformly in [0, 2π]. x = r j cos(φ) (3) y = r j sin(φ) (4) y = rjsin(ϕ)(4) Deposition Density Distribution of the 2-Inch Target Sputtering Figure 3 displays the variation in the thickness distribution of the sputtered film deposited from the 2-inch target with the target-substrate distance.In Figure 3, the experimentally measured film thickness distributions are depicted by dotted curves, while the calculated deposition density distributions are graphed by colored solid curves.Herein, the deposition density is defined as the number of deposited Cu atoms on the substrate per unit area.To evaluate the radial deposition density distribution on the substrate in the simulation, the circular substrate plane was partitioned into 13 regions, including 1 circular region and 12 concentric annular regions.The radius of the circular region and the radial width of the annular regions were set to 1 and 2 mm, respectively.In each simulation, the number of Cu atoms deposited in the j-th region was recorded and then divided by the area of the j-th region.Accordingly, the relationship between the film thickness and deposition density can be expressed as follows: T j = 𝑚n 𝜌𝐴 = k n A j =kD j (5) where Tj is the film thickness in the j-th region, m is the mass of Cu atom, nj is the number of Cu atoms deposited in the j-th region, ρ is the coating density, Aj is the area of the j-th region, k is the ratio of m and ρ, and Dj is the deposition density.Therefore, the film thickness is proportional to the deposition rate.To intuitively represent the variation in the film thickness uniformity, all the calculated deposition density values within the 13 regions at various target-substrate distances were normalized by that in the circular region when the target-substrate distance was set to 30 mm.Accordingly, Figure 3 represents the relative film thickness distributions for the 2-inch target sputtering.To compare the experimentally measured results with the simulated results, the measured film thickness values at various target-substrate distances were normalized by that in the circular region when the target-substrate distance was set to 30 mm.It is clear that the calculated distributions are consistent with the experimentally measured distributions at all target-substrate distances.It can be seen from Figure 3 that, when the target-substrate distance is 30 mm, the film thickness distribution has a bell-shaped profile, where the film thickness at the substrate center is much greater than that near the substrate margin.With the increase in the target-substrate distance from 30 to 120 mm, the bell-shaped film thickness distribution gradually varies to an arch-shaped one and, ultimately, to a line-shaped one.It can be found that the increase in the target-substrate distance is beneficial for improvements in film thickness uniformity but at the expense of the deposition rate. Deposition Density Distribution of the 2-Inch Target Sputtering Figure 3 displays the variation in the thickness distribution of the sputtered film deposited from the 2-inch target with the target-substrate distance.In Figure 3, the experimentally measured film thickness distributions are depicted by dotted curves, while the calculated deposition density distributions are graphed by colored solid curves.Herein, the deposition density is defined as the number of deposited Cu atoms on the substrate per unit area.To evaluate the radial deposition density distribution on the substrate in the simulation, the circular substrate plane was partitioned into 13 regions, including 1 circular region and 12 concentric annular regions.The radius of the circular region and the radial width of the annular regions were set to 1 and 2 mm, respectively.In each simulation, the number of Cu atoms deposited in the j-th region was recorded and then divided by the area of the j-th region.Accordingly, the relationship between the film thickness and deposition density can be expressed as follows: T j = mn j ρA j = k n j A j = kD j(5) where T j is the film thickness in the j-th region, m is the mass of Cu atom, n j is the number of Cu atoms deposited in the j-th region, ρ is the coating density, A j is the area of the j-th region, k is the ratio of m and ρ, and D j is the deposition density.Therefore, the film thickness is proportional to the deposition rate.To intuitively represent the variation in the film thickness uniformity, all the calculated deposition density values within the 13 regions at various target-substrate distances were normalized by that in the circular region when the target-substrate distance was set to 30 mm.Accordingly, Figure 3 represents the relative film thickness distributions for the 2-inch target sputtering.To compare the experimentally measured results with the simulated results, the measured film thickness values at various target-substrate distances were normalized by that in the circular region when the targetsubstrate distance was set to 30 mm.It is clear that the calculated distributions are consistent with the experimentally measured distributions at all target-substrate distances.It can be seen from Figure 3 that, when the target-substrate distance is 30 mm, the film thickness distribution has a bell-shaped profile, where the film thickness at the substrate center is much greater than that near the substrate margin.With the increase in the target-substrate distance from 30 to 120 mm, the bell-shaped film thickness distribution gradually varies to an arch-shaped one and, ultimately, to a line-shaped one.It can be found that the increase in the target-substrate distance is beneficial for improvements in film thickness uniformity but at the expense of the deposition rate. Deposition Density Distribution of the 4-Inch Target Sputtering Figure 4 displays the variation in the thickness distributions of sputtered Cu films deposited from the 4-inch target with the target-substrate distance.In Figure 4, the experimentally measured film thickness distributions are depicted by colored dotted curves, while the calculated deposition density distributions are graphed by colored solid curves.To compare the experimentally measured results with the simulated results, the measured film thickness values and calculated deposition density values were normalized by the same scheme mentioned in Section 2.2.Accordingly, Figure 4 plots the relative film thickness distribution.It is clear that the calculated distributions are accordant with the experimentally measured distributions at all target-substrate distances.It can be found from Figure 4 that, when the target-substrate distance is 30 mm, the distribution profile of the film thickness is a saddle-shaped curve, where the film thickness at the substrate center is lower than that near the substrate margin.With the increase in the target-substrate distance from 30 to 120 mm, the saddle-shaped film thickness distribution gradually transforms to an arch-shaped one and, further, to a line-shaped one.It also can be seen that the increase in the target-substrate distance leads to a reduction in the deposition rate. Deposition Density Distribution of the 4-Inch Target Sputtering Figure 4 displays the variation in the thickness distributions of sputtered Cu films deposited from the 4-inch target with the target-substrate distance.In Figure 4, the experimentally measured film thickness distributions are depicted by colored dotted curves, while the calculated deposition density distributions are graphed by colored solid curves.To compare the experimentally measured results with the simulated results, the measured film thickness values and calculated deposition density values were normalized by the same scheme mentioned in Section 2.2.Accordingly, Figure 4 plots the relative film thickness distribution.It is clear that the calculated distributions are accordant with the experimentally measured distributions at all target-substrate distances.It can be found from Figure 4 that, when the target-substrate distance is 30 mm, the distribution profile of the film thickness is a saddle-shaped curve, where the film thickness at the substrate center is lower than that near the substrate margin.With the increase in the target-substrate distance from 30 to 120 mm, the saddle-shaped film thickness distribution gradually transforms to an arch-shaped one and, further, to a line-shaped one.It also can be seen that the increase in the target-substrate distance leads to a reduction in the deposition rate. Deposition Density Distribution of the 4-Inch Target Sputtering Figure 4 displays the variation in the thickness distributions of sputtered Cu films deposited from the 4-inch target with the target-substrate distance.In Figure 4, the experimentally measured film thickness distributions are depicted by colored dotted curves, while the calculated deposition density distributions are graphed by colored solid curves.To compare the experimentally measured results with the simulated results, the measured film thickness values and calculated deposition density values were normalized by the same scheme mentioned in Section 2.2.Accordingly, Figure 4 plots the relative film thickness distribution.It is clear that the calculated distributions are accordant with the experimentally measured distributions at all target-substrate distances.It can be found from Figure 4 that, when the target-substrate distance is 30 mm, the distribution profile of the film thickness is a saddle-shaped curve, where the film thickness at the substrate center is lower than that near the substrate margin.With the increase in the target-substrate distance from 30 to 120 mm, the saddle-shaped film thickness distribution gradually transforms to an arch-shaped one and, further, to a line-shaped one.It also can be seen that the increase in the target-substrate distance leads to a reduction in the deposition rate. Film Thickness Uniformity of 2-Inch and 4-Inch Target Sputtering The thickness uniformity of a sputtered film (U) can be evaluated using the following equation [32]: U = 100% − 2(H max − H min ) H max + H min ×100%(6) where the H max and H min represent the maximum and minimum film thickness, respectively.Figure 5 displays the thickness uniformity of sputtered films deposited by 2-inch and 4-inch targets under different target-substrate distances.In Figure 5, variation curves of film thickness uniformity for 2-inch and 4-inch target sputtering are depicted by red and blue lines, respectively.It can be seen that, as the target distance increases from 30 to 120 mm, the film thickness uniformity of 2-inch target sputtering increases monotonically, while the film thickness uniformity of 4-inch target sputtering decreases first and then increases.The film thickness uniformity of 4-inch target sputtering is superior to that of 2-inch target sputtering.As the target-substrate distance increases from 60 to 120 mm, the film thickness uniformity of 2-inch and 4-inch target sputtering gradually increases.It is known that sputtered atoms have an anisotropic initial emission angular distribution [33]. As the sputtering pressure is 0.5 Pa, the mean free paths of sputtered atoms with 2 eV and 30 eV kinetic energy are 41.6 mm and 113.7 mm, respectively, and the percentage of these sputtered Cu atoms is around 70% [24].This suggests that, at a target-substrate distance of 30 mm, most of the sputtered atoms undergo no collision before they arrive at the substrate surface.Accordingly, the anisotropic ejection of sputtered atoms results in the bell-shaped and saddle-shaped film thickness profiles shown in Figures 3 and 4 under a target-substrate distance of 30 mm.When the target-substrate distance is set to 120 mm, it exceeds the mean free path of sputtered atoms possessing energy of 30 eV.Thus, most of the sputtered atoms will experience scattering collisions before they arrive at the substrate surface.The flying angles of sputtered atoms gradually vary due to the increase in the scattering collision frequency with the target-substrate distance, leading to a quasi-isotropic incident angular distribution of sputtered atoms when they approach the substrate surface [15,34].Accordingly, since the anisotropy of the incident angular distribution gradually decreases with the target-substrate distance, the arch-shaped or saddle-shaped film thickness distribution is gradually transformed into the line-shaped one shown in Figures 3 and 4, resulting in an improvement in the film thickness uniformity. Molecules 2023, 28, x FOR PEER REVIEW 6 of 13 Film Thickness Uniformity of 2-Inch and 4-Inch Target Sputtering The thickness uniformity of a sputtered film (U) can be evaluated using the following equation [32]: U = 100% − 2(H max − H min ) H max +H min ×100%(6) where the Hmax and Hmin represent the maximum and minimum film thickness, respectively.Figure 5 displays the thickness uniformity of sputtered films deposited by 2-inch and 4-inch targets under different target-substrate distances.In Figure 5, variation curves of film thickness uniformity for 2-inch and 4-inch target sputtering are depicted by red and blue lines, respectively.It can be seen that, as the target distance increases from 30 to 120 mm, the film thickness uniformity of 2-inch target sputtering increases monotonically, while the film thickness uniformity of 4-inch target sputtering decreases first and then increases.The film thickness uniformity of 4-inch target sputtering is superior to that of 2-inch target sputtering.As the target-substrate distance increases from 60 to 120 mm, the film thickness uniformity of 2-inch and 4-inch target sputtering gradually increases.It is known that sputtered atoms have an anisotropic initial emission angular distribution [33]. As the sputtering pressure is 0.5 Pa, the mean free paths of sputtered atoms with 2 eV and 30 eV kinetic energy are 41.6 mm and 113.7 mm, respectively, and the percentage of these sputtered Cu atoms is around 70% [24].This suggests that, at a target-substrate distance of 30 mm, most of the sputtered atoms undergo no collision before they arrive at the substrate surface.Accordingly, the anisotropic ejection of sputtered atoms results in the bellshaped and saddle-shaped film thickness profiles shown in Figures 3 and 4 under a target-substrate distance of 30 mm.When the target-substrate distance is set to 120 mm, it exceeds the mean free path of sputtered atoms possessing energy of 30 eV.Thus, most of the sputtered atoms will experience scattering collisions before they arrive at the substrate surface.The flying angles of sputtered atoms gradually vary due to the increase in the scattering collision frequency with the target-substrate distance, leading to a quasi-isotropic incident angular distribution of sputtered atoms when they approach the substrate surface [15,34].Accordingly, since the anisotropy of the incident angular distribution gradually decreases with the target-substrate distance, the arch-shaped or saddle-shaped film thickness distribution is gradually transformed into the line-shaped one shown in Figures 3 and 4, resulting in an improvement in the film thickness uniformity. Discussion More interestingly, for the 2-inch target sputtering, sputtered Cu atoms were initially emitted from the annular erosion region of the target surface in our simulation.However, under a target-substrate distance of 30 mm, the film thickness distribution had a bell- Discussion More interestingly, for the 2-inch target sputtering, sputtered Cu atoms were initially emitted from the annular erosion region of the target surface in our simulation.However, under a target-substrate distance of 30 mm, the film thickness distribution had a bellshaped profile, in which the maximum value of the film thickness appeared at the substrate center (R = 0 mm).As the target diameter increased from 2 to 4 inches, this bell-shaped film thickness distribution was varied to a saddle-shaped one, leading to an improvement in the film thickness uniformity.To understand the underlying mechanism, the deposition behavior of Cu atoms sputtered from fan-shaped sputtering sources on 2-inch and 4-inch targets was further studied via supplemental simulations, respectively. Figure 6a-c show the deposition density distribution nephograms of a left fan-shaped sputtering source, a right fan-shaped sputtering source, and two symmetrical fan-shaped sputtering sources on the 2-inch target surface under a target-substrate distance of 30 mm.Furthermore, Figure 6d displays the radial deposition density distributions of the three kinds of sputtering sources, which are plotted by the green, blue, and red solid curves, respectively.In Figure 6d, all the deposition density values are normalized by the maximum value in the radial deposition density distribution of two symmetrical fan-shaped sputtering sources.For the sputtering of the left (right) fan-shaped source, the radial deposition density distribution is an arch-shaped curve, whose peak value appears at the projection on the substrate of the point with the maximum sputtering possibility in the fan-shaped source.It can be found that, as the number of fan-shaped sputtering sources increases from 1 to 2, the arch-shaped deposition density distribution is varied to a bell-shaped one, which is analogous to the red solid curve shown in Figure 3.This suggests that the sputtered particle flow from all infinitesimal fan-shaped sputtering sources of the erosion groove overlaps near the substrate center.This strong superposition effect near the substrate center accounts for the bell-shaped deposition density distribution of the 2-inch sputtering target at a short target-substrate distance. Molecules 2023, 28, x FOR PEER REVIEW 7 of 13 shaped profile, in which the maximum value of the film thickness appeared at the substrate center (R = 0 mm).As the target diameter increased from 2 to 4 inches, this bellshaped film thickness distribution was varied to a saddle-shaped one, leading to an improvement in the film thickness uniformity.To understand the underlying mechanism, the deposition behavior of Cu atoms sputtered from fan-shaped sputtering sources on 2inch and 4-inch targets was further studied via supplemental simulations, respectively.Figure 6a-c show the deposition density distribution nephograms of a left fanshaped sputtering source, a right fan-shaped sputtering source, and two symmetrical fanshaped sputtering sources on the 2-inch target surface under a target-substrate distance of 30 mm.Furthermore, Figure 6d displays the radial deposition density distributions of the three kinds of sputtering sources, which are plotted by the green, blue, and red solid curves, respectively.In Figure 6d, all the deposition density values are normalized by the maximum value in the radial deposition density distribution of two symmetrical fanshaped sputtering sources.For the sputtering of the left (right) fan-shaped source, the radial deposition density distribution is an arch-shaped curve, whose peak value appears at the projection on the substrate of the point with the maximum sputtering possibility in the fan-shaped source.It can be found that, as the number of fan-shaped sputtering sources increases from 1 to 2, the arch-shaped deposition density distribution is varied to a bell-shaped one, which is analogous to the red solid curve shown in Figure 3.This suggests that the sputtered particle flow from all infinitesimal fan-shaped sputtering sources of the erosion groove overlaps near the substrate center.This strong superposition effect near the substrate center accounts for the bell-shaped deposition density distribution of the 2-inch sputtering target at a short target-substrate distance.7e shows the radial deposition density distributions of the four kinds of sputtering sources, which are depicted by the green, blue, purple, and red solid curves, respectively.In Figure 7e, all the deposition density values are normalized by the maximum value in the radial deposition density distribution of four symmetrical fan-shaped sputtering sources.It can be seen that, as the number of fan-shaped sputtering sources increases from 1 to 4, the radial deposition density distribution gradually transforms from an arch-shaped curve to a saddle-shaped curve, which resembles the red solid curve shown in Figure 4. Accordingly, it can be concluded that, in the vicinity of the substrate center, the superposition of the arch-shaped deposition density distributions of infinitesimal fan-shaped sources results in the saddled-shape deposition density distribution of the 4-inch sputtering target when the target-substrate distance is set to 30 mm.Furthermore, comparing Figure 7d with Figure 7e, it can be found that, as the target diameter increases from 2 to 4 inches, both peak points of the arch-shaped deposition density distributions of the left and right fan-shaped sputtering sources move towards the substrate margin, which is mainly due to the increase in the diameter of the annular erosion groove.The enlargement of the spacing distance between these peak points weakens the superposition effect of deposition density near the substrate center.Consequently, deposition density values near the substrate center are less than those near the peak points of the deposition density distributions of left and right fan-shaped sputtering sources, leading to a saddle-shaped deposition density distribution of the 4-inch sputtering target at a short target-substrate distance. Molecules 2023, 28, x FOR PEER REVIEW 8 of 13 inch target surface under a target-substrate distance of 30 mm. Figure 7e shows the radial deposition density distributions of the four kinds of sputtering sources, which are depicted by the green, blue, purple, and red solid curves, respectively.In Figure 7e, all the deposition density values are normalized by the maximum value in the radial deposition density distribution of four symmetrical fan-shaped sputtering sources.It can be seen that, as the number of fan-shaped sputtering sources increases from 1 to 4, the radial deposition density distribution gradually transforms from an arch-shaped curve to a saddle-shaped curve, which resembles the red solid curve shown in Figure 4. Accordingly, it can be concluded that, in the vicinity of the substrate center, the superposition of the arch-shaped deposition density distributions of infinitesimal fan-shaped sources results in the saddledshape deposition density distribution of the 4-inch sputtering target when the target-substrate distance is set to 30 mm.Furthermore, comparing Figure 7d with Figure 7e, it can be found that, as the target diameter increases from 2 to 4 inches, both peak points of the arch-shaped deposition density distributions of the left and right fan-shaped sputtering sources move towards the substrate margin, which is mainly due to the increase in the diameter of the annular erosion groove.The enlargement of the spacing distance between these peak points weakens the superposition effect of deposition density near the substrate center.Consequently, deposition density values near the substrate center are less than those near the peak points of the deposition density distributions of left and right fan-shaped sputtering sources, leading to a saddle-shaped deposition density distribution of the 4-inch sputtering target at a short target-substrate distance.Figure 8 presents a schematic diagram to explain the underlying mechanism for the variation in film thickness distribution with the diameter of the erosion groove.Given the central symmetry of the spatial distribution of sputter particle flow, for simplification, Figure 8 only displays the superposition of sputtered particle streams on a cross-section.In Figure 8, the deposition density distributions of left and right infinitesimal sputtering sources are plotted with red and blue dash curves, respectively, while the entire deposited density distributions on the substrate are depicted with green solid curves.The aniso- Figure 8 presents a schematic diagram to explain the underlying mechanism for the variation in film thickness distribution with the diameter of the erosion groove.Given the central symmetry of the spatial distribution of sputter particle flow, for simplification, Figure 8 only displays the superposition of sputtered particle streams on a cross-section.In Figure 8, the deposition density distributions of left and right infinitesimal sputtering sources are plotted with red and blue dash curves, respectively, while the entire deposited density distributions on the substrate are depicted with green solid curves.The anisotropic ejection of sputtered atoms eventuates an arch-shaped deposition density distribution of an infinitesimal sputtering source.The increase in the scattering collision frequency with the target-substrate distance leads to a quasi-isotropic flying angle distribution of sputtered atoms.Consequently, the uniformity of the entire film thickness distribution is gradually improved.In addition, since the possibility of sputtered atoms moving outside the substrate region increases with the collision frequency, the film deposition rate gradually decreases with the target-substrate distance.For the sputtering of the target with a small diameter, in the vicinity of the substrate center, the strong superposition of the sputtering particle flow from infinitesimal sputtering sources results in a bell-shaped entire film thickness distribution at a short target-substrate distance.This bell-shaped film thickness distribution was also reported in ref. [35], in which the diameter of the sputtering target was 50 mm.With the increase in the target-substrate distance, the superposition of more uniform deposition density distributions of infinitesimal sputtering sources gives rise to an increasingly flat entire film thickness distribution on the substrate, which is consistent with the variations shown in Figure 3. Furthermore, as the diameter of the target increases, the entire film thickness distribution might have a saddle-shaped profile at a short targetsubstrate distance due to the reduction in the superposition effect of sputtering particle flow near the substrate center.With the increase in the target-substrate distance, the film thickness distribution becomes comparatively uniform owing to the enchantment in the scattering effect, which coincides with the variations shown in Figure 4.The variation in the film thickness profile with the target-substrate distance shown in Figure 8b was also reported in Refs.[7,36]. Molecules 2023, 28, x FOR PEER REVIEW 9 of 13 tropic ejection of sputtered atoms eventuates an arch-shaped deposition density distribution of an infinitesimal sputtering source.The increase in the scattering collision frequency with the target-substrate distance leads to a quasi-isotropic flying angle distribution of sputtered atoms.Consequently, the uniformity of the entire film thickness distribution is gradually improved.In addition, since the possibility of sputtered atoms moving outside the substrate region increases with the collision frequency, the film deposition rate gradually decreases with the target-substrate distance.For the sputtering of the target with a small diameter, in the vicinity of the substrate center, the strong superposition of the sputtering particle flow from infinitesimal sputtering sources results in a bell-shaped entire film thickness distribution at a short target-substrate distance.This bell-shaped film thickness distribution was also reported in ref. [35], in which the diameter of the sputtering target was 50 mm.With the increase in the target-substrate distance, the superposition of more uniform deposition density distributions of infinitesimal sputtering sources gives rise to an increasingly flat entire film thickness distribution on the substrate, which is consistent with the variations shown in Figure 3. Furthermore, as the diameter of the target increases, the entire film thickness distribution might have a saddle-shaped profile at a short target-substrate distance due to the reduction in the superposition effect of sputtering particle flow near the substrate center.With the increase in the target-substrate distance, the film thickness distribution becomes comparatively uniform owing to the enchantment in the scattering effect, which coincides with the variations shown in Figure 4.The variation in the film thickness profile with the target-substrate distance shown in Figure 8b was also reported in Refs [7,36]. Materials and Methods Experiment of Cu Film Deposition and Film Thickness Measurement In the experiment, two copper targets with a purity of 99.99% were used as sputtering sources, whose diameters were 50.8 and 101.6 mm, respectively.The (001) silicon wafer with a diameter of 50.8 mm was chosen as the substrate.The substrate was placed in acetone and cleaned via ultrasonication for 3 min.Then, it was washed using deionized water.A multifunctional magnetron sputtering system, including 2-inch and 4-inch balanced magnetron sputtering sources produced by Sky Technology Development Co., Ltd.(Hunnan, Shenyang, China), was employed to prepare sputtered Cu films.The target voltage was set to −400 V.The pressure of the background gas was maintained at 0.5 Pa.The deposition time of the sputtered film was set to 10 min.The target-substrate distance was varied from 30 mm to 120 mm. Materials and Methods Experiment of Cu Film Deposition and Film Thickness Measurement In the experiment, two copper targets with a purity of 99.99% were used as sputtering sources, whose diameters were 50.8 and 101.6 mm, respectively.The (001) silicon wafer with a diameter of 50.8 mm was chosen as the substrate.The substrate was placed in acetone and cleaned via ultrasonication for 3 min.Then, it was washed using deionized water.A multifunctional magnetron sputtering system, including 2-inch and 4-inch balanced magnetron sputtering sources produced by Sky Technology Development Co., Ltd.(Hunnan, Shenyang, China), was employed to prepare sputtered Cu films.The target voltage was set to −400 V.The pressure of the background gas was maintained at 0.5 Pa.The deposition time of the sputtered film was set to 10 min.The target-substrate distance was varied from 30 mm to 120 mm. Figure 9 shows the measurement scheme of the film thickness.Prior to the preparation of Cu film, four rotational symmetrically arranged rectangular regions on the substrate surface were masked by polyimide films with a width of 3 mm, such that eight steps in the sputtered Cu film can be formed.To qualify the Cu film thickness, the step height between the masked and unmasked region of the substrate was measured using a Bruker DektakXT surface profilometer (Billerica, MA, USA) [9,37].Thirteen measurement points numbered from 1 to 13 were chosen on each step.Therefore, the eight measurement points marked by the same number i (1 ≤ i ≤ 13) on the eight steps were located on an identical circle with a radius of R i .Then, the mean value of the film thickness at these eight measurement points was calculated and recorded as the film thickness value at R i , such that the radial film thickness distribution could be obtained. Figure 9 shows the measurement scheme of the film thickness.Prior to the preparation of Cu film, four rotational symmetrically arranged rectangular regions on the substrate surface were masked by polyimide films with a width of 3 mm, such that eight steps in the sputtered Cu film can be formed.To qualify the Cu film thickness, the step height between the masked and unmasked region of the substrate was measured using a Bruker DektakXT surface profilometer (Billerica, MA, USA) [9,37].Thirteen measurement points numbered from 1 to 13 were chosen on each step.Therefore, the eight measurement points marked by the same number i (1 ≤ i ≤ 13) on the eight steps were located on an identical circle with a radius of Ri.Then, the mean value of the film thickness at these eight measurement points was calculated and recorded as the film thickness value at Ri, such that the radial film thickness distribution could be obtained. MC Simulation of Magnetron Sputtering Discharge DC magnetron sputtering discharge was simulated using the MC method.In the MC simulation, the target and substrate of the magnetron sputtering system are in a parallel and coaxial configuration.The target voltage was set to −400 V.The pressure of the background gas was maintained at 0.5 Pa.Since the effect of gas heating can be neglected during the DC magnetron sputtering discharge under pressure of less than 1 Pa [38], the temperature of the background gas was set to 300 K. In this MC simulation of the DC magnetron sputtering discharge, the magnetic and electric fields were assumed to be time-independent and location-dependent [39].At the beginning of the simulation, it was supposed that electrons were uniformly ejected from the target surface with an initial energy of zero.Then, they underwent cycloidal spiral oscillation under the action of electrostatic field force and Lorentz force in the non-uniform electromagnetic field.Their motion equation can be expressed by dv dt = q m (E+v×B)(7) where E and B represent the intensity of the electric field and magnetic field, respectively, and v, m, and q denote the velocity, mass, and charge of the electron, respectively.It was postulated that argon gas was in a thermal equilibrium state.Argon ions were generated by the collisions between argon atoms and energetic electrons.In the present simulation, elastic scattering, excitation, and ionization collisions were taken into consideration, whose occurrences were determined by the corresponding momentum transfer cross-sections [40].The energy loss of an electron in the excitation and ionization collisions was set to 11.6 eV and 15.8 eV [30], respectively.The movement of primary and secondary elec- MC Simulation of Magnetron Sputtering Discharge DC magnetron sputtering discharge was simulated using the MC method.In the MC simulation, the target and substrate of the magnetron sputtering system are in a parallel and coaxial configuration.The target voltage was set to −400 V.The pressure of the background gas was maintained at 0.5 Pa.Since the effect of gas heating can be neglected during the DC magnetron sputtering discharge under pressure of less than 1 Pa [38], the temperature of the background gas was set to 300 K. In this MC simulation of the DC magnetron sputtering discharge, the magnetic and electric fields were assumed to be time-independent and location-dependent [39].At the beginning of the simulation, it was supposed that electrons were uniformly ejected from the target surface with an initial energy of zero.Then, they underwent cycloidal spiral oscillation under the action of electrostatic field force and Lorentz force in the non-uniform electromagnetic field.Their motion equation can be expressed by dv dt = q m (E + v × B)(7) where E and B represent the intensity of the electric field and magnetic field, respectively, and v, m, and q denote the velocity, mass, and charge of the electron, respectively.It was postulated that argon gas was in a thermal equilibrium state.Argon ions were generated by the collisions between argon atoms and energetic electrons.In the present simulation, elastic scattering, excitation, and ionization collisions were taken into consideration, whose occurrences were determined by the corresponding momentum transfer cross-sections [40]. The energy loss of an electron in the excitation and ionization collisions was set to 11.6 eV and 15.8 eV [30], respectively.The movement of primary and secondary electrons was traced during the simulation, and the specific simulation procedure was previously described in ref. [41].In particular, the electric potential between the target and substrate was assumed to obey the distribution introduced in ref. [41].A Hall sensor was adopted to measure the horizontal and vertical components of the magnetron field [42].During the movement of the electron, if an ionization collision between the electron and argon atom occurred, the coordinate of the collision point was recorded and further used to calculate the ionization density distribution.The electron would be killed when it moved outside the simulation domain or its energy was lower than the ionization threshold energy of 15.8 eV. MC-MD Simulation Method of Sputtered Particle Transport The initial emission positions of sputtered atoms were determined by the sputtering possibility distribution on the target surface calculated based on the MC simulation results, and the specific procedures were introduced in Section 2.1.The initial emission energy of sputtered atoms was assumed to obey the Thompson distribution [43] and sampled via the rejection algorithm [15,44].Yamamura's angular distribution [33] was used to choose the initial emission polar angles of sputtered atoms.The emission azimuth angles of sputtered atoms, due to symmetry, were supposed to be distributed uniformly between 0 and 2π.After that, the transport processes of sputtered atoms could be modeled using the coupled MC-MD method, whose simulation procedures were introduced in our previous paper [24].Ultimately, the deposition density distributions of 2-inch target sputtering and 4-inch target sputtering can be calculated, respectively. Conclusions In this work, 2-inch and 4-inch circular planar magnetron cooper targets were utilized to prepare Cu thin films at different target-substrate distances, respectively.Simultaneously, the MC method was used to simulate the magnetron sputtering discharge and calculate the ionization density distribution near the target surface.Then, the transport processes of sputtered atoms were modeled by a coupled MC-MD method based on the calculated results of the MC simulation.Experimental results suggested that, with the increase in the target-substrate distance, the film thickness uniformity was gradually improved, while the deposition rate gradually decreased.In particular, as the target-substrate distance increased from 30 to 120 mm, the bell-shaped film thickness distribution of the 2-inch target sputtering was gradually changed to a line-shaped one, while the saddle-shaped film thickness distribution of the 4-inch target sputtering was gradually varied to a lineshaped one.The simulation results agreed well the with experimental results.The MC simulation results suggested that the peak points of the V-shaped radial ionization density distributions of the 2-inch and 4-inch target sputtering were located at radial coordinates of 16 and 30 mm, respectively.Then, the radial sputtering possibility distributions of the 2-inch and 4-inch targets were obtained based on the calculated ionization density distributions.The MC-MD simulation results revealed that, when the target-substrate distance was set to 30 mm, the strong superposition of the sputtering particle flow near the substrate center led to a bell-shaped film thickness distribution of the 2-inch target sputtering, and the reduction in this superposition effect of the sputtering particle flow, resulting from the increased diameter of the erosion groove, resulted in a saddle-shaped film thickness distribution of the 4-inch target sputtering.In addition, the scattering collision frequency increased with the target-substrate distance, eventuating a quasi-isotropic incident angle distribution of deposited atoms and, thus, a comparatively uniform film thickness profile.This work proposed an integrated simulation scheme for the magnetron sputtering discharge and sputtered particle transport, which facilitates studying the dependence of the sputtered film uniformity on sputtering conditions. Figure 1 . 1 Figure 1.Distribution nephograms of ionization density for (a) 2-inch sputtering and (b) 4-inch target sputtering. Figure 1 . 1 Figure 1.Distribution nephograms of ionization density for (a) 2-inch sputtering and (b) 4-inch target sputtering. Figure 2 . 2 Figure 2. The radial distribution of sputtering possibility for (a) 2-inch target and (b) 4-inch target. Figure 2 . 2 Figure 2. The radial distribution of sputtering possibility for (a) 2-inch target and (b) 4-inch target. Figure 3 . 3 Figure 3. Relative film thickness distribution of Cu atoms sputtered from the 2-inch target under different target-substrate distances. Figure 4 . 4 Figure 4. Relative film thickness distribution of Cu atoms sputtered from the 4-inch target under different target-substrate distances. Figure 3 . 3 Figure 3. Relative film thickness distribution of Cu atoms sputtered from the 2-inch target under different target-substrate distances. 13 Figure 3 . 133 Figure 3. Relative film thickness distribution of Cu atoms sputtered from the 2-inch target under different target-substrate distances. Figure 4 . 4 Figure 4. Relative film thickness distribution of Cu atoms sputtered from the 4-inch target under different target-substrate distances. Figure 4 . 4 Figure 4. Relative film thickness distribution of Cu atoms sputtered from the 4-inch target under different target-substrate distances. Figure 5 . 5 Figure 5. Thickness uniformity of sputtered films deposited on 2-inch substrate for 2-inch and 4inch target sputtering under different target-substrate distances. Figure 5 . 5 Figure 5. Thickness uniformity of sputtered films deposited on 2-inch substrate for 2-inch and 4-inch target sputtering under different target-substrate distances. Figure 6 . 6 Figure 6.The deposition density distribution nephograms of (a) a left fan-shaped sputtering source, (b) a right fan-shaped sputtering source, and (c) two symmetrical fan-shaped sputtering sources on the 2-inch target, and (d) the radial deposition density distribution profiles of the three kinds of sputtering sources. Figure Figure 7a-d display the deposition density distribution nephograms of a left fanshaped sputtering source, a right fan-shaped sputtering source, two symmetrical fanshaped sputtering sources, and four symmetrical fan-shaped sputtering sources on the 4- Figure 6 . 6 Figure 6.The deposition density distribution nephograms of (a) a left fan-shaped sputtering source, (b) a right fan-shaped sputtering source, and (c) two symmetrical fan-shaped sputtering sources on the 2-inch target, and (d) the radial deposition density distribution profiles of the three kinds of sputtering sources. Figure Figure 7a-d display the deposition density distribution nephograms of a left fanshaped sputtering source, a right fan-shaped sputtering source, two symmetrical fanshaped sputtering sources, and four symmetrical fan-shaped sputtering sources on the 4-inch target surface under a target-substrate distance of 30 mm. Figure 7e shows the Figure 7 . 7 Figure 7.The deposition density distribution nephograms of (a) a left fan-shaped sputtering source, (b) a right fan-shaped sputtering source, (c) two symmetrical fan-shaped sputtering sources, and (d) four symmetrical fan-shaped sputtering sources on the 4-inch target, and (e) the radial deposition density distribution profiles of the four kinds of sputtering sources. Figure 7 . 7 Figure 7.The deposition density distribution nephograms of (a) a left fan-shaped sputtering source, (b) a right fan-shaped sputtering source, (c) two symmetrical fan-shaped sputtering sources, and (d) four symmetrical fan-shaped sputtering sources on the 4-inch target, and (e) the radial deposition density distribution profiles of the four kinds of sputtering sources. Figure 8 . 8 Figure 8.The influence mechanism of the diameter of the erosion groove on the film thickness distribution and the variation of the film thickness distribution with the target-substrate distance for (a) 2-inch target sputtering and (b) 4-inch target sputtering. Figure 8 . 8 Figure 8.The influence mechanism of the diameter of the erosion groove on the film thickness distribution and the variation of the film thickness distribution with the target-substrate distance for (a) 2-inch target sputtering and (b) 4-inch target sputtering. Figure 9 . 9 Figure 9. Measurement points on the eight steps in the deposited film.These points are marked by red dots and numbered from 1 to 13.The points located on an identical dashed circle are numbered by the same number. Figure 9 . 9 Figure 9. Measurement points on the eight steps in the deposited film.These points are marked by red dots and numbered from 1 to 13.The points located on an identical dashed circle are numbered by the same number. Molecules 2023, 28, x FOR PEER REVIEW Data Availability Statement: Data are contained within the article.Conflicts of Interest:The authors declare no conflict of interest.Funding: This work was mainly supported by the Hunan Provincial Natural Science Foundation of China (2022JJ30114) and, in part, supported by excellent youth funding from the Hunan Provincial Education Department (22B0783). Formation and Properties of Thermistor Chips Based on Semiconductor 3D Metal Oxide Films Obtained by RF-Magnetron Sputtering. V Novozhilov, A Belov, 10.3390/ijms24010742Int. 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Social determinants of health as drivers of fungal disease Jeffrey D Jenks jeffrey.jenks@duke.edu Durham County Department of Public Health DurhamNCUnited States of America Division of Infectious Diseases Department of Medicine Duke University DurhamNCUnited States of America Juergen Prattes Division of Infectious Diseases ECMM Excellence Center for Medical Mycology Department of Internal Medicine Medical University of Graz GrazAustria BioTechMed, GrazAustria Sebastian Wurster Division of Internal Medicine Department of Infectious Diseases Anderson Cancer Center Infection Control and Employee Health MD University of Texas HoustonTXUnited States of America Rosanne Sprute Faculty of Medicine and University Hospital Cologne Institute of Translational Research University of Cologne Cologne Excellence Cluster on Cellular Stress Responses in Aging -Associated Diseases (CECAD) CologneGermany Faculty of Medicine Department I of Internal Medicine Center of Integrated Oncology Aachen Bonn Cologne Duesseldorf (CIO ABCD) and Excellence Center of Medical Mycology (ECMM) University Hospital Cologne University of Cologne CologneGermany German Centre for Infection Research (DZIF) Partner Site Bonn-CologneCologneGermany Danila Seidel Faculty of Medicine and University Hospital Cologne Institute of Translational Research University of Cologne Cologne Excellence Cluster on Cellular Stress Responses in Aging -Associated Diseases (CECAD) CologneGermany Faculty of Medicine Department I of Internal Medicine Center of Integrated Oncology Aachen Bonn Cologne Duesseldorf (CIO ABCD) and Excellence Center of Medical Mycology (ECMM) University Hospital Cologne University of Cologne CologneGermany Matteo Oliverio Faculty of Medicine and University Hospital Cologne Institute of Translational Research University of Cologne Cologne Excellence Cluster on Cellular Stress Responses in Aging -Associated Diseases (CECAD) CologneGermany Department I of Internal Medicine University of Cologne CologneGermany Matthias Egger Division of Infectious Diseases ECMM Excellence Center for Medical Mycology Department of Internal Medicine Medical University of Graz GrazAustria BioTechMed, GrazAustria Carlos Del Rio Emory Center for AIDS Research Emory University School of Medicine AtlantaGAUnited States of America Hatim Sati Department of Global Coordination and Partnership on Antimicrobial Resistance World Health Organization GenevaSwitzerland Oliver A Cornely Faculty of Medicine and University Hospital Cologne Institute of Translational Research University of Cologne Cologne Excellence Cluster on Cellular Stress Responses in Aging -Associated Diseases (CECAD) CologneGermany Faculty of Medicine Department I of Internal Medicine Center of Integrated Oncology Aachen Bonn Cologne Duesseldorf (CIO ABCD) and Excellence Center of Medical Mycology (ECMM) University Hospital Cologne University of Cologne CologneGermany German Centre for Infection Research (DZIF) Partner Site Bonn-CologneCologneGermany Faculty of Medicine Clinical Trials Centre Cologne (ZKS Koln) University Hospital Cologne University of Cologne CologneGermany George R Thompson University of California Davis Center for Valley Fever SacramentoCAUnited States of America Division of Infectious Diseases Department of Internal Medicine University of California Davis Medical Center SacramentoCAUnited States of America Department of Medical Microbiology and Immunology University of California Davis DavisCAUnited States of America Dimitrios P Kontoyiannis Division of Internal Medicine Department of Infectious Diseases Anderson Cancer Center Infection Control and Employee Health MD University of Texas HoustonTXUnited States of America Martin Hoenigl hoeniglmartin@gmail.com Division of Infectious Diseases ECMM Excellence Center for Medical Mycology Department of Internal Medicine Medical University of Graz GrazAustria BioTechMed, GrazAustria Division of Infectious Diseases Department of Medicine Hanes House 315 Trent Drive27710DurhamNCUnited States of America Corresponding author. Division of Infectious Diseases Department of Internal Medicine Medical University of Graz Auenbruggerplatz 158036GrazAustria Social determinants of health as drivers of fungal disease DED9053E2D2A3E85F3ADCF29E717027010.1016/j.eclinm.2023.102325eClinicalMedicine 2023;66: 102325Social determinants of healthFungal infectionsWorking conditionsHealth care accessStructural conflict Disparities in social determinants of health (SDOH) play a significant role in causing health inequities globally.The physical environment, including housing and workplace environment, can increase the prevalence and spread of fungal infections.A number of professions are associated with increased fungal infection risk and are associated with low pay, which may be linked to crowded and sub-optimal living conditions, exposure to fungal organisms, lack of access to quality health care, and risk for fungal infection.Those involved and displaced from areas of armed conflict have an increased risk of invasive fungal infections.Lastly, a number of fungal plant pathogens already threaten food security, which will become more problematic with global climate change.Taken together, disparities in SDOH are associated with increased risk for contracting fungal infections.More emphasis needs to be placed on systematic approaches to better understand the impact and reducing the health inequities associated with these disparities. Introduction Despite efforts to improve access to quality health care, health inequities persist globally.Many factors influence disparities in health outcomes, including race, ethnic background, sex, gender identity, and sexual orientation, among others.For example, compared to white Americans, data shows that Black, Indigenous, and people of color (BIPOC) throughout the U.S. experience higher rates of morbidity and mortality from a diverse spectrum of chronic health conditions, including diabetes mellitus, hypertension, heart disease, cancer, obesity, asthma, mental health disorders, and acute conditions, such as infections. 1,2The ongoing coronavirus disease 2019 (COVID-19) pandemic provides a stark illustration of how underlying health inequities can be amplified by superimposed events like pandemics, leading to further increase in disease burden and health disparities. 3,4DOH refer to non-medical factors that shape individuals' health outcomes and well-being.They encompass a broad range of influences and systems that impact the circumstances of people's everyday lives, including working conditions, income, housing, access to affordable and quality health services, early childhood development, education, job security, food security, social inclusion, and structural conflict. 5In this context, social and economic factors including education, employment, and income have a significant impact on our health behaviors and access to quality health care. 6hese factors are further influenced and shaped by biased economic policies, development agendas, social norms, social policies, and political systems. Globally, significant health inequities exist both within and between countries, contributing to wide gaps in life expectancy.Average life expectancy ranges from 52 years in Sierra Leone and the Central African Republic to 84 years in Japan and Hong Kong-a gap of 32 years. 7Even within a country, there can be striking differences in life expectancy. 8For instance, in Scotland, the average life expectancy for males in the less advantaged parts of Glasgow is 66 years compared to 82 years in more advantaged areas. 9Globally, children from the lowest income households are twice as likely to die by the age of 5 years as children born into the highest income households. 9nvasive fungal infections (IFIs) occur primarily in individuals with underlying immunocompromising conditions such as human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS), such as those with hematologic malignancies, solid organ transplant recipients, or those critically ill in the intensive care unit (ICU), [10][11][12][13][14][15] and are overrepresented in males. 16While genetic and immunologic factors as well as long-term chemotherapy in individuals with cancer may play a role in the increased risk for certain IFIs in some racial and ethnic groups, such as with coccidioidomycosis in individuals with Black African or Filipino descent, 2 it is largely believed that risk factors for IFIs are predominantly influenced by SDOH and differences in exposure to them. 17However, this relationship is still not fully understood. Here, we review the literature examining how SDOH, using examples suggested by the World Health Organization (WHO) 5 (Table 1), impact the risk for fungal infections (Fig. 1). Methods Search strategy and selection criteria Data for this Review were identified by searches of MEDLINE, PubMed, Google Scholar, and references from relevant articles using the search terms "fungal infections", "invasive fungal infections", "superficial fungal infections", "occupation", "income", "housing", "healthcare access", "incarceration", "substance use", "structural conflict", and "food insecurity".We selected relevant case reports, case series, or other studies describing cases or outbreaks of fungal infections that were related to SDOH and published in English.Searches were conducted from database inception until 14 June 2023. Role of the funding source This study was unfunded. Social determinants of health and fungal infections Working conditions Occupational factors and associated working conditions represent one of the key SDOH.Occupational exposures that increase the hazard of inhalation of fungal propagules, such as construction and agricultural work, have been shown to increase the risk for mycoses caused by dimorphic fungi in endemic regions of the U.S. [18][19][20][21][22] Multiple studies have found an association between occupational exposure and outbreaks of coccidioidomycosis infection, including work on military bases 23,24 or during military training exercises, 25,26 employment in prisons, 27 excavation at archeological sites, [28][29][30] construction work, [31][32][33] agricultural farm work, 34 combating wildland fires, 18 and work in cotton mills. 35imilarly, outbreaks of occupational histoplasmosis have been linked to work at an agricultural processing plant in Nebraska, U.S., 20 exposure to Histoplasma conidium in an air conditioning unit at a medical school campus, 36 and work at a landfill and repair of a bridge in Illinois, U.S. 37 In Alberta, Canada, an outbreak of histoplasmosis was associated with renovation of a golf course, during which soil was disturbed. 38A large outbreak associated with aerosol-generating tasks in caves occurred among workers in La Habana province in Cuba 39 and among workers in the Dominican Republic who removed large amounts of bat guano from tunnels without proper respiratory protection. 40Occupational exposure to Blastomyces spp.has been reported as well.One study evaluating forestry workers in Minnesota and Wisconsin, U.S., found a high prevalence of previous subclinical infection. 21Other workers in endemic areas who may be at increased risk for blastomycosis include veterinarians, 41 pathologists, 42 or other laboratory workers. 43In 2023, an outbreak at a Michigan paper mill resulted in more than 100 cases of blastomycosis and at least one death. 44Two cases that occurred in Colorado, U.S., were associated with relocation of infected prairie dogs. 45aracoccidioides brasiliensis can cause paracoccidioidomycosis, which most commonly occurs in countries in South America and mainly affects young and middle-aged men who work outdoors as farmers, miners, and hunters. 46,47One outbreak of paracoccidioidomycosis involving eight individuals in Rio de Janeiro, Brazil was thought to be due to deforestation and earth removal during construction of a highway. 22nfections due to Talaromyces marneffei, which is endemic to Southeast Asia, have also been associated with soil exposure from agricultural work and farming. 48lthough Aspergillus spp.are ubiquitous in the environment and humans inhale at least several hundred Aspergillus conidia daily, [49][50][51] high-inoculum exposures have been associated with allergic pulmonary manifestations 52 and long-term changes to the adaptive immune environment. 53Occupations such as mining that involve excavation, drilling or tunneling pose a significant risk as these activities often expose workers to high concentrations of fungal propagules in the dust generated during these operations. Examples of social determinants of health Occupational exposure to aflatoxin in poultry workers in Portugal has been associated with Aspergillus section Flavi contaminating indoor air in workspaces. 54oreover, there have been rare cases of invasive lung infections after occupational exposures to Aspergillus, even in immunocompetent persons, such as from agricultural work 55 and gardening. 56Cryptococcosis has been associated with outdoor occupations, such as construction and landscaping, 57 and mucormycosis with dust exposure in a warehouse, 58 construction 59 and after farm work accidents. 60Entomophthoramycosis, which is caused by infections from fungi belonging to the order Entomophthorales, can occur following direct inoculation such as during farm work. 61ndividuals in certain occupations may be prone to soft tissue fungal infections acquired through direct, traumatic inoculation.Sporotrichosis, caused by Sporothrix spp.has been associated with work in mines, 62 forestry and tree nursery work, [63][64][65] armadillo hunting in Uruguay and Brazil, 66,67 and veterinary work. 68Feline sporotrichosis, caused by Sporothrix brasiliensis, is endemic in Brazil and neighboring countries and is a risk for veterinarians, animal caretakers, and even the general public.Infection can occur following a scratch or bite from an infected cat, although even touching an infected cat can result in infection. 69Infections from fungi causing mycetoma and chromoblastomycosis have been linked to farm work, agricultural work, or other outdoor manual labor. 47,70In endemic regions such as Sudan, fungi causing mycetoma (eumycetoma) is often found to disproportionately affect manual workers, including farmers, laborers, and herdsmen. 71dditionally, students in limited resource areas who frequently walk to schools without appropriate footwear or barefoot are also at higher risk. 72][75] Direct inoculation through gardening has been implicated as the cause of subcutaneous mucormycosis 76 and mycetoma caused by Acremonium spp. 77ythiosis, an infection caused by Pythium insidiosum, has been associated with agricultural work. 78A number of fungi causing traumatic keratitis from funguscontaminated plant material, including Fusarium spp., Aspergillus spp., Chrysosporium spp., and Curvularia spp., has also been related to agricultural work. 79,80n the U.S., work that poses the greatest risk for occupational exposure to fungi employ a high percentage of BIPOC individuals, such as landscapers, groundskeepers, nursery workers, farm workers, agricultural workers, and construction workers. 81Notably, occupations associated with increased risk for fungal exposures and infection predominantly represent the low-wage sector 82,83 as these workers are often unable to advocate for better working conditions or adhere to public health recommendations to prevent fungal diseases due to lack of support from supervisors.Workers who are employed in such conditions are often exposed to high rates of occupational hazards, inadequate ventilation, limited access to proper sanitation and hygiene facilities, and a lack of proper workwear and personal protective equipment (PPE). W O R K IN G C O N D IT IO N S INCOME HOUSING, BASIC AMENITIES & THE ENVIRONMENT S U B S T A N C E U S E & C R IM E FO OD INS EC UR ITY ST R U C TU R A L C O N FL IC T A C C E S S T O A F F O R D A B L E H IG H -Q U A L IT Y H E A L T H S E R V IC E S Thus, there is a clear association between a range of fungal infections, occupations, working conditions, and the workers' socioeconomic background. Income Income, another key SDOH, is closely related to working conditions, as discussed above, as well as housing, neighborhood conditions, and access to health care.In the U.S., life expectancy has been shown to increase with income. 84n 2019, of 35.5 million hospitalizations in the U.S., those living in areas with the lowest quartile median income had disproportionately high hospitalization rates.IFIs overall were 1.2 times more likely to be diagnosed in individuals living in a Q1 postal code (median household income < US$48,000) compared to those in a Q4 postal code (median household income ≥ US$82,000), driven by cryptococcosis (twice as likely), and histoplasmosis (1.7 times more likely).Only aspergillosis was diagnosed more frequently in Q4 postal codes compared to Q1 postal codes, possibly related to higher rates of autoimmune diseases, hematologic disorders, and access to advanced cancer treatment and procedures such as organ transplants among hospitalized patients living in higher income areas.IFIs were also more common in hospitalizations where Medicaid was billed compared to hospitalizations where private insurance was billed. 85lthough poorly studied on a global level, individuals belonging to lower socioeconomic groups have also been shown to be at greater risk for superficial fungal infections.For example, in a case series of consecutive patients diagnosed with tinea capitis in India, 90% of cases occurred in children younger than 15 years and 75% of cases among individuals who belonged to lower income socioeconomic groups. 86In Nigeria, among schoolchildren diagnosed with superficial fungal infections, infection was more likely to occur in children from families of lower socioeconomic status, with 66% of cases reported, whereas those from higher socioeconomic status were overrepresented (57%) in the control group. 87In the U.S., lower socioeconomic status has been shown to be associated with increased risk of onychomycosis. 88 Housing, basic amenities, and the environment The residential environment, encompassing both housing conditions and community characteristics, exerts a substantial influence on individuals well-being and health outcomes.In most settings the physical built environment has a significant impact on public health and health outcomes, as it influences factors such as physical activity levels, access to healthy food, good quality indoor air, and water, social interactions, medical services, and overall safety and well-being. Currently, three billion people-40% of the global population-lack facilities at home to wash their hands with soap and water, and nearly half of schools and 43% of healthcare settings lack these amenities. 9In addition, over 90% of people globally breathe outdoor air with pollution levels exceeding WHO air quality guideline values, which contributes to over 7 million deaths annually. 9Even within industrialized Western countries, the residential environment is a major determinant of health outcomes.][91][92] Higher social vulnerability has also been shown to be associated with unsafe housing environments. 93here is a relationship between population density and risk for developing an IFI.Rates of histoplasmosis tend to be higher in individuals who live in rural lower income areas, as previously discussed.There is likely an interplay between living in rural areas, lower income, and environmental as well as occupational exposures to fungi. 85,94Conversely, some IFIs are diagnosed more frequently in urban areas, including aspergillosis, coccidioidomycosis, and pneumocystosis. 85,95Aspergillosis and pneumocystosis may be diagnosed more frequently in urban areas due to the risk factors for this infection, such as hematologic malignancy, immunosuppressive conditions, transplantation, and closer proximity to tertiary medical centers treating patients with these conditions.Outbreaks of IFIs, particularly aspergillosis, have been associated with construction work in the vicinity of hospitals, which can aerosolize conidia, 96 further contributing to the risk for IFIs from aerosolized conidia in urban areas. Globally, higher rates of dermatophytoses have been observed in areas with higher population density or overcrowded areas such as in homes and schools.In Iran, higher rates of tinea capitis were associated with larger families and larger class size in schools. 97Similarly, among school-age children, sharing a bed with more than three people was associated with higher rates of superficial fungal infections of the skin in southern Tanzania. 98Poor living conditions were associated with higher rates of superficial fungal infections in South Western Nigeria, 87 and in another study in Nigeria, diagnosis of tinea capitis occurred more frequently in areas of frequent interaction with soil and animals. 99n India, which represents the country with the highest reported rate of mucormycosis, studies have shown that some individuals acquired COVID-19associated mucormycosis (CAM) at home in their bedrooms, where higher Mucorales spore counts were measured in the rooms of individuals convalescing from COVID-19 infection compared to homes of individuals not affected by CAM. 100,101n addition to invasive infection, outdoor and indoor air pollution in residential environments, including high concentrations of fungal pathogens, has a significant adverse effect on human health by enhancing the production of allergens. 102Stachybotrys chartarum growth can occur on water-damaged areas of buildings and has been associated with acute idiopathic pulmonary hemorrhage in infants, although this association is controversial. 103Some fungal propagules contain proinflammatory and pro-allergenic compounds which can cause allergic response.For example, Aspergillus fumigatus contains eighty allergic proteins capable of inducing IgE responses, Alternaria spp.contains twelve, and Cladosporium herbarum eight allergens. 104It is estimated that 6% of the general population and 20-30% of allergy-predisposed individuals are allergic to environmental fungi. 105In addition, an estimated eight to ten percent of individuals with cystic fibrosis experience allergic bronchopulmonary aspergillosis. 104Lastly, Exophiala dermatitidis colonizes the respiratory tract of up to nineteen percent of individuals with cystic fibrosis 106,107 and can cause infections as well. 108esides the residential environment, recreational activities can contribute to fungal exposure and infection.For instance, outbreaks of blastomycosis infections have been associated with environmental exposure during recreational activities.One study in Wisconsin, U.S. documented a high incidence of blastomycosis infection associated with visitation to rivers or associated waterways, suspected to reflect recreational exposure. 109Another cluster was documented among two school groups who visited an environmental camp in northern Wisconsin, with soil at a beaver pond near the camp thought to be the cause. 110Lastly, overcrowded housing may have contributed to a large outbreak of blastomycosis in Wisconsin clustered among households which primarily affected people with Hmong ethnicity, for whom larger average household sizes have been documented. 111In addition, an outbreak of Cryptococcus gattii occurred on Vancouver Island in British Columbia, Canada.C. gattii was cultured from a number of environmental samples, with the highest concentration occurring at a park where almost half of those involved in the outbreak had visited within the prior 12 months. 112 Access to affordable and high-quality health services A lack of timely access to affordable health services is associated with worse health outcomes.Living far from a health care center, fear of deportation if living in a county without documentation, high medical costs, or lack of understanding of how to access health care are among the more common reasons why individuals may lack access to high-quality health care.In the U.S., adults without health insurance are less likely to receive preventive services for chronic medical conditions that predispose to IFI such as poorly controlled diabetes mellitus, especially in the setting of widespread glucocorticoid use, 113 cardiovascular disease, and cancer care. 114Conversely, health insurance is associated with improved access to health services and basic clinical services, 115,116 increased rates of detection of diabetes mellitus, lower rates of depression, and reduced financial strain. 116Globally, approximately half of the population lacks access to health care, with an additional 100 million driven into extreme poverty by the cost of health care. 117Additionally, many communities globally affected by communicable diseases such as neglected tropical diseases (NTDs) are of low socioeconomic status and lack access to health services. 118tudies have shown that expanding health coverage decreases health disparities, 119,120 and it is likely that health disparities are lower in settings with universal access to health care compared to settings that lack health care access for all. Higher incidence of histoplasmosis has been associated with underlying medical conditions such as poorly controlled diabetes mellitus, autoimmune conditions such as rheumatoid arthritis, inflammatory bowel disease, psoriasis, and solid organ or hematopoietic stem cell transplant. 94Poorly controlled diabetes mellitus is a well-known risk factor for a number of IFIs, including mucormycosis. 85,121Severe COVID-19 infection, especially in the setting of widespread glucocorticoid use, 113 has emerged as another major risk factor for mucormycosis 121 and invasive aspergillosis, 12 including a large mucormycosis outbreak during the COVID-19 delta wave in India. 121,122In one study, access to advanced COVID-19 treatment-including supplemental oxygen, remdesivir therapy, and ICU admission-was associated with lower CAM risk in resource-limited settings. 123There is also a clear association between prior pulmonary tuberculosis with cavitation and subsequent chronic pulmonary aspergillosis, which is a significant health problem in tuberculosis-endemic settings. Recently, an outbreak of fungal meningitis among patients who received procedures under epidural anesthesia at two clinics has been reported in Matamoros, Mexico.Fusarium solani species complex has been identified as the causative pathogen, although the exact source is still unclear. 125Many of these affected individuals were from the U.S. and involved in medical tourism-seeking lower-cost procedures with shorter waiting times.Increased risk for candidemia has been associated with poor health care infrastructure and access in lower income countries. 126Overcrowded intensive care units, insufficient air conditioning/ventilation systems, and lack of efficient infection control services predispose individuals from lower income countries to a greater risk for hospital and ICU outbreaks of IFIs, as recently shown for COVID-19-associated pulmonary aspergillosis, CAM, and CNS fusariosis outbreaks in Mexico, 123,127,128 as well as invasive Candida infections caused by fluconazole resistant C. parapsilosis 129 and C. auris. 130Cryptococcosis is associated with advanced HIV infection as well as other medical comorbidities such as diabetes mellitus, corticosteroid use, and malignancy. 131,132Improved primary prevention, affordable access to early diagnosis, and treatment of chronic medical conditions would likely reduce the risk for IFIs. Substance use, crime, and incarceration Substance use has been associated with a number of IFIs.Injection drug use (IDU) is a common cause of bacterial and, less frequently, fungal endocarditis.The latter is mainly caused by Candida spp., followed by Aspergillus spp.5][136] Cigarette smoking has been associated with an increased risk for coccidioidomycosis, 137,138 paracoccidioidomycosis, 139 and cryptococcosis 57 .1][142] In one study, persons who use cannabis were 3.5 times more likely to develop an IFI compared to non-cannabis users. 141Electronic cigarette use or vaping has been associated with invasive pulmonary aspergillosis infection 143 and Candida albicans growth in the gingiva of the oral cavity. 144ultiple studies have shown increased risk for coccidioidomycosis infection for incarcerated individuals in areas endemic for Coccidioides spp., such as in the Central Valley in California [145][146][147] as well as state prisons in other regions in California. 148[152] Structural conflict Structural conflict such as armed conflict and/or the occupation by foreign armed forces can result in the displacement of populations, loss of income and shelter, the destruction of social networks and physical infrastructure, and violence and human rights abuses, exposing underlying inequities in SDOH.For instance, in Afghanistan, thousands of individuals were injured by unexploded ordinance associated with the ongoing war.One study showed that the vast majority were men and boys, with most injuries occurring in children playing and tending to animals, and adults engaged in economically necessary activities such as farming and traveling. 153n a study of combat casualties in Afghanistan during a 5-year period, it was estimated that IFIs accounted for up to 13% of associated morbidity and mortality.Risk factors included sustaining a dismounted blast injury, experiencing a traumatic transfemoral amputation, and requiring large-volume blood transfusions. 154igher risk for IFIs has also been shown to be related to lower elevations, warmer temperatures, and greater isothermality, such as in southern Afghanistan where molds grew from 61% of wound cultures. 155Mucorales are the most frequent cause of fungal infections complicating combat-related injuries, followed by Aspergillus spp.and Fusarium spp. 156,157In addition, species not commonly seen in sinopulmonary mucormycosis are more common in theaters of war, such as Apophysomyces spp., Saksenaea spp., and Lichtheimia spp. 158Treatment of combat related IFIs can be particularly challenging, often requiring extensive debridement 159 due to imperfect penetration of antifungals into wounds and often limited medical infrastructure in conflict zones. 160Lastly, the ongoing conflict in Sudan has led to the suspension of activities at the Mycetoma Research Center in Khartoum, the only institution in the world that specializes in the treatment of this disease.Thousands of patients now lack access to treatment. 161eople displaced from structural conflict are also at an increased risk for fungal infections.For instance, superficial dermatophytoses were common among displaced Rohingya in a refugee camp in Bangladesh, 162 refugees from camps in Sudan, 163 Syrian refugees in a refugee camp Jordan, 164 and refugees settling in the U.S. 165 Food insecurity Food insecurity is defined as a household-level economic or social condition of limited or uncertain access to adequate food. 166Globally, between 702 and 828 million people-approximately 10% of the global population-were affected by hunger in 2021.This number grew by 150 million since the start of the COVID-19 pandemic, with global conflict, climate extremes, economic instability, and growing inequality also contributing. 167In the U.S., 13.5 million households were food insecure at some point in 2021. Food insecurity and malnutrition can be a predisposing factor for IFIs.As an example, Pneumocystis jirovecii infection has been associated with low body weight and protein deficiency in children. 168n addition, fungal plant pathogens pose a significant threat to food security and contribute to the US$220 billion estimated annual crop losses caused by pathogens and other pests. 169For instance, the plant fungus Claviceps purpurea can contaminate rye, wheat, and other cereals and cause ergot, which can manifest in a diverse array of symptoms in humans, including tremors, delusions, seizures, muscle spasms, and hallucinations.The plant pathogen Phytophthora infestans causes an estimated US$6 billion in potato losses and management costs annually. 170Pyricularia oryzae is a major rice pathogen, causing 10-35% of loss to harvests, and Puccinia graminis can cause up to 70% of wheat crop losses.Numerous Fusarium spp.are pathogenic to a diverse array of fruits and vegetables, 171 causing up to 50% yield losses in fruit and vegetable crops such as bananas, pineapples, lentils, tomatoes, and peas. 172osema spp.have been implicated in colony collapse disorder and declining honeybee populations, which has had a significant impact on crops pollinated by the honeybee. 173,174Overall, fungi destroy a third of all food crops annually, resulting in significant economic loss and contributing to global poverty. 175A significant fungal outbreak involving one or more crops could have devastating consequences for the global food supply, which would most impact those from lower income settings.Lastly, although fungicides may help prevent crop loss, they can also lead to the emergence of antifungal resistance, as has been shown against all classes of antifungal drugs. 176,177terventions to reduce the risk for fungal infections Reducing the risk for fungal infections involves addressing underlying disparities in SDOH that increase the risk for acquiring these infections.Some of these interventions may be applied generally, while specific interventions may be needed to target individual risk factors.Social inequities, such as poverty, limited access to food and health care, inadequate housing, and educational disparities can contribute to an increased risk for developing fungal infections, and addressing these iniquities is multi-faceted (Fig. 2). Susceptible individuals may develop IFIs after traumatic inoculation of fungal propagules in wounds.Thus, at-risk individuals should avoid tissue trauma when working in surroundings likely to have high concentrations of fungal propagules, such as in soil.PPE such as protective clothing and gloves can help to protect the skin from contact with mycoses that can potentially cause infection. 178n addition, addressing housing issues such as leaks, mold growth, or poor ventilation can also decrease the risk for acquiring a fungal infection, and this is particularly important for immunocompromised individuals.Mitigating actions to reduce the risk for developing fungal infections during construction work may help reduce outbreaks in hospitals.Such actions may include educating construction workers and hospital staff on preventative strategies to reduce dust production during construction activities, implementing physical barriers to minimize dust spread, and implementing filter systems to reduce fungal burden in the air.High-efficiency particular air (HEPA) filters, for example, have been proven to efficiently filter particles with a diameter of more than 0.3 μm 179 and can prevent airborne IFIs in critical areas, such as stem cell transplantation units. 180he implementation and enforcement of well-funded laws and regulations are crucial to address the discussed issues.Governments, along with regulatory bodies, have a responsibility to ensure their implementation and that strong labor laws and protections exist to protect workers from fungal infections.Stakeholders, including professional associations, tenants, landlords and private sector industries such as housing and agriculture, should prioritize these concerns by conducting regular checkups and air quality testing to safeguard the well-being of residents and workers.Employers must uphold the highest occupational safety and health standards, minimizing hazards through the provision of necessary mitigation strategies.These actions are vital in creating healthier and safer living and working environments for individuals and communities. Access to affordable and quality health care is critical for individuals with underlying conditions associated with increased susceptibility to IFIs.In addition, health care institutions should have access to appropriate fungal diagnostics and antifungal treatment.Unfortunately, these resources are not equitably distributed globally. 181For example, 90% of health care institutions in Europe but only 70% of health care institutions in the Asia/Pacific region reported access to echinocandins to treat IFIs such as candidiasis and aspergillosis. 182,183Access to antifungals for the treatment of implantation mycoses, such as eumycetoma, actinomycetoma, cutaneous sporotrichosis, and chromoblastomycosis, continues to be problematic in lower and middle-income countries globally. 184Inequities in antifungal drug agents and diagnostics can lead to suboptimal management of IFIs in resource limited settings. Awareness and education on the signs and symptoms of fungal infections is important for both the general population as well as health care workers, to ensure timely and effective management of these infections.Several guidelines, such as those published on the management of cryptococcosis, 185,186 mucormycosis, 187 endemic mycoses, 188 and rare mold infections 189 offer guidance on the management of these infections, including in resource-limited settings.In addition, in the oncology setting recommendations on how to avoid excessive fungal exposure have been published. 190Still, there remains a lack of guidelines for neglected fungal tropical diseases such as mycetoma and chromoblastomycosis, and the guidelines that currently exist need to be widely available and freely accessible to healthcare workers around the globe.More research should be done on IFIs associated with occupational health hazards and combat injuries, including better tracking of these fungal infections so those who manage these injuries are better able to diagnose and offer appropriate antifungal treatment.Lastly, although a number of surveillance networks already exist to monitor existing communicable and noncommunicable diseases, including for emerging pathogens, [191][192][193] more widespread global surveillance of fungi is warranted to mitigate the impact of these pathogens on the health of those most at risk. Discussion SDOH play a significant role in global health inequity and are associated with increased risk for developing superficial and IFIs, as discussed in this review (Fig. 1).This is particularly true for working conditions, where the risk for developing an infection from fungi is highly associated with certain types of work, including construction, farming, landscaping, and other agricultural work.Lower income is related with increased risk for certain IFIs globally and, along with more crowded housing, is associated with risk for superficial fungal infections globally.Lack of access to quality health care, and the underlying medical conditions associated with it, is associated with increased risk for IFIs.Structural and armed conflicts are associated with risk for IFIs following traumatic injury, and displacement of people fleeing conflict is associated with superficial and likely subcutaneous fungal infections.7][198][199][200][201] These global changes are expected to predominantly affect those who are already at an increased risk for fungal infections due to inequities in SDOH.Of note, the current published literature likely significantly underrepresents the association between SDOH and IFIs outside of the U.S., where the risks likely mirror those found within the U.S. To decrease the morbidity and mortality associated with fungal infections, more research needs to be dedicated to implementing interventions that can help decrease the acquisition of fungal infections in those most at risk.The gaps in evidence need to be addressed and a more systematic approach to understanding and addressing the impact of SDOH on IFIs globally-with a specific focus on low-resource settings-needs to be implemented.Well-funded regulations that hold employers, landlords, and companies accountable could help address SDOH, if they are enforced.In addition, more emphasis needs to be placed on decreasing the health inequities between low-income and high-income countries and within higher-income countries such as the U.S. Outstanding questions An important question is how to best provide surveillance of fungal infections globally, particularly in resource-limited settings where the prevalence of fungal infections is likely grossly underestimated.Another challenge is how to capture data on SDOH at a global level so the association between SDOH and fungal infections can be better understood.From a programmatic level, what role do agencies such as the WHO play in monitoring SDOH and fungal diseases and the association between the two? amenities, and the environment Substance use, crime, and incarceration Early childhood development Social inclusion and non-discrimination Structural conflict Access to affordable health services of decent quality Fig. 1 : 1 Fig. 1: Social determinants of health and fungal infections. Fig. 2 : 2 Fig. 2: Strategies to decrease risk of fungal infections by SDOH. Table 1 : 1 Examples of social determinants of health as defined by the World Health Organization.5 www.thelancet.com Vol 66 December, 2023 www.thelancet.com Vol 66 December, 2023 ContributorsJDJ and MH conceived and designed the study.JDJ, JP, and MH wrote the initial draft.RS, MO, and ME produced figures.SW, RS, DS, ME, CDR, HS, OAC, GRT, and DPK provided critical comments.All authors read and approved the final manuscript.Declaration of interestsof Interest and Sources of Funding: JDJ received research funding from Astellas, F2G, and Pfizer-all outside of the submitted work.JP has received speakers' fees from Gilead Sciences, Pfizer, Swedish Orphan Biovitrum, Associated of Cape Cod, served at advisor boards for Gilead Sciences and Pfizer and holds stocks of Novo Nordisk and AbbVie Inc-all outside of the submitted work.RS received speaker fees and travel support from Pfizer-all outside of the submitted work.DS received speaker fees from Pfizer-all outside of the submitted work.OAC reports grants or contracts from BMBF, Cidara, EU-DG RTD (101 037 867), F2G, Gilead, MedPace, MSD, Mundipharma, Octapharma, Pfizer, Scynexis; Consulting fees from Abbvie, AiCuris, Biocon, Cidara, Gilead, IQVIA, Janssen, Matinas, MedPace, Menarini, Moderna, Molecular Partners, MSG-ERC, Noxxon, Octapharma, Pfizer, PSI, Scynexis, Seres; Honoraria for lectures from Abbott, Abbvie, Al-Jazeera Pharmaceuticals/Hikma, Gilead, Grupo Biotoscana/United Medical/Knight, ISHAM Working Group, MedScape, MedUpdate, Merck/MSD,Noscendo, Pfizer, Shionogi, streamedup!; Payment for expert testimony from Cidara; Participation on a Data Safety Monitoring Board or Advisory Board from Boston Strategic Partners, Cidara, IQVIA, Janssen, MedPace, PSI, Pulmocide, Shionogi, The Prime Meridian Group; A patent at the German Patent and Trade Mark Office (DE 10 2021 113 007.7);Stocks from CoRe Consulting, EasyRadiology; Other interests from Wiley.GRT received research and consulting fees from Astellas, Cidara, F2G, Mayne, Melinta, Mundipharma, and served on the DRC for Pfizer-all outside of the submitted work.DPK received honoraria and research support from Gilead Sciences, Merck, United Medical, and Astellas Pharma.He received consultant fees from Astellas Pharma, Amplyx Pharmaceuticals, Ciadara Therapeutics, Mayne Pharma, and is a member of the Data Review Committee of Cidara Therapeutics, AbbVie, Scynexis, and the Mycoses Study Group-all outside of the submitted work.MH received research funding from Gilead, Astellas, Euroimmune, MSD, IMMY, Mundipharma, Scynexis, and Pfizer-all outside of the submitted work.All authors declare no conflict of interest. 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Rathke's cleft cysts causing Cushing's disease: Two unique cases and review of the literature 17 November 2023 Krisna Maddy 0000-0002-2007-7668 Evan Luther evanluthermd@gmail.com Katherine Berry Victor M Lu Ashish Shah ashah@med.miami.edu Michael E Ivan mivan@med.miami.edu Ricardo J Komotar rkomotar@med.miami.edu MDNancy E Epstein Departments of 1 Neurosurgery and 2 Neurological Surgery, University of Miami Miller School of Medicine, Miami, Florida, United States. Miami, Florida, United States. State U. of NY at Stony Brook. Ishikawa, Japan Department of Neurosurgery, University of Miami Miller School of Medicine, Professor of Clinical Neurosurgery, School of Medicine, Kanazawa University, Rathke's cleft cysts causing Cushing's disease: Two unique cases and review of the literature 17 November 2023846E7A727302B62A9D2879BA2E619D1010.25259/SNI_616_2023Received: 23 July 2023 Accepted: 19 October 2023Cushing's diseaseNeurosurgeryPituitary adenomaRathke's cleft cyst Background: e presentation of isolated Rathke's cleft cysts (RCC) without any associated pituitary adenoma in patients with symptoms consistent with Cushing's disease (CD) remains exceedingly rare.As such, we aim to present two cases of RCC presenting with CD with a resultant resolution of their CD following surgical resection.Case Description: Here, we present two cases of RCCs presenting with symptoms suggestive of CD.A functional pituitary microadenoma was the presumed diagnosis based on initial clinical presentation and diagnostic imaging suggesting a pituitary lesion.However, pathology results demonstrated no evidence of adenoma but cysts lined with columnar epithelia consistent with RCC.Complete surgical resection was achieved in both patients through endoscopic endonasal pituitary resection with postoperative symptomatic resolution and normalization of cortisol levels.In addition, we discuss the literature on this rare presentation and suggest a pathological mechanism for this unique presentation of RCC-causing CD.Conclusion:Surgical resection of RCC may provide a "biochemical cure" for patients presenting with CD, as demonstrated by these two unique cases.e clinical features, histological findings, and possible pathological mechanisms for this unique presentation of RCC causing CD discussed lay the groundwork for future studies into the pathophysiology of RCC and CD. INTRODUCTION Rathke's cleft cysts (RCC) arise from the epithelial remnants of Rathke's pouch, which persists in adult life between the pars anterior and pars posterior of the pituitary. [5]ese cysts are benign sellar and suprasellar lesions with a peak incidence at 30-50 years of age. [8]Most RCCs are between 10 mm and 20 mm in diameter and contain mucoid or gelatinous material encapsulated in a thin cyst wall of simple or pseudostratified cuboidal or columnar epithelium. [8]RCCs are usually found incidentally in adults, with an incidence of about 12-33% in autopsy cases. [6]Functionally, these cysts are mostly asymptomatic.Progressive enlargement can result in compression of the optic chiasm, pituitary stalk, and gland, resulting in symptomatic visual disturbances, and endocrinopathies. [6]Cushing's disease (CD) is an endocrinopathy defined by autonomous/ dysregulated secretion of adrenocorticotropic hormone (ACTH) and excess cortisol production. [7]t is primarily caused by pituitary ACTH-secreting tumors, namely pituitary adenomas.e presence of RCC concurrently with pituitary adenoma is rare, however reported in the literature. [12,21]One study reported that RCC was associated with 1.7% pituitary adenoma cases. [13]However, minimal data exist discussing isolated RCCs without any associated pituitary adenoma causing CD. [17] Here, we present two cases of RCCs presenting symptoms suggestive of CD.We discuss the clinical features and histological findings and suggest a pathological mechanism for this unique presentation of RCC-causing CD. CASE PRESENTATION #1 A 72-year-old male with a history of growth hormone deficiency symptomatic for generalized fatigue and weakness and on somatropin replacement therapy (1 mg daily) had an incidentally found pituitary lesion identified two decades prior on magnetic resonance imaging (MRI) following hospitalization for stroke.His comorbidities included obesity (body mass index, BMI 30) and diabetes.At that time, the lesion was assumed to be a microadenoma measuring 1.0 cm and abutting the optic chiasm.At the time, he had no visual deficits and was managed conservatively.In 2021, he presented to his outside endocrinologist with an elevated cortisol level of 2.3 mcg/dL from a normal value one year prior and the absence of overt changes in clinical features, suggesting subclinical CD.In addition, the low-dose (1 mg) dexamethasone suppression study did not suppress cortisol levels, indicating CD versus Cushing's syndrome.Repeat imaging demonstrated the growth of the lesion, now measuring 1.3 cm, with no compression of the optic apparatus [Figure 1]. As a result of these findings, surgery was recommended.He underwent endoscopic endonasal transsphenoidal resection of the lesion without issue, and postoperative MRI demonstrated no residual mass [Figure 2].Postoperative AM serum cortisol was 12.4 mcg/dL one day after surgery and 1.7 mcg/dL two days after surgery.At one year followup, the patient's insulin-like growth factor 1 level was decreased to 181 ng/mL from 281 ng/mL prior.Complete postoperative endocrinological laboratory results for patient 1 are unavailable due to reliance on endocrinological workup from an external hospital, despite efforts to access the records.Pathology demonstrated multiple fragments of the pituitary gland with normal nesting growth patterns.In addition, pituitary transcription factors, including growth hormone factor 1 (Pit-1), T-box transcription factor Tpit, and steroidogenic factor-1 (SF-1) showed normal distribution patterns in the parenchyma.Adjacent to these fragments was a colloid nodule and epithelium suggestive of RCC.e patient's postoperative course was uneventful with his RCC resection.Approximately one week after surgery, for symptoms of nausea, fatigue, and muscle weakness, his workup was benign aside from hyponatremia, which was corrected during his hospitalization.One month postoperatively, the patient exhibited excellent recovery and no complications from the operation.On a longer-term follow-up with endocrinology, the patient reported improved fatigue and generalized muscle weakness. CASE PRESENTATION #2 A 61-year-old female initially presented in 2015 to her physician with a several-year history of symptoms of hirsutism, proximal muscle weakness, thinning skin, and easy bruising.Her comorbidities included obesity (BMI 37.6), diabetes, and hypertension.In June 2018, the patient's serum ACTH was elevated at 77 pg/mL, and in February 2019, the patient's urine-free cortisol was elevated at 134 mcg/dL.e patient reported a trial of mifepristone for several months but could not tolerate the medication.MRI in 2019 showed a 5 mm hypoenhancing lesion in the pituitary gland, presumed to be a pituitary microadenoma measuring approximately 3.5 mm [Figure 3].e pathology report of the specimen suggested RCC with Crooke's hyaline changes.Noted on histology were several islands of eosinophilic acellular material, strips of ciliated epithelium with adjacent intact native pituitary parenchyma with all hormonal markers in a nonneoplastic distribution with no evidence of adenoma.e patient's postoperative course was uneventful.One month postoperatively, the patient exhibited excellent recovery and no complications from the operation.On longer-term follow-up with endocrinology, the patient reported improvement in hirsutism and proximal muscle weakness. DISCUSSION To our knowledge, these are the first reported cases of RCC causing CD without concurrent pituitary adenoma or pituitary hyperplasia, as confirmed on histology.RCCs are considered embryological remnants of Rathke's pouch, presenting typically as asymptomatic benign lesions. [16]ey rarely present in association with pituitary adenomas in patients presenting with CD.In the literature, to date, there are several reported cases of pituitary adenoma and hyperplasia presenting in association with RCC. [2,17,21]ese typically present as nonfunctional pituitary adenomas and prolactinomas coexisting with RCC in patients presenting with endocrinological imbalances and compressive effects, which resolved after transsphenoidal resection. [21]Even more rare is the presentation of RCC causing similar endocrinological symptomology without evidence of concurrent pituitary adenoma on histology.We present two cases where histology demonstrated completely benign adjacent pituitary tissue to the RCC in these patients with symptomatic CD. Typically, RCCs present on MRI as well-demarcated homogeneous lesions with a variable intensity highly dependent on cyst contents, ranging from clear cerebrospinal fluid-like fluid to thick mucoid material. [6]ey are typically hypointense on T1-weighted images and hyperintense on T2-weighted images (serous) or hyperintense on T1-weighted images and isointense on T2-weighted images (mucoid). [4]A small area of hypo-or iso intensity may indicate the thickening of the cyst wall or aggregation of cellular debris.In our patients, the lesions were presumed to be pituitary microadenomas.Given the clinical correlation with the presenting symptoms, this is not unusual. RCCs are typically thought to not have functional disturbances compared to pituitary microadenomas; thus, the radiological correlation will suggest the latter's presentation in a syndromic CD patient.Typically, an ACTH-producing adenoma would be suspected.e shared component of MRI presentation in both of our patients was the hypoenhanced/non-enhancing presentation of the lesion.Pituitary microadenomas also typically present as relatively hypointense lesions within an intensely enhanced pituitary gland. [3]Given the similarity of presentation on MRI, the diagnosis of RCC in our patients relied on histological findings. e histopathological intricacies in patients with other sellar lesions are also worth noting, as this is pivotal for accurate diagnosis, treatment planning, and prognostication, underscoring the significance of ongoing research efforts in this domain.Pituitary adenomas, the most prevalent sellar lesions, exhibit diverse histological patterns, as highlighted by the comprehensive classification by the World Health Organization, using markers of cytodifferentiation as the principal classifier. [9]Furthermore, craniopharyngiomas present characteristic histopathological features such as keratinized epithelium and surrounding fibrovascular stroma. [18]ey are categorized as adamantinomatous, characterized by mutations in the β-catenin gene as a specific molecular pathogenic mechanism, and papillary. [1]e complexity of sellar lesions extends beyond these common entities, encompassing rare pathologies such as sellar chordomas, which are characterized by large, vacuolated cells known as "physaliphorous" cells enclosed within a mucinous matrix.ese cells are arranged in interconnected cords or sheets, occasionally exhibiting mitotic activity.Tumors express specific markers, including S100, low-molecularweight keratins, and epithelial membrane antigens.Brachyury, or transcription factor T, [20] and of interest to the discussion on the presented cases, RCC is characterized by a monolayer of cuboidal cells with microvilli, scattered columnar, and goblet cells.e cyst contents typically comprise amorphous eosinophilic material.Squamous metaplasia, a transformation of the lining epithelium, is frequent and may pose a diagnostic challenge, resembling craniopharyngioma.In addition, xanthogranulomas with chronic inflammation and cholesterol crystals may be present in this context. [19]derstanding the histological presentation of RCCs may elucidate the pathophysiological mechanisms by which these lesions may have led to CD in our patients.CD typically occurs from a pituitary gland tumor or pituitary hyperplasia, resulting in the overproduction of ACTH and resultant overproduction of cortisol by the adrenal glands. [10]ne report of a patient presenting with CD and pituitary microadenoma also demonstrated Crooke's changes in pituitary cells on histology. [2]In one case of CD in a female patient in her 3 rd decade, corticotroph cell hyperplasia/ ACTH-positive cells penetrating the wall of the RCC were seen on histology. [17]Findings demonstrated an RCC, numerous ACTH+ cells, and, to a lesser extent, thyroidstimulating hormone (TSH)+ cells. [17]In our patients, biopsy demonstrated unremarkable pituitary tissue with adjacent islands of eosinophilic acellular material and strips of ciliated epithelium, a colloid nodule, and a fibrous membrane/wall.Biopsy findings in our second patient described Crooke's hyaline changes.In 75-80% of patients with chronic hypercortisolism of any etiology, the normal corticotrophs undergo changes in which the cytoplasmic granules are replaced with homogeneous hyaline material, known as Crooke's changes. [14]e absence of these changes does not confirm the absence of CD; however, the presence of these changes is associated with the degree of hypercortisolism in the patient. [14]boratory values reflecting endocrine dysfunction presented similarities and differences between these two cases [Table 1].First, patient two presented with elevated ACTH preoperatively at 60.4 pg/mL with associated symptoms of CD.Patient 1, however, presented with low ACTH levels at 4.1 pg/mL.is patient had a long-reported history of growth hormone deficiency and symptoms consistent with CD, but given the low ACTH value preoperatively, his disease likely was cyclical.Both patients presented with markedly elevated insulin-like growth factor 1 levels, 281 ng/mL and 245 ng/mL, respectively.Prolactin, follicle-stimulating hormone, TSH, and free T4 levels were within normal limits for both patients.Interestingly, the growth hormone in our first case was elevated at 22.5 ng/mL but low in the second patient at 0.1 ng/mL.Another report of a patient presenting with CD secondary to concurrent pituitary microadenoma and RCC found a similar resolution of elevated ACTH levels following complete resection, normalization of blood cortisol levels, and improved CD symptomatic presentation. [2] measuring pre-and postoperative cortisol changes in both patients, we measured the degree of improvement that surgical resection of the RCC provided [ F/u: Follow-up, N/A: Not available of cases with overt signs of RCC compression causing symptomatic clinical presentation, there was demonstrated improvement in clinical symptoms after surgery. [6]In particular, transsphenoidal endoscopic surgery is the first choice for surgical treatment of RCC, intending to drain the cyst contents and remove the surrounding capsule. Suprasellar involvement of RCC can prove to be a neurosurgical challenge.Our first patient case presented with an abutment of the optic chiasm.It has been shown that compared with sellar-based RCCs, RCC's visual dysfunction is more difficult to eliminate, recurs more frequently, and is associated with higher postoperative endocrine morbidity, and their preoperative visual dysfunction and headache less frequently improve with surgery. [15]Avoidance of optic nerve damage due to its location above the surgical corridor through traversing the tuberculum sella and the planum sphenoidale also offers a factor of difficulty to the surgical management of these RCCs. [11]Surgically, both cases presented without any postoperative complications. What remains to be understood in these cases is the pathophysiological mechanism by which these RCCs resulted in CD presentation, typically resulting from pituitary microadenomas.One possible explanation is that the RCC resulted in an overproduction of ACTH in neighboring cells surrounding the cyst.is suggests an effect of elevated circulating corticosteroids on the anterior pituitary parenchyma, resulting in the presentation of CD.In addition, the resolution of symptoms in both patients, including fatigue and generalized weakness, clinical and reduction of cortisol levels, and improvement in endocrinological laboratories postoperatively, suggests that the surgical removal of the RCC in these patients provided a "biochemical cure." However, the pathophysiologic mechanism of how the cystic cells resulted in symptomatic CD remains unclear.e absence of a concurrent pituitary microadenoma and benign pituitary tissue leaves no clear explanation of the cellular level changes that may have occurred.Nevertheless, the apparent symptomatic resolution of CD in these patients suggests that neurosurgical intervention was worthwhile.In cases of CD secondary to the presence of RCC, neurosurgical intervention should be considered for symptomatic improvement; however, further research should be undertaken to understand the underlying mechanism of this presentation. CONCLUSION Here, we present two cases of isolated RCCs presenting symptoms suggestive of CD.Based on the initial clinical presentation in these two patients and diagnostic imaging suggesting a pituitary lesion, pituitary microadenoma was the presumed diagnosis.However, there was no evidence of adenoma histopathologically, with findings more consistent with RCC.Maximal surgical resection was achieved in both patients through endoscopic endonasal pituitary resection with postoperative symptomatic resolution and normalization of cortisol levels.e clinical features, histological findings, and possible pathological mechanisms for this unique presentation of RCC-causing CD have been discussed and laid the groundwork for future studies into the pathophysiology of RCC and CD. Declaration of patient consent Patients' consent not required as patients' identities were not disclosed or compromised. Financial support and sponsorship Nil. Conflicts of interest ere are no conflicts of interest. Use of artificial intelligence (AI)-assisted technology for manuscript preparation e author confirms that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI. the lesion, and postoperative Figure 1 :Figure 2 : 12 Figure 1: (a and b) Patient 1 preoperative imaging demonstrating solid appearing, hypo-enhancing suprasellar mass immediately anterior to midline pituitary stalk with abutment of the undersurface of the optic chiasm.ab Figure 3 :Figure 4 : 34 Figure 3: (a and b) Patient 2 preoperative imaging demonstrating ovoid, hypo-enhancing lesion in the superior aspect of the pituitary gland just to the left of midline.a b Table 2 ] 2 . Our first patient with a PM cortisol level of 2.3 mcg/dL preoperatively, an AM cortisol of 12.4 mcg/dL on postoperative day 1, and an AM cortisol of 1.7 mcg/dL on postoperative day 2, an overall downward trend in cortisol levels.Our second patient presented with an elevated PM cortisol level at 22.8 mcg/dL.Postoperatively, her AM cortisol was 4.3 mcg/ dL on postoperative day 1, and an AM cortisol of 5.6 mcg/dL on postoperative day 2, an overall downward trend in cortisol levels.is decline in cortisol levels following RCC resection led us to consider this a biochemical cure to the hormone imbalance causing this CD.e surgical management of RCC is indicated in symptomatic presentations such as that in our patients.In previous studies Table 1 : 1 Pre-operative laboratory results in patients presenting with Cushing's disease. PreoperativePostoperativePatient 1 Patient 2 Patient 1 Patient 2TSH (mlu/L)0.1370.208-0.82FT4 (ng/dL)0.841.16-1.3Prolactin (ng/mL)7.014.8-18.3LH (mlu/mL)1.533.5-13FSH (mlu/mL)357.1-27.8ILGF1 (ng/mL)281245181-Growth hormone22.50.1-1.9(ng/mL)ACTH (pg/mL)4.160.4-25TSH: yroid-stimulating hormone, FT4: Free thyroxine, LH: Luteinizinghormone, FSH: Follicle-stimulating hormone, ILGF1: Insulin-like growthfactor 1, ACTH: adrenocorticotropic hormone Table 2 : 2 Trends in cortisol level (mcg/dL) in patients pre and post-operatively. Patient 1Patient 2Preoperative2.3 (PM)22.8 (PM)Postoperative day 112.4 (AM)4.3 (AM)Postoperative day 21.7 (AM)5.6 (AM)One month F/u visitN/A8.9 (AM) My approach to pathology of the pituitary gland. Al-Brahim Ny , Asa Sl, J Clin Pathol. 592006 A case of Cushing's disease accompanied by Rathke's cleft cyst: e usefulness of cavernous sinus sampling in the localization of microadenoma. K Arita, T Uozumi, A Takechi, T Hirohata, B Pant, K Kubo, Surg Neurol. 421994 Imaging of the pituitary: Recent advances. V Chaudhary, S Bano, Indian J Endocrinol Metab. 1532011Suppl Rathke's cleft cyst. W B Crenshaw, F S Chew, AJR Am J Roentgenol. 15813121992 A cyst of Rathke's cleft. B Fairburn, I M Larkin, J Neurosurg. 211964 Clinical manifestations of Rathke's cleft cysts and their natural progression during 2 years in children and adolescents. J E Jung, Jin J Jung, M K Kwon, A Chae, H W Kim, D H , Ann Pediatr Endocrinol Metab. 222017 Pathophysiology and treatment of subclinical Cushing's disease and pituitary silent corticotroph adenomas. K Kageyama, Y Oki, T Nigawara, T Suda, M Daimon, . Endocr J. 612014 Rathke's cleft cyst. S Larkin, N Karavitaki, O Ansorge, Handb Clin Neurol. 1242014 e new WHO classification of pituitary tumors: Highlights and areas of controversy. E R LawsJr, M B Lopes, Acta Neuropathol. 1112006 Cushing's disease: Pathobiology, diagnosis, and management. R R Lonser, L Nieman, E H Oldfield, J Neurosurg. 1262017 Endoscopic endonasal resection of Rathke cleft cysts: Clinical outcomes and surgical nuances. R Madhok, D M Prevedello, P Gardner, R L Carrau, C H Snyderman, A B Kassam, J Neurosurg. 1122010 Pituitary adenoma combined with Rathke's cleft cyst--case report. A Miyagi, M Iwasaki, T Shibuya, G Kido, H Kushi, M Miyagami, Neurol Med Chir (Tokyo). 331993 Pituitary tumors composed of adenohypophysial adenoma and Rathke's cleft cyst elements: A clinicopathological study. S Nishio, J Mizuno, D L Barrow, Y Takei, G T Tindall, Neurosurgery. 211987 Crooke's changes in cushing's syndrome depends on degree of hypercortisolism and individual susceptibility. E H Oldfield, M L Vance, R G Louis, C L Pledger, Jane Ja LopesJr, M B , J Clin Endocrinol Metab. 1002015 Suprasellar Rathke cleft cysts: Clinical presentation and treatment outcomes. M B Potts, A Jahangiri, K R Lamborn, L S Blevins, S Kunwar, M K Aghi, Neurosurgery. 692011 Clinical features, management and recurrence of symptomatic Rathke's cleft cyst. D M Raper, M Besser, J Clin Neurosci. 162009 Cushing's syndrome in a patient with Rathke's cleft cyst and ACTH cell hyperplasia detected by (11)C-methionine PET imaging-a case presentation. K P Sagan, E Andrysiak-Mamos, L Sagan, P Nowacki, B Małkowski, A Syrenicz, Front Endocrinol (Lausanne). 114602020 Craniopharyngiomas of adamantinomatous type harbor beta-catenin gene mutations. S Sekine, T Shibata, A Kokubu, Y Morishita, M Noguchi, Y Nakanishi, Am J Pathol. 1612002 Cystic lesions of the pituitary: Clinicopathological features distinguishing craniopharyngioma, Rathke's cleft cyst, and arachnoid cyst. J L Shin, S L Asa, L J Woodhouse, H S Smyth, S Ezzat, J Clin Endocrinol Metab. 841999 Building a global consensus approach to chordoma: A position paper from the medical and patient community. S Stacchiotti, J Sommer, Lancet Oncol. 162015Chordoma Global Consensus Group Pituitary adenoma associated with Rathke's cleft cyst: Report of 15 cases. W Wu, G Jia, W Jia, G Li, J Zhang, L Zhang, Can J Neurol Sci. 452018

Incidence and associated factors of developing second pelvic malignant neoplasms among prostate cancer patients treated with radiotherapy 17 November 2023 Youbiao Wang Department of Urology The Second People's Hospital of Jingdezhen City Jingdezhen, JiangxiChina Ru Chen Department of Urology The First Affiliated Hospital of Nanchang University NanchangJiangxiChina Xinxi Deng Department of Urology Jiujiang First People's Hospital Jiujiang, JiangxiChina Xinghua Jiang Department of Urology The Second People's Hospital of Jingdezhen City Jingdezhen, JiangxiChina Zsofia Kote-Jarai Fu Jin Noorwati Sutandyo Institute of Cancer Research (ICR) United Kingdom Chongqing University China Dharmais Hospital National Cancer Center Indonesia Incidence and associated factors of developing second pelvic malignant neoplasms among prostate cancer patients treated with radiotherapy 17 November 2023F7257AB28F41D325858F11E56665B02D10.3389/fonc.2023.1260325RECEIVED 17 July 2023 ACCEPTED 03 November 2023prostate cancerpelvic malignant neoplasmsbladder cancerrectal cancerradiotherapySEER Objective: To identify risk factors of secondary pelvic malignant neoplasms (SPMNs) among prostate cancer (PCa) patients treated with radiotherapy.Simultaneously, population-based data were used to validate the high risk of SPMNs in PCa patients with radiotherapy.Materials and methods:We identified male patients diagnosed with PCa (localized and regional) as the first primary cancer and pelvic malignant neoplasm (including bladder and rectal cancer) as secondary cancer from Surveillance, Epidemiology, and End Results database ).An external validation cohort was obtained from the First Affiliated Hospital of Nanchang University.The Fine-Gray competing risk regression and Poisson regression were utilized to evaluate the risk of SPMNs development.Poisson regression was also performed to calculate the standardized incidence ratio (SIR).The Kaplan-Meier method was used to assess the overall survival (OS) of patients with SPMNs.Results: 89397 PCa patients treated with radiotherapy were enrolled.We identified associated factors of SPMNs, including age at diagnosis, race, year of diagnosis, marital status, radiation strategy and latency.In the multivariable competing risk regression model and Poisson regression model, a significantly higher risk of SPMNs development was observed in patients over 50 years (P<0.05),white patients(P<0.001),unmarried patients and treated with brachytherapy combined with external beam radiotherapy or brachytherapy (P<0.05).Patients treated with radiotherapy had a higher bladder and rectal cancer incidence than the general population.Patients who developed SPMNs showed poorer OS.Conclusion:We identified several risk factors associated with SPMNs and confirmed a relatively higher incidence of bladder and rectal cancer among PCa patients with radiotherapy.These results help tailor treatment and surveillance strategies. Introduction It is estimated that in 2019, nearly three million men in the United States were newly diagnosed with prostate cancer (PCa) (1).PCa was the most prevalent of all male cancers.While early-stage diagnosis and treatment have proven successful in managing PCa, reducing long-term mortality and improving quality of life remains a top priority.However, it is important to note that the high prevalence of PCa may be in part due to observation-based management techniques as well as the widespread use of PSA testing (2). Radiotherapy (RT) and radical prostatectomy (RP) are standard treatment options for active treatment of localized PCa, and there is evidence that they have similar long-term disease-free survival rates (3).However, observational data with low to moderate risk of bias indicate that radiotherapy may be associated with a higher risk of overall and prostate cancer-specific mortality when compared with surgery (4).A particularly worrying potential effect of prostate radiotherapy is radiation-induced second malignancy.There have been many previous studies on the risk analysis of secondary malignant neoplasm (SMN) for PCa after radiotherapy, but the results were not consistent (1,3,(5)(6)(7).The results of most studies suggested that prostate irradiation increased the risk of developing secondary pelvic malignant neoplasms (SPMNs), which included bladder cancer (BC) and rectum cancer (RC).A retrospective study suggested that PCa patients who received RT were more prone to developing a second primary cancer compared to those who did not receive the therapy, with a higher risk over time.Despite a lower incidence and risk of second primary cancer (8).A recent metaanalysis to evaluate the second malignancies after radiotherapy for PCa suggested that radiotherapy was associated with an increased risk of secondary BC and RC compared with patients who did not treat with radiotherapy (7).However, most of the controversy was that the included studies are retrospective, so the reliability of the results was still limited.In theory, large prospective studies aimed at minimizing the effects of possible confounding factors would address the real risk of SMN after prostate irradiation.However, it seems unlikely that such trials will be carried out in the near future for logistical reasons. One hypothesis based on this study is that radiotherapy for PCa will increase the risk of developing secondary pelvic neoplasms incorporating BC and RC.This study intended to explore relevant risk factors of SPMNs development for PCa patients treated with radiotherapy using contemporary data in a large population-based cohort.In addition, we used a ratio which was expressed with standardized incidence ratio (SIR) to evaluate the risk of SPMNs development in PCa patients treated with radiotherapy and without. Methods Database and study population We identified male patients diagnosed with prostate cancer as the first primary cancer from the Surveillance, Epidemiology and End Results (SEER) database (1975-2020; 9 registries) (Figure 1).Then, we created a study population that included cases diagnosed with bladder cancer (BC) and rectal cancer (RC) as secondary cancers from the same database.All cancer sites were identified based on the case list of "The International Classification of Diseases for Oncology, Third Edition (Site recode ICD-O-3)".We enrolled PCa patients who received radiotherapy only and excluded patients undergoing surgery.The diagnostic confirmation method was restricted to positive histology.The other exclusion criteria are as follows: (1) tumor stage as distant; (2) survival months unknown;(3) survival months less than 60 months (4) survival status unknown; (5) Race unknown; (6) tumor grade unknown, (7) Diagnosis after 2013 year. We have obtained an external validation cohort consisting of patients diagnosed with prostate cancer at the First Affiliated Hospital of Nanchang University between 2010 and 2019.These patients received surgery or RT.Demographic and clinical data, including age, marital status, smoking habits, alcohol consumption, Prostate-Specific Antigen (PSA) levels, distant metastasis, and the number of patients observed with bladder or rectal cancer after treatment, were collected. Variable definition The following data were collected: age at diagnosis (<50 years, 50-70 years, >70 years); race [white, black, others (American/ Indian/Alaska/Native and Asian/Pacific Islander)]; year of diagnosis, marital status (married, unmarried, unknown); Gleason biopsy (6-10); clinical T stage (cT1, cT2, cT3, cT4, unknown); AJCC Stage Group (II, III, IV, unstaged); summary stage (localized, regional, unknown); tumor grade (grade I or grade II, grade III or grade IV); radiation strategy [external beam radiotherapy(EBRT); external beam radiotherapy-brachytherapy (EBRT+BT); brachytherapy(BT)]; survival month; survival status. Definition and follow-up of SPMNs The primary outcome of this study was the development of an SPMN, which was defined as bladder cancer or rectal cancer occurring more than 5 years after PCa patients received radiotherapy in consideration of the incubation period of at least 5 years from radiation exposure to a solid tumor (9).The SEER program followed the guidelines of the third edition of the International Classification of Oncology Diseases to distinguish between SPMN and recurrent diseases.The cancer history is obtained according to the " Sequence number " case list, which lists the order in which all the primary tumors can be reported in the patient's life.The follow-up for SPMN began 5 years after PCa diagnosis and ended at the date of diagnosis of BC or RC, all-cause death, or the last follow-up, whichever occurred first.The last follow-up data was December 31, 2020. Statistical analysis Fine-Gray competing risk regression analysis was utilized to calculate the cumulative incidence of SPMN development. Experiencing end of follow-up or death from all-cause were considered competing events.The multivariable competing risk model was established by using a backward selection procedure with variables which were statistically significant in the univariable analyses. The results were presented as hazard ratios (HRs) and 95%CIs.Poisson regression analysis was used to estimate the association between different incorporated factors and the risk of SPMN development, and the results exhibited as SPMN associated risk and 95%CIs.The missing values were imputed using multiple imputation method for conducting sensitivity analysis.Meanwhile, Poisson regression analysis was also performed to calculate the standardized incidence ratio (SIR) and 95%CIs.The SIR was defined as a ratio of the observed SPMN incidence rate in PCa survivors to the pelvic malignant neoplasm (PMN) incidence rate in the U.S. general population.The SIRs were calculated with SEER*Stat 8.4.0(ID: 20420-Nov2020).Then, we calculated the SIRs stratified by year of diagnosis, age at diagnosis, and latency to furtherly evaluate the incidence of SPMNs associated with radiotherapy. To evaluate the prognosis of SPMNs, survival analyses were performed with the Kaplan-Meier method and log-rank tests to calculate the overall survival (OS) for patients who developed SPMNs and with only primary PCa.The only primary PCa was defined as a patient who had only been diagnosed with PCa and had no other cancer throughout his lifetime.Propensity score matching (PSM) was performed to adjust the potential baseline matched 1:1 for survival comparison (caliper set at 0.02). All statistical analyses were performed by R software (version 4.1.3).P-values less than 0.05 were statistically significant. Results Patient characteristics 89397 PCa patients who received radiotherapy were enrolled in our study.There were 2125(2.38%)PCa survivors who developed SPMN, and there were 1758(1.97%)cases who developed bladder cancer and 367(0.41%)developed rectal cancer, respectively (Table 1). FIGURE 1 Flow-chart showing the procedure used to identify male patients diagnosed with prostate cancer as the first primary cancer bladder cancer (BC) and rectal cancer (RC) as secondary cancers from the Surveillance, Epidemiology and End Results (SEER) database (1975-2018; 9 registries). We finally identified 116 PCa patients who received surgery (67, 57.75%) and RT (49, 42.25%), respectively from the First Affiliated Hospital of Nanchang University (Supplementary Table 1).During the follow-up period, we observed 3 cases of BC or RC among patients in the surgery group and 9 cases among patients in the radiation therapy group(P=0.001). Cumulative incidences of SPMNs We presented the cumulative incidences of SPMNs in PCa patients treated with radiotherapy by different characteristics.The cumulative incidences of SPMNs in patients aged less than 50 years were significantly lower than in patients aged 50-70 years and more than 70 years (Figure 2A) (P=0.008).For different race patients, white patients showed a higher cumulative incidence than black and other races (Figure 2B) (P<0.001).Married patients had a statistically higher incidence than those unmarried (Figure 2C) (P<0.001).We did not obtain statistical differences in cumulative incidences distribution when stratifying the study population by summary stage, AJCC stage, Gleason biopsy and tumor grade (Figures 2D-G) (all P>0.05).For patients who received different radiotherapy strategy, we observed a relatively lower cumulative incidence of SPMNs in patients who received EBRT when compared with those with EBRT+BT or BT (Figure 2H) (P<0.001). Risk factors associated with SPMNs We performed competing risk regression and Poisson regression to identify risk factors associated with SPMNs development.We found statistically significant variables, including age at diagnosis, race, year of diagnosis, marital status, tumor grade, radiation strategy and latency in the univariate competing risk regression and univariate Poisson regression (Table 2).In the multivariable competing risk regression model, a significantly higher risk of SPMNs development was observed in 50-70 years (HR:1.78,95%CI:1.16-2.73)and >70 years (HR: 2.05,95% CI: 1.33-3.15)patients compared with those age less than 50 years.White patients showed a relatively higher risk than black and other patients (P<0.001).Patients diagnosed after 2000 exhibited a descending risk compared with those diagnosed between 1995-1999 (P<0.001).Unmarried PCa patients had a relatively higher risk Dynamic incidence evaluation for SPMNs We evaluated the SIRs of BC and RC for PCa patients treated with radiotherapy and patients undergoing surgery, respectively (Table 3).For PCa patients treated with radiotherapy, we observed a significantly increased incidence of BC and RC compared with the US general population (SIR: 1.44; 95%CI: 1.38-1.5;P<0.05 for BC; SIR: 1.48; 95%CI: 1.35-1.62;P<0.05 for RC).In the subgroup analysis by year of diagnosis, we found the increasing incidence of BC after 1995 and incidence of RC after 2000.In analyses of SIRs for different age, a relatively higher incidence than US general population was observed in PCa patients over 60 years old for BC and in PCa patients over 70 years old for RC.For PCa patients who survived longer than 5 years, we obtained a significantly increased incidence of BC and RC.For patients undergoing surgery, no increased incidences of BC and RC were observed in PCa patients when compared with US general population.Similar results were obtained when we stratified the PCa patients by year of diagnosis and latency.We just observed the increasing incidences of BC in PCa patients aged 60-64 years and 65-69 years. Survival outcome of SPMNs We compared the OS of PCa patients with only primary tumor and patients who developed SPMNs to evaluate the prognosis of SPMNs.Survival curves revealed that patients with only PCa had a significantly better OS than those who developed SPMNs for PCa patients with a survival time longer than 140 months (Supplementary Figure 1A).This difference in survival outcome became more prominent after PSM (Supplementary Figure 1B).In addition, we assessed the prognosis of BC or RC development, respectively.We failed to obtain a statistical difference in OS between patients with only primary tumor and developed BC (Supplementary Figure 1C).However, patients with BC development showed poorer survival outcomes than those without after PSM(P<0.001).Patients with only PCa had a significantly better OS than patients with RC development before and after PSM (Supplementary Figures 1D-F). Discussion In this study, we identified several associated factors of SPMNs development, including age at diagnosis, race, year of diagnosis, marital status, radiation strategy, and latency in PCa patients treated with radiotherapy by multiple statistical methods.Meanwhile, we found a higher incidence of subsequent BC and RC in PCa with radiotherapy than in the general US population, and no increase in the incidence of SPMNs was observed in PCa undergoing surgery.Finally, we observed a relatively poorer OS in PCa patients who developed SPMNs when compared with those with only primary PCa. Normal aging had been reported as a risk factor for secondary malignancies by many previous studies (10).For example, ANURAG K et al. found increasing age was accompanied by a risk of BC development for patients diagnosed with PCa as first cancer (11).Moreover, for the general population, old age has also been proved to be an essential factor in the occurrence of BC and RC (12,13).Our study also obtained similar results and identified a higher risk of SPMNs development among older PCa patients treated with radiotherapy.Meanwhile, older patients may have lower sensitivity to radiation therapy and often require higher radiation doses during the radiotherapy process.Older prostate cancer patients typically have more traditional cancer risk factors, such as familial inheritance, overeating, smoking, etc.These risk factors may have a composite effect, leading to an increase in the incidence of other cancers.These results suggested that the surveillance of SPMNs in elderly patients with PCa receiving radiotherapy might be a more practical method.An interesting finding is a decrease in risk of SPMNs development in PCa patients diagnosed after 2000 years.This might be due to the decline in the age of diagnosis caused by PSA screening and early detection of preclinical cancer (10).It was consistent with the lower incidence of SPMNs in the young PCa patient.Another similar result was a significant increase in the risk of SPMNs development in patients with PCa who had survived longer than 10 years.The possible explanation for this result was that a longer survival time might increase the exposure probability of carcinogens (14, 15).It is not easy to make a reasonable explanation for the risk difference of SPMNs development between different races and marital status.The possible explanation for this phenomenon is discrepancies in living habits (including smoking), living environment, carcinogens exposure, and radiation sensitivity (2,14).For example, married individuals are more likely to adopt healthy lifestyle habits, such as maintaining a regular diet and exercise routine, undergoing regular check-ups, and practicing smoking cessation and alcohol moderation.These healthy habits may potentially reduce the risk of developing cancer.At the same time, certain racial groups may face more economic pressures and disadvantages in society, including low income, limited health insurance coverage, and fewer medical resources.This may result in late cancer diagnosis and fewer treatment options, thereby increasing the risk of developing other types of cancer.However, because we lacked this information and were limited by retrospective studies, more relevant evidence was needed to explain it in the future.Notably, we observed a relatively lower risk of SPMNs development in patients with EBRT than in those with EBRT+BT or BT.This result was not consistent with some previous results.For instance, Moon et al. observed an increased risk of BC after EBRT compared with brachytherapy, and the data was also from the SEER database (16).Generally, intensity modulated radiotherapy and charged particle therapy might reduce the risk of SMNs by reducing the number of tissues exposed to high doses of radiation (15).Kishan J. Pithadia et al. based on SEER found patients treated with intensity-modulated radiotherapy (IMRT) had no significant differences with those with three-dimensional conformal radiation therapy (3DCRT) (17).However, the radiation dose of radioactive implants to the pelvis was still relatively high, even higher than EBRT to some extent (18).Therefore, it was no surprise that an increased incidence of SPMNs was shown in PCa patients treated with EBRT+BT or BT.Meanwhile, considering the differences in the definition of SPMN, the choice of the incubation period, the length of follow-up, the methods of the cohort population and the sample size between studies, we might need more studies to explain these inconsistent results. Although there had been studies focusing on the association between radiotherapy and SPMNs, the conclusions were conflicting (2,3,10).Considering that the patient selection bias caused by the retrospective study might affect the reliability of the results partly, we introduce epidemiological indicators (SIR) to evaluate the difference in subsequent SPMNs development among PCa patients treated with radiotherapy and without.This kind of data based on a large population will be more representative for most US population.Meanwhile, because the calculation of SIR is based on the data observed by the population, this index reduces the artificial bias to a great extent (19).In addition, we took the incidence of SPMNs in the general population as a control, while previous studies mainly had compared the risk of SPMNs development among PCa patients in two cohorts, including patients who received radiotherapy and prostatectomy.We think that setting the incidence outside our study population as a comparison might make our results more convincing (20). It is no surprise that PCa patients with subsequent SPMNs showed a poorer OS than those with only primary PCa because the prognosis of PCa was significantly better than BC and RC, and the 5year relative survival rate of local or regional PCa approached 100% after aggressive treatment (1).Notably, our study began five years after the diagnosis of PCa, not at the time of SPMNs diagnosis.Therefore, this difference in survival was not significant during the initial study period but became more prominent with the extension of follow-up time.Recent research findings demonstrate that even for low/medium risk patients, those undergoing ultra-conformal hypofractionated RT face a heightened risk of mortality from second cancers, surpassing the risk posed by prostate cancer itself (21).These findings serve as a crucial reminder for physicians to exercise greater caution during the evaluation of treatment necessities for prostate cancer patients, taking into account the potential risks associated with developing a secondary malignancy.In the case of patients with mild or low-risk disease presentations, prioritizing the avoidance of unnecessary treatment becomes paramount in order to minimize the potential for additional health hazards. The strengths of this study include a long follow-up period to discover potential SPMNs as well as the utilization of a large, population-based database, which allowed for the application of the results across the USA.In addition, we used external cohort data to validate our results, thus increasing the scientific validity of our results.However, our study was not devoid of limitations.First, we tried to include all available factors in our research for analysis.Still, due to the limitations of the database, we lacked some crucial factors like smoking, lifestyle, genetic background, psychosocial factors, more detail information of tumor stage and Gleason score, and radiation dose to prevent us from adjusting our analysis to understand the potential effect of these confounding factors (14).Secondly, considering PCa patients with secondary RC were insufficient for further investigation, we set our positive event in this study as SPMNs development.Then, we excluded patients with PCa for whom radiotherapy information was unknown, and this population represented a larger number of patients in the overall study population, which may to some extent undercut the validity of our results.Last but not least, because of the limitations of the SEER database, specific information on radiotherapy, such as more detailed radiotherapy modalities, is lacking to obtain more detailed information on outcomes.Still, we think this effect on our conclusions was negligible. Conclusion This study comprehensively evaluated the risk factors of SPMNs development in PCa patients receiving radiotherapy.In addition, we demonstrated a high incidence of SPMNs in PCa patients treated with radiotherapy by comparing with the general population.Increased risk of BC or RC in PCa patients with radiotherapy might have implications for public health, cancer surveillance and patient counseling.Perhaps most importantly, the study confirmed the belief that for patients with low-risk prostate cancer who did not need treatment at all, a second malignant tumor should be added to the already long list of avoidable risks associated with treatment. 2 2 FIGURE 2 The cumulative incidences of Second Pelvic Malignant Neoplasm in prostate cancer (PCa) patients treated with radiotherapy by different characteristics: (A): for age; (B) for race; (C) for marital status (D) for tumor stage; (E) for AJCC Stage Group; (F) for Gleason biopsy; (G) for tumor grade; (H) radiotherapy strategy. TABLE 1 1 Baseline characteristics of patients with prostate cancer treated with radiotherapy. No. (%)CharacteristicRadiotherapy (n= 80006)Age at diagnosis<50 years1752(2.19%)50-70 years43795(54.74%)>70 years34459(43.07%)RaceWhite62404(78%)black12112(15.14%)others 15490(6.86%)Year of diagnosis1995-199916305(20.38%)2000-200424538(30.67%)2005-200924105(30.13%)2010-201415058(18.82%)Marital statusMarried16121(20.15%)Single37521(46.89%)Widowed/Divorced20051(25.07%)Unknown6313(7.89%)Gleason biopsy64040(5.05%)77872(9.84%)8-102776(3.47%)Unknown65318(81.64%)Clinical T stagecT126762(33.45%)cT215817(19.77%)cT31280(1.6%)cT496(0.12%)Unknown36051(45.06%)AJCC Stage GroupII40651(50.81%)III1920(2.4%)IV400(0.5%)Unstage37035(46.29%)Lymph node statusN040083(50.1%)(Continued) TABLE 1 Continued 1No. (%)CharacteristicRadiotherapy (n= 80006)Nx12800(16%)unknown27123(33.9%)Summary stageLocalized67413(84.26%)Regional4208(5.26%)Unknown8385(7.48%)GradeGrade I or Grade II49283(61.6%)Grade III or Grade IV30723(38.4%)Radiation strategyEBRT44259(55.32%)EBRT+BT14705(18.38%)BT21042(26.3%)Developed SPMN2125(2.38%)Developed BC1758(1.97%)Developed RC367(0.41%) EBRT, external beam radiotherapy; EBRT+BT, interstitial brachytherapy or a combination of external beam radiotherapy; BT, brachytherapy; SPMN, second pelvic malignant neoplasm; BC, bladder cancer; RC, rectal cancer; AJCC, American Joint Committee on Cancer.Other: American/Indian/Alaska/Native and Asian/Pacific Islander. TABLE 2 2 Risk factors of developing SPMN (bladder cancer or rectum cancer) after prostate cancer diagnosis among patients receiving radiotherapy by Statistical Method. CharacteristicCompeting risk regressionPoisson regressionUnivariateP-MultivariableP-UnivariateP-MultivariableP-analysisvalueanalysisvalueanalysisvalueanalysisvalueHR (95% CI)HR (95% CI)HR (95% CI)HR (95% CI)Age at diagnosis<50 yearsRef.Ref.Ref.Ref.50-70 years1.89(1.23-2.90)0.014 ※1.78(1.16-2.73)0.027 ※1.97(1.32-3.12)0.009 ※1.83(1.22-2.9)0.02 ※>70 years2.08(1.35-3.18)0.005 ※2.05(1.33-3.15)<0.001 ※2.32(1.56-3.68)0.001 ※2.18(1.45-3.46)0.02 ※RaceWhiteRef.Ref.Ref.Ref.black0.68(0.61-0.77)<0.001 ※0.75(0.66-0.84)<0.001 ※0.63(0.56-0.71)<0.001 ※0.75(0.66-0.84)<0.001 ※others 10.66(0.55-0.78)<0.001 ※0.67(0.57-0.79)<0.001 ※0.65(0.55-0.77)<0.001 ※0.67(0.56-0.79)<0.001 ※Year of diagnosis1995-1999Ref.Ref.Ref.Ref.2000-20040.86(0.79-0.93)<0.001 ※0.83(0.75-0.9)<0.001 ※0.79(0.68-0.9)<0.001 ※0.77(0.7-0.85)<0.001 ※2005-20090.69(0.63-0.76)<0.001 ※0.72(0.64-0.81)<0.001 ※0.43(0.39-0.48)<0.001 ※0.5(0.45-0.56)<0.001 ※2010-20140.67(0.56-0.8)<0.001 ※0.96(0.78-0.99)<0.001 ※0.16(0.13-0.18)<0.001 ※0.27(0.22-0.33)<0.001 ※Marital statusMarriedRef.Ref.Ref.Ref.Unmarried1.29(1.17-1.42)<0.001 ※1.19(1.08-1.31)0.004 ※1.34(1.22-1.48)<0.001 ※1.18(1.07-1.3)0.005 ※Gleason biopsy6Ref.Ref.71.03(0.7-1.5)0.890.99(0.68-1.47)0.988-101.2(0.76-1.9)0.531.29(0.81-2.03)0.35AJCC Stage GroupIIRef.Ref.III0.95(0.63-1.4)0.830.96(0.62-1.4)0.86IV0.47(0.18-1.2)0.190.48(0.15-1.08)0.2Summary stageLocalizedRef.Ref.Regional0.83(0.66-1.04)0.180.84(0.66-1.04)0.19GradeGrade I or Grade IIRef.Ref.Ref.Grade III or Grade0.85(0.79-0.92)<0.001 ※0.99(0.91-1.07)0.830.61(0.57-0.66)<0.001 ※0.98(0.9-1.07)0.768IVRadiation strategyEBRTRef.Ref.Ref.Ref.EBRT+BT1.14(1.04-1.25)0.023 ※1.15(1.04-1.26)0.02 ※1.17(1.07-1.29)0.004 ※1.14(1.03-1.25)0.02 ※BT1.17(1.07-1.27)0.002 ※1.16(1.06-1.26)0.005 ※1.15(1.05-1.24)0.007 ※1.14(1.04-1.24)0.01 ※(Continued) than married cases (HR: 1.19; 95%CI: 1.08-1.31).Patients who received EBRT+BT or BT showed a higher risk in comparison to patients with EBRT (HR: 1.15; 95%CI: 1.04-1.26forEBRT+BT;HR: 1.16; 95%CI: 1.06-1.26forBT).For patients with latency more than 10 years, they showed a significantly higher risk than patients who had survival time between 5 and 10 years (P<0.001).After using the multiple imputation technique to fill in the missing values, similar results were obtained as before.Supplementary Table2recorded In the multivariable Poisson regression, we similarly identified the statistically significant variables associated with SPMNs development, including age at diagnosis, race, year of diagnosis, marital status, radiation strategy and latency. TABLE 2 Continued 2 EBRT, external beam radiotherapy; EBRT+BT, interstitial brachytherapy or a combination of external beam radiotherapy; BT, brachytherapy; SPMN, second pelvic malignant neoplasm; AJCC, American Joint Committee on Cancer; HR, hazard ratio; CI, confidence interval. CharacteristicCompeting risk regressionPoisson regressionUnivariateP-MultivariableP-UnivariateP-MultivariableP-analysisvalueanalysisvalueanalysisvalueanalysisvalueHR (95% CI)HR (95% CI)HR (95% CI)HR (95% CI)Latency5-10 yearsRef.Ref.Ref.11-15 years1.47(1.34-1.62)<0.001 ※1.55(1.39-1.72)<0.001 ※2.31(2.1-2.55)<0.001 ※1.76(1.59-1.97)<0.001 ※16-20 years1.61(1.46-1.78)<0.001 ※1.57(1.39-1.76)<0.001 ※3.38(3.06-3.75)<0.001 ※2.02(1.8-2.28)<0.001 ※21-25 years1.6(1.38-1.86)<0.001 ※1.43(1.2-1.69)<0.001 ※3.89(3.32-4.54)<0.001 ※2.04(1.7-2.43)<0.001 ※1 Other: American/Indian/Alaska/Native and Asian/Pacific Islander. ※ : statistical significance. TABLE 3 3 Standardized incidence ratio of bladder cancer and rectum cancer after prostate cancer diagnosis among patients receiving radiotherapy or without radiotherapy. Bladder cancerRectum cancerAdjusted SIR (95% CI)P-valueAdjusted SIR (95% CI)P-valueRT vs US general populationAll patients1.44(1.38-1.50)<0.051.48(1.35-1.62)<0.05Year of diagnosis1995-19991.37(1.18-1.57)<0.051.21(0.90-1.58)NS2000-20041.44(1.29-1.60)<0.051.32(1.05-1.65)<0.052005-20091.34(1.21-1.48)<0.051.58(1.28-1.93)<0.052010-20151.54(1.41-1.68)<0.051.49(1.19-1.85)<0.05Age at diagnosis<60 yearsNANANANA60-64 years2.08(1.44-2.91)<0.051.20(0.55-2.27)NS65-69 years1.72(1.41-2.07)<0.051.45(0.98-2.06)NS70-74 years1.33(1.16-1.52)<0.051.35(1.03-1.73)<0.0575-79 years1.36(1.24-1.49)<0.051.44(1.18-1.74)<0.0580-84 years1.38(1.27-1.50)<0.051.47(1.22-1.75)<0.0585+ years1.53(1.42-1.65)<0.051.66(1.38-1.98)<0.05Survival months5-10 years1.27(1.19-1.35)<0.051.18(1.02-1.35)<0.05>10 years1.61(1.52-1.70)<0.051.87(1.64-2.11)<0.05Undergoing surgery vs US general populationAll patients1.00(0.98-1.02)NS0.92(0.89-0.95)<0.05Year of diagnosis1995-19990.92(0.86-0.97)<0.050.93(0.84-1.02)NS2000-20040.98(0.93-1.03)NS0.92(0.84-1.01)NS2005-20091.03(0.99-1.08)NS0.90(0.83-0.98)NS2010-20151.00(0.96-1.04)NS0.97(0.89-1.05)NSAge at diagnosis<60 yearsNANANANA60-64 years1.32(1.22-1.42)<0.050.98(0.87-1.11)NS65-69 years1.07(1.01-1.14)<0.050.85(0.77-0.94)<0.0570-74 years0.97(0.93-1.02)NS0.88(0.81-0.96)<0.0575-79 years0.96(0.92-1.01)NS0.90(0.83-0.98)<0.0580-84 years0.95(0.91-0.99)<0.050.85(0.78-0.92)<0.0585+ years0.94(0.91-0.98)<0.050.94(0.87-1.01)NSSurvival months5-10 years1.00(0.98-1.02)NS0.97(0.92-1.02)NS>10 years0.96(0.94-0.99)<0.050.88(0.84-0.93)<0.05 SIR, standardized incidence ratio; RT, radiotherapy; NA, not applicable; NS: no significance. Frontiers in Oncology frontiersin.org AcknowledgmentsThis statement is to certify that all authors have approved the manuscript being submitted, have contributed significantly to the work, attest to the validity and legitimacy of the data and its interpretation.Data availability statementThe original contributions presented in the study are included in the article/Supplementary Material.Further inquiries can be directed to the corresponding authors.FundingThe author(s) declare that no financial support was received for the research, authorship, and/or publication of this article.Ethics statementEthical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements.Written informed consent from the participants was not required to participate in this study in accordance with the national legislation and the institutional requirements.Author contributionsConflict of interestThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Publisher's noteAll claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.Supplementary materialThe Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc.2023.1260325/full#supplementary-material SUPPLEMENTARY FIGURE1Overall survival between patients with only prostate cancer and developing Second Pelvic Malignant Neoplasm(SPMN): (A) all SPMNs before PSM; (B) all SPMNs after PSM; (C) For bladder cancer before PSM; (D) For bladder cancer after PSM (E) For rectal cancer before PSM; (F) For rectal cancer after PSM. 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EDITED AND REVIEWED BY Rufin Vanrullen Ron Kupers kupers@sund.ku.dk Maurice Ptito Centre National de la Recherche Scientifique (CNRS) France Department of Neuroscience Panum Institute University of Copenhagen École d'optométrieCopenhagenDenmark Université de Montréal MontrealQCCanada EDITED AND REVIEWED BY E03766A31251C725E7DAB42A9C83E18CRECEIVED October ACCEPTED November PUBLISHED November CITATION Kupers R and Ptito M (visionauditionbrain plasticitybrain imagingsomethesiscortexrehabilitation There is now ample evidence that both congenital blindness (CB) and acquired or late-onset blindness (LB) trigger a myriad of brain neuroplastic changes.With the advent of modern non-invasive brain imaging techniques such as positron emission tomography (PET), (functional) magnetic resonance imaging (f)MRI, magnetoencephalography (MEG) and diffusion imaging, the scientific study of the mechanisms mediating brain plasticity following sensory loss has gathered momentum over the past decades.The first brain imaging studies focused largely on blindness and revealed that the visually deprived occipital cortex at rest is metabolically hyperactive.Ensuing studies showed that tactile input (e.g., Braille reading) and other forms of non-visual input activate the occipital cortex in CB and to a lesser extent also in LB subjects.Brain morphometric and diffusion imaging studies have further shed light on the associated brain structural changes.Although most of the initial work strongly focused on the absence of vision, later studies also dealt with brain plastic changes following loss of auditory input, and to a lesser extent following loss of smell, taste, and somatosensory input.With this Research Topic, we wanted to further our understanding of the mechanisms underlying brain plasticity following sensory loss, to highlight similarities and differences between sensory loss in different sensory domains (e.g., vision, audition and somesthesis), and to identify major challenges for novel avenues for therapeutic progress.Finally, studies of the sensory-deprived brain also help us to shed new light on normal brain development and function. Therefore, this Research Topic of Frontiers in Neuroscience is timely and brings a collection of ground-breaking novel research on the effects of sensory loss on neuroplastic processes in humans and in animal models.We present nine original articles and two systematic reviews that will contribute to expand our knowledge of brain reorganization following sensory loss. Description of the contents The majority of the papers in this Research Topic deal with alterations in the visual system.Arend et al. report that blind individuals have changes in cortical gyrification, an anatomical measure that has not been previously reported in this context.The authors show an increase in gyrification in several brain areas of CB individuals and, importantly, The results showed activation of the fusiform gyrus and other face-responsive-regions of the ventral visual stream.The authors concluded that there is a predisposition for sensory-independent and computation-specific processing in specific cortical regions that is independent of previous perceptual experience and that is pertained following sensory deprivation.Nadvar et al. studied resting state functional connectivity (rsFC) of area V1 following sight restoration in patients with retinitis pigmentosa who were implanted with the Argus II retinal prosthesis which partially restores vision.The aim was to test whether sight restoration with this treatment would reverse, in full or partly, the plastic changes induced by the vision loss.Their results showed that the decrease in rsFC between V1 and the post-central gyrus in CB participants was partially reversed by vision restoration.The authors suggest that rsFC between the occipital and somatosensory cortices could provide a biomarker for functional plastic changes following vision recovery.Maimon et al. report on visual perception in a small but unique group of children who had undergone vision-restoring cataract removal surgery as part of the Himalayan Cataract Project.Some of the children in the study were born with cataracts and gained a sense of sight for the first time, whereas others suffered late-onset blindness in one eye alone.The authors discuss their findings in the context of Molyneux's problem, i.e., the ability to correlate vision with touch quickly following sight restoration in blind individuals, and Hubel and Wiesel's theory of critical periods.Two papers relate to plasticity following auditory deprivation, one in humans and a second one in animals.Grégoire et al. performed a meta-analysis of the literature on brain plastic changes following hearing loss at birth or later in life.Hearing loss is a growing problem in modern Western societies due to an aging population.Moreover, knowledge of brain neuroplastic changes could help to understand some disappointing results with cochlear implants, and therefore could improve hearing rehabilitation.The literature research revealed that the most consistent finding in deaf individuals was a volumetric decrease in gray matter around the auditory cortex.In deaf children, an additional volumetric decrease was reported in both gray and white matter at the level of the visual cortex.Grégoire et al. further discuss the role of confounding factors that could affect brain plasticity in deaf individuals such as the use of sign language and hearing aids, and frequently observed associated vestibular dysfunction or neurocognitive impairments.Using kittens rendered deaf, Mitzetlfelt et al. investigated the stimulus-driven neural activity associated with visual localization.The researchers recorded visual evoked potentials (VEPs) in response to visual stimuli presented at various eccentricities in the visual field.Their results showed no significant changes in VEPs in deaf cats that could explain the previously observed behavioral advantage.The authors concluded that cross-modal plasticity in deafness does not play a major role in cortical processing of the peripheral visual field. Two studies report on patient groups with either central or peripheral lesions.Araneda et al. used diffusion MRI to study changes in white matter (WM) architecture in the geniculostriate pathway in 40 children with unilateral spastic cerebral palsy (USCP).The authors report several alterations in diffusion imaging parameters of the optic radiations on the lesional compared with the non-lesional hemisphere.Both the nature and the side of the lesion (left or right hemisphere) had an impact on the type and magnitude of the WM changes.In USCP with periventricular and right-hemispheric lesions, the diffusion imaging parameters correlated with the patients' visuospatial assessment.Dedry et al. studied three unique patients with unilateral vocal fold paralysis.The patients were followed for 1 year with multiparametric voice assessments and longitudinal fMRI during a sustained phonation task and rsfMRI.One patient received an augmentation injection in the paralyzed vocal fold.This patient showed a bilateral activation of the voice-related nuclei in the brainstem during sustained phonation.In addition, rsFC between the voice motor/sensory brainstem nuclei and other voice-related ROIs correlated with mean airflow measures in this patient.This observation supports the hypothesis that promoting proprioceptive feedback, by temporarily rehabilitating glottic closure, can enhance the neural recovery process. Finally, the study by Vaessen et al. addressed the question whether there is an abstract representation of emotions in the brain that is shared across stimulus types (face, body, voice) and sensory origin (visual, auditory).Thereto, the authors studied fMRI responses to ecological types of emotion expressions of different types and modalities.Using multivariate statistical analyses, the authors showed that there is a specific brain organization for affective signals which depends on stimulus category and modality.These findings are consistent with the notion that emotion expressions conveyed by different stimulus types have different functional roles in triggering rapid adaptive behavior. We hope that the papers presented in this Research Topic of Frontiers in Neuroscience will contribute to a better understanding of the mechanisms of cross-modal plasticity following different forms of sensory loss and of sensory substitution negative correlation between gyrification and cortical thickness in several different cortical areas.The authors discuss the impact of their results in relation to brain development and plasticity.Yizhar et al. used fMRI to study the role of the extrastriate body area (EBA) in action-related functions in CB individuals.Their findings indicate that the absence of visual experience does not favorize the development of action-related responses in the EBA.Moreover, CB participants showed a decrease in functional connectivity of the EBA with sensorimotor cortices, whereas connectivity with perception-related visual occipital cortices remained high.The authors further demonstrated that action-related functions and connectivity of the visual cortex are dependent on visuomotor experience.Bleau et al. present a meta-analysis of the neural substrates of spatial processing and navigation in blind individuals through touch and audition.The meta-analysis reveals that most studies agree that CB individuals recruit the same neural pathways as sighted controls when processing non-visual spatial information.The meta-analysis further shows that the primary visual cortex and associative occipital areas are involved in visuospatial processing via cross-modal plasticity mechanisms.The authors discuss the results in terms of the amodality hypothesis of spatial representations.Arbel et al. present novel data on face recognition in CB individuals.The authors trained a group of CB participants to use a visual-to-auditory sensory substitution device to recognize faces, whereafter they participated in an fMRI study. ./fnins.. a Frontiers in Neuroscience frontiersin.org ./fnins. .and other restorative therapies that may lead to restoration of the lost functions.FundingThe author(s) declare financial support was received for the research, authorship, and/or publication of this article.This work was supported by grants from the Canadian Institutes of Health Research (grant No. 451125).Conflict of interestThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Publisher's noteAll claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Genomics of Yoonia sp. Isolates (Family Roseobacteraceae) from Lake Zhangnai on the Tibetan Plateau 20 November 2023 Marina P Isaeva Lyudmila A Romanenko Valeriya Kurilenko Xiaoyuan Feng Shenzhen Research Institute Chinese University of Hong Kong 518000ShenzhenChina State Key Laboratory of Lake Science and Environment Institute of Geography and Limnology Chinese Academy of Sciences 210008Nanjing, NanjingChina Peng Xing pxing@niglas.ac.cn 0000-0002-4917-5181 State Key Laboratory of Lake Science and Environment Institute of Geography and Limnology Chinese Academy of Sciences 210008Nanjing, NanjingChina Genomics of Yoonia sp. Isolates (Family Roseobacteraceae) from Lake Zhangnai on the Tibetan Plateau 20 November 20234DA023E25BC2F981F08647C7FDE4009110.3390/microorganisms11112817Received: 25 October 2023 Revised: 18 November 2023 Accepted: 18 November 2023RoseobacteraceaeYooniaTibetan Plateau lakeshabitat transitionextreme environment adaptation Understanding the genomic differentiation between marine and non-marine aquatic microbes remains a compelling question in ecology.While previous research has identified several lacustrine lineages within the predominantly marine Roseobacteraceae family, limited genomic data have constrained our understanding of their ecological adaptation mechanisms.In this study, we isolated four novel Yoonia strains from a brackish lake on the Tibetan Plateau.These strains have diverged from their marine counterparts within the same genus, indicating a recent habitat transition event from marine to non-marine environments.Metabolic comparisons and ancestral genomic reconstructions in a phylogenetic framework reveal metabolic shifts in salinity adaptation, compound transport, aromatics degradation, DNA repair, and restriction systems.These findings not only corroborate the metabolic changes commonly observed in other non-marine Roseobacters but also unveil unique adaptations, likely reflecting the localized metabolic changes in responses to Tibetan Plateau environments.Collectively, our study expands the known genomic diversity of non-marine Roseobacteraceae lineages and enhances our understanding of microbial adaptations to lacustrine ecosystems. Introduction Although aquatic environments exhibit many shared ecological parameters, microbial transitions between marine and non-marine habitats are considered to be infrequent events [1].As a result, microbes in these two types of aquatic systems are often phylogenetically distinct and often segregate into well-defined marine and non-marine clusters [2].Notable exceptions to this pattern at the species level are scarce, with Escherichia coli serving as one of the few examples detected in both aquatic ecosystems.Instead, the transition from marine to lacustrine environments has been observed in several microbial lineages at higher taxonomic levels, such as Nitrososphaerales [3], SAR11 [4], Methylophilaceae [5], Cyanobacteria [6], and Flavobacteriaceae [7].In support of this, studies have identified significant differences in isoelectric points (pI) at the level of global amino acid composition between marine and lacustrine microbes, suggesting that a considerable evolutionary timescale is requisite for such transitions [8].Furthermore, these transition events are often accompanied by both genomic and phenotypic adaptations, encompassing alterations in salinity tolerance, transport functions, and environmental information processing [9]. The alphaproteobacterial Roseobacter group provides another illustrative example of ecological transitions between marine and lacustrine habitats.Comprising up to 20% of bacterial communities in coastal areas and 3-5% in pelagic oceans, the Roseobacter group plays a pivotal role in global carbon and sulfur cycling [10].Recent taxonomic revisions have reclassified some marine Roseobacters into a novel Roseobacteraceae family, while their non-marine counterparts such as Paracoccus and Rhodobacter have been categorized under the Rhodobacteraceae family [11].Intriguingly, non-marine lineages like Rubellimicrobium, Ketogulonicigenium [12], Loktanella, and Yoonia [13] are also phylogenetically placed within the Roseobacteraceae family.Phylogenetic analyses reveal a mixed structure between marine Roseobacteraceae and their non-marine relatives, thereby offering an ideal setting for studying microbial transitions between marine and non-marine aquatic ecosystems.Non-marine members have evolved high-affinity transporters to cope with lower sulfate concentrations and have lost genes associated with reduced sodium chloride and organohalogen concentrations in their habitats [14].Additionally, these organisms have lost pathways related to mercury antitoxin, carbon monoxide oxidation, and de novo cobalamin synthesis, which have become largely dispensable in lacustrine environments [14].Nevertheless, such transitions are rarely reported within the genus level [13]. In this study, we sequenced four novel genomes from Lake Zhangnai (also called Zhangnaicuo or Zhangnai Co) on the Tibetan Plateau.These genomes are taxonomically classified under the marine Yoonia genus, suggesting a habitat transition from marine to non-marine ecosystems.Metabolic comparisons and ancestral reconstructions elucidate metabolic shifts in salinity adaptation and compound transport.They further gain genes related to aromatic compound degradation and defense systems, likely reflecting localized adaptations to the unique conditions of the Tibetan Plateau.Our findings uncover a marine-to-lacustrine transition within the genus level, thereby offering a valuable genomic repertoire for advancing our understanding of adaptations to lacustrine environments. Methods Sample Collection, Bacterial Cultivation, and Sequencing Lake Zhangnai is situated on the Tibetan Plateau with a maximal aquatic depth of 2.7 m (Figure 1).Water temperature, conductivity, salinity, and pH were measured using a 6600 Multi-Parameter Water Quality Sonde (YSI Inc., Yellow Springs, OH, USA).The lake surface waters were collected in 2017 and subjected to pre-filtration using a 20 µm mesh to exclude large particles and eukaryotic organisms.Bacterial isolation and genomic sequencing were conducted following our previously established protocol [15].Briefly, the collected lake water was sprayed onto plates containing modified LB medium consisting of 2 g/L tryptone, 1 g/L yeast extract, 7 g/L NaCl, and 20 g/L agar.The four novel Yoonia strains were isolated through serial dilutions and cultured on modified LB plates.Genomic DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) and subsequently sequenced on the Illumina Miseq platform (Shanghai Biozeron Biotechnology Co., Ltd., Shanghai, China). Genomic Assembly and Annotation Raw reads derived from Illumina sequencing were quality-trimmed using Trimmomatic v0.39 [16] with the parameters 'SLIDINGWINDOW:4:15 MAXINFO:40:0.9MINLEN:40 .Assembly was performed using SPAdes v3.14.0 [17] with the '-careful' parameter.Contigs exceeding 1000 bp in length and 5× in sequencing depth were retained for further analysis.The genome size, GC content, and coding density were estimated using CheckM v1.1.3[18].To minimize biases from varying gene prediction software employed in previous studies, protein-coding genes were re-predicted using Prokka v1.14.6 [19] for the four novel Yoonia isolates and 99 reference Roseobacter genomes.Functional annotation of these protein sequences was conducted against the KEGG database using the BlastKOALA (https://www.kegg.jp/blastkoala/,accessed on in 1 June 2023) [20]. Genomic Assembly and Annotation Raw reads derived from Illumina sequencing were quality-trimmed using Trimmomatic v0.39 [16] with the parameters 'SLIDINGWINDOW:4:15 MAXINFO:40:0.9MINLEN:40′.Assembly was performed using SPAdes v3.14.0 [17] with the '-careful' parameter.Contigs exceeding 1000 bp in length and 5× in sequencing depth were retained for further analysis.The genome size, GC content, and coding density were estimated using CheckM v1.1.3[18].To minimize biases from varying gene prediction software employed in previous studies, protein-coding genes were re-predicted using Prokka v1.14.6 [19] for the four novel Yoonia isolates and 99 reference Roseobacter genomes.Functional annotation of these protein sequences was conducted against the KEGG database using the BlastKOALA (https://www.kegg.jp/blastkoala/,accessed on in 1 June 2023) [20] . Construction of Phylogenomic Trees For the construction of Roseobacter phylogeny, a set of 120 single-copy genes (bac120) were identified, aligned, and trimmed (Supplemental Text S1) using GTDB-tk v1.7.0 [21].These bac120 genes are universally conserved and have undergone minimal recombination events, thus providing a reliable framework for phylogenetic analysis [22].GTDB-tk identified the bac120 genes through a hidden Markov model (HMM) searching against a defined reference database.The identified gene sequences were then aligned with a pre-existing alignment of over 62,293 bacterial genomes, followed by a trimming process [21].The phylogenomic tree was built using IQ-TREE v2.2.0 [23], with Construction of Phylogenomic Trees For the construction of Roseobacter phylogeny, a set of 120 single-copy genes (bac120) were identified, aligned, and trimmed (Supplemental Text S1) using GTDB-tk v1.7.0 [21].These bac120 genes are universally conserved and have undergone minimal recombination events, thus providing a reliable framework for phylogenetic analysis [22].GTDB-tk identified the bac120 genes through a hidden Markov model (HMM) searching against a defined reference database.The identified gene sequences were then aligned with a pre-existing alignment of over 62,293 bacterial genomes, followed by a trimming process [21].The phylogenomic tree was built using IQ-TREE v2.2.0 [23], with ModelFinder [24] assigning the most appropriate substitution model for IQ-TREE analysis (LG + R9).A total of 1000 bootstrap replicates were sampled to assess the robustness of the phylogeny.Additionally, the phylogeny was validated using RAxML v8.2.12 [25], with ModelTest-NG [26] assigning the most appropriate substitution model for RAxML analysis (LG + I + G4 + F).Pairwise comparisons of average nucleotide identity (ANI) were computed using FastANI v1.3 [27]. Metabolic Comparisons The four novel Yoonia isolates are postulated to have transitioned from marine environments to lake ecosystems on the Tibetan Plateau.To elucidate the metabolic adaptations accompanying this ecological shift, we reconstructed the metabolic profiles of the ancestral nodes relevant to the Loktanella, Cognatiyoonia, and Yoonia genera and also forecasted KEGG orthology (KO) acquisition and deletion events for each ancestral node.This analysis was performed using BadiRate v1.35 [28] with the parameters '-anc-bmodel FR-rmodel BDI-ep CSP'.A pruned phylogenetic subtree and the KO count per genome were employed as input data.The metabolic comparisons were further performed between marine and non-marine lineages within the Loktanella, Cognatiyoonia, and Yoonia genera by using the 'binaryPGLMM' function in the 'ape' R package v5.6 [29]. Results and Discussion Genomic Characterization of Novel Roseobacteraceae Strains Four novel Roseobacteraceae strains were isolated in 2017 from the surface waters of Lake Zhangnai on the Tibetan Plateau (Figure 1).These strains were taxonomically classified within the Yoonia genus (Figure 2A), which was a recently reclassified genus originally part of the Loktanella genus [13].The phylogenetic topology of these strains was confirmed using both IQ-TREE v2.2.0 (Figure 2A) and RAxML v8.2.12 (Figure S1).It is worth noting that many published Roseobacteraceae strains have been misidentified within their genus classification due to the poor resolution of 16S rRNA genes in the Roseobacteraceae phylogeny [13].To mitigate this issue, we used the genome IDs from the NCBI database and marked the corrected genus assignments [13] for reference genomes in the phylogenetic tree (Figure 2B,C).The novel strains formed two distinct clusters.Within each cluster, the genomes exhibited a nearly identical 16S rRNA gene identity and an average nucleotide identity (ANI) (Figure 2B,C), suggesting the homogenetic wild population in lacustrine environments.These four isolates exhibited a 16S rRNA gene identity ranging from 99.1% to 99.5% and an ANI ranging from 82.9% to 89.3% when compared to previously published genomes.The latter metric fell below the established ANI threshold of 95% that delineates a novel bacterial species [27]. The draft genomes of these isolated strains comprised 10-80 contigs, with N50 values spanning from 231 Kb to 770 Kb (Table 1).Strains designated as Yoonia sp.72 and Yoonia sp.76 possessed genome sizes of approximately 3.66 Mb, a GC content of 61.7%, and a coding density of 91.6% (Figure 2A and Table 1).These genomes encoded around 3522 genes, which formed ~1678 KEGG orthologs (KOs) according to the KEGG database.In contrast, strains Yoonia sp.67 and Yoonia sp.67-2 exhibited slightly larger genomes (~3.8 Mb) and a lower GC content (90.6% and 91.3%) and coding density (90.6% and 91.3%).These two genomes contained 3673 and 3717 genes that encoded 1700 and 1714 KOs, respectively.Collectively, these genomic attributes aligned well with the ranges reported for published Yoonia genomes, which have genome sizes of 3.1-4.7 Mb, GC content of 53.4-61.8%,and coding densities of 86.5-92.4%. Habitat Transition from Marine to Lacustrine Environments Roseobacteraceae members, including those from the Loktanella, Cognatiyoonia, and Yoonia genera, are known for their ecological preference for marine and intertidal environments [30].However, several isolates have also been discovered in non-marine habitats, such as inland saline waters and Antarctic lakes, indicating a possible ecological transition from marine to lacustrine habitats [31].The four novel Yoonia isolates presented herein were sampled from a brackish lake on the Tibetan Plateau, thereby expanding the genomic resources available for non-marine lineages.To identify metabolic functions enriched in either marine or non-marine lineages, we employed a 'binaryPGLMM' analysis that accounts for the phylogenetic branching order and evolutionary history in the metabolic comparisons [29].Our analysis revealed two genes related to the degradation of malonyl-CoA and taurine that were enriched in marine lineages (Figure 3).Conversely, eighteen genes or operons were found to be enriched in lacustrine members, many of which were associated with the utilization of various compounds such as cellulose, peptide, arginine, hydroxyproline, and chlorobenzene.Intriguingly, the enrichment of genes related to chlorobenzene degradation, specifically 2,4-dichlorophenol 6-monooxygenase (tfdB), maleylacetate reductase(tfdF), and hydroxyquinol 1,2-dioxygenase (chqB), contrasted with the previously reported loss of 2-haloacid dehalogenase in non-marine Roseobacters [14].This discrepancy might be attributed to the prevalent presence of organohalogens in cold environments like Antarctic lakes [32].Collectively, our findings suggest that the differentiated utilization of nutritional substrates is a key adaptive feature in lacustrine lineages within the Loktanella, Cognatiyoonia, and Yoonia genera. Some of these metabolic enrichments in non-marine lineages were largely attributed to the KO presence in the four novel Yoonia isolates.For example, four genes were identified only in novel Yoonia isolates but not in previous published reference genomes, including those encoding toxin resistance two-component system (adeSR), endoglucanase for cellulose degradation (K01179), restriction system (mrr), and fusion protein for cell signaling (secDF).Next, we further explored the metabolic adaptations accompanying the ecological transition for these novel isolates.We analyzed the ancestral genomes of the Loktanella, Cognatiyoonia, and Yoonia genomes.The last common ancestor (LCA) of strains Y. sp.72 and Y. sp.76 (NODE41 in Figure 3) was estimated to harbor 1678 KOs and subjected to a net gain of 68 KOs (a gain of 77 KOs and a loss of 9 KOs).A similar trend of net KO gain was also identified at the LCA of strains Y. sp.67 and Y. sp.67-2 (NODE45 in Figure 3), which possessed 1699 KOs and experienced a net gain of 89 KOs (a gain of 133 KOs and a loss of 44 KOs). One of the most crucial differences between marine and non-marine aquatic ecosystems is the variation in salinity levels.Such ecological transitions often involve alterations in genes linked to osmoregulation and ion transport [9].For example, sodium antiporters have been documented to be consistently lost in many non-marine Rhodobacteraceae, while the biosynthesis pathway for ectoine is significantly enriched in marine Roseobacteraceae [14].In the case of the newly identified Yoonia strains, only two salinity-related genes were found to be lost in either NODE41 or NODE45, while no metabolic alterations related to sodium and potassium transport were identified.Intriguingly, the gene clusters ectABC and treYZ-otsAB, which are responsible for the synthesis of ectoine and trehalose, were estimated to be acquired at NODE45 and NODE41, respectively.Both ectoine and trehalose function as compatible solutes that confer resilience to extreme osmotic stress [33].Given that these novel Yoonia strains were isolated from brackish environments with a salinity of 0.27% and given the prevalence of brackish and saline lakes across the Tibetan Plateau [34], these metabolic acquisitions likely equipped these strains to withstand a wider range of salinity conditions, potentially enabling them to colonize a more diverse array of lake environments on the Plateau.2. Metabolic comparisons between marine and non-marine genomes were performed using 'binaryPGLMM' function in the 'ape' R package v5.6.Ancestral genomic reconstructions were conducted using BadiRate v1.35.Each ancestral node is labeled with a triplet of numbers, denoting the aggregate count of KEGG orthologs (KOs) at that node, as well as the numbers of KOs gained and lost.Ancestral nodes of strains sp.72 and sp.76 and of strains sp.67 and sp.67-2 are labeled as NODE41 and NODE45, respectively.Isolates sampled from Lake Zhangnai on the Tibetan Plateau, lacustrine environments, and marine environments are colored in red, green, and blue in their genome IDs, respectively.The lower panel illustrates the phyletic distribution of selected genes, where filled and Figure 3. Ancestral metabolic profiling of Loktanella, Cognatiyoonia, and Yoonia genomes.The phylogenomic tree displayed in the upper panel is a replication of that presented in Figure 2. Metabolic comparisons between marine and non-marine genomes were performed using 'binaryPGLMM' function in the 'ape' R package v5.6.Ancestral genomic reconstructions were conducted using BadiRate v1.35.Each ancestral node is labeled with a triplet of numbers, denoting the aggregate count of KEGG orthologs (KOs) at that node, as well as the numbers of KOs gained and lost.Ancestral nodes of strains sp.72 and sp.76 and of strains sp.67 and sp.67-2 are labeled as NODE41 and NODE45, respectively.Isolates sampled from Lake Zhangnai on the Tibetan Plateau, lacustrine environments, and marine environments are colored in red, green, and blue in their genome IDs, respectively.The lower panel illustrates the phyletic distribution of selected genes, where filled and open circles signify the presence and absence, respectively, of the corresponding KOs.KOs enriched in marine and non-marine lineages are colored in blue and green, respectively, in the gene abbreviations.Gains and losses of KOs in novel Yoonia genomes in the ancestral reconstructions are also marked.The gene abbreviations and the corresponding functions are listed in Table S1. Transport functions stand as another key metabolic divergence between marine and lacustrine microbes [9].In the case of LCA NODE45, the gene cluster cysAUWP, which is responsible for sulfate transport, was gained, which aligned well with previous research [9] and the significantly lower sulfate concentrations in lake environments [35].Additionally, NODE45 acquired gene cluster lhpMNOP, which is responsible for hydroxyproline transport.Hydroxyproline is a major component of animal collagen, which may be more readily available in terrestrial niches.In contrast, NODE45 and NODE41 each lacked gene cluster somEFG, which is associated with polyol transport, and gene cluster ABC.FEV, which is responsible for iron complex transport, the latter of which was consistent with the generally higher bioavailability of iron in lacustrine ecosystems [36]. The comparative genomic analysis between marine Roseobacteraceae and lacustrine Rhodobacteraceae has previously identified other metabolic pathways typically lost in the latter, such as mercury antitoxin, carbon monoxide oxidation, and de novo cobalamin synthesis [14].However, these metabolic changes were not identified in the 'binaryPGLMM' comparison or in the ancestral genomes of the newly identified Yoonia strains.Instead, strains Y. sp.72 and Y. sp.76 from Tibetan Plateau lakes exhibited enhanced metabolic capabilities for degrading aromatic compounds, as evidenced by the acquisition of the gene clusters ligBM, galBCD, and xylCE.These compounds are likely introduced into the lake water from the surrounding soil and plant residues through precipitation or glacial melt [37].Additionally, all four novel strains acquired genes associated with restriction and defense systems, which may facilitate their adaptation to the extreme conditions of the Tibetan Plateau [38].Taken together, these metabolic shifts diverged from other previous research, suggesting that adaptation to lacustrine environments may be phylogenetically specific and depend on the scope of genome samplings. Conclusions Given the large population sizes and high dispersal abilities of aquatic microbes, the genomic differentiation between marine and non-marine habitats remains one of the most enigmatic questions in ecological fields [9].In this study, we isolated four novel Yoonia strains from a brackish lake on the Tibetan Plateau.Diverging from their marine counterparts within the same genus, these strains have transitioned from their original marine habitats to lacustrine niches.Serving as a supplement to previously reported non-marine Loktanella and Yoonia lineages, these new strains enrich the existing genomic repertoire for studying the microbial adaptations to lacustrine ecosystems within the genus level.These novel strains share certain metabolic changes commonly observed in other non-marine Rhodobacteraceae (e.g., specialized compound transport) and in non-marine Loktanella and Yoonia lineages (e.g., sulfate transport).Additionally, they display unique metabolic adaptations, such as aromatic compound degradation and defense systems.These specialized adaptations may reflect localized responses to Tibetan Plateau environments.Taken together, our study broadens the known genomic information of non-marine Roseobacter lineages and illuminates their ecological transitions characterized by both shared and unique metabolic adaptations. Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms11112817/s1, Figure S1.Phylogenomic tree constructed using RAxML v8.2.12.Nodes with bootstrap support exceeding 80% are indicated by solid circles.Table S1.Metabolic alterations leading to the last common ancestor (LCA) of strains Y. sp.72 and Y. sp.76 (NODE41) and the LCA of strains Y. sp.67 and Y. sp.67-2 (NODE45).Text S1: Sequence of the bac120 alignment. Author Contributions: Software, X.F.; Formal analysis, X.F.; Resources, P.X.; Writing-original draft, X.F.; Writing-review & editing, X.F. and P.X.; Visualization, X.F.; Project administration, P.X.; Funding acquisition, X.F. and P.X.All authors have read and agreed to the published version of the manuscript. Microorganisms 2023 , 10 Figure 1 . 2023101 Figure 1.Geographical location and ecological characteristics of Lake Zhangnai on the Tibetan Plateau. Figure 1 . 1 Figure 1.Geographical location and ecological characteristics of Lake Zhangnai on the Tibetan Plateau. Figure 2 . 2 Figure 2. Phylogenomic placement of novel Yoonia strains.The phylogenomic tree was constructed based on 120 conserved single-copy genes using IQ-TREE v2.2.0 with the 'LG + R9′ substitution model.Nodes with bootstrap support exceeding 95% are indicated by solid circles.(Panel A) presents the Roseobacter phylogeny.Sampling locations of Loktanella, Cognatiyoonia, and Yoonia genomes are marked next to their genome IDs.Isolates sampled from Lake Zhangnai on the Tibetan Figure 2 . 2 Figure 2. Phylogenomic placement of novel Yoonia strains.The phylogenomic tree was constructed based on 120 conserved single-copy genes using IQ-TREE v2.2.0 with the 'LG + R9 substitution model.Nodes with bootstrap support exceeding 95% are indicated by solid circles.(Panel A) presents the Roseobacter phylogeny.Sampling locations of Loktanella, Cognatiyoonia, and Yoonia genomes are marked next to their genome IDs.Isolates sampled from Lake Zhangnai on the Tibetan Plateau, lacustrine environments, and marine environments are colored in red, green, and blue in their genome IDs, respectively.(Panel B) shows the pairwise comparisons of 16S rRNA gene similarities among Yoonia and Loktanella genomes.Genus reassignments [13] are annotated alongside the similarity heatmaps.(Panel C) illustrates the pairwise comparisons of average nucleotide identities (ANIs) with values below 80% not shown. Figure 3 . 3 Figure 3. Ancestral metabolic profiling of Loktanella, Cognatiyoonia, and Yoonia genomes.The phylogenomic tree displayed in the upper panel is a replication of that presented in Figure2.Metabolic comparisons between marine and non-marine genomes were performed using 'binaryPGLMM' function in the 'ape' R package v5.6.Ancestral genomic reconstructions were conducted using BadiRate v1.35.Each ancestral node is labeled with a triplet of numbers, denoting the aggregate count of KEGG orthologs (KOs) at that node, as well as the numbers of KOs gained and lost.Ancestral nodes of strains sp.72 and sp.76 and of strains sp.67 and sp.67-2 are labeled as NODE41 and NODE45, respectively.Isolates sampled from Lake Zhangnai on the Tibetan Plateau, lacustrine environments, and marine environments are colored in red, green, and blue in their genome IDs, respectively.The lower panel illustrates the phyletic distribution of selected genes, where filled and Table 1 . 1 Genomic features of four novel Yoonia isolates. Genome IDContigsN50 (bp)Genome Size (bp)GC ContentCoding DensityGenesYoonia sp. 7210770,9703,664,10961.7%91.6%3524Yoonia sp. 7616704,7013,665,67261.7%91.6%3522Yoonia sp. 67-280299,2403,855,34959.2%90.6%3717Yoonia sp. 6741231,9473,767,97159.4%91.3%3673 Microorganisms 2023, 11, x FOR PEER REVIEW Microorganisms 2023,11, 2817 Data Availability Statement:The assembled contigs of the four novel Yoonia strains are available in the NCBI database under the accession number PRJNA1026722 and the NODE database (https://www.biosino.org/node/)accessed on 13 November 2023 under the accession number OED866054-OED866057.Conflicts of Interest:The authors declare no conflict of interest.Funding: This study was supported by the National Natural Science Foundation of China U2102216 (P.X.) and 92251304 (P.X.); the Second Tibetan Plateau Scientific Expedition and Research Program (STEP) 2019QZKK0503 (P.X.) and 2021QZKK0100 (P.X.); the China Postdoctoral Science Foundation 2022M712195 (X.F.); and the Guangdong Basic and Applied Basic Research Foundation 2023A1515012162 (X.F). 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Fluctuations in Upper and Lower Body Movement during Walking in Normal Pressure Hydrocephalus and Parkinson's Disease Assessed by Motion Capture with a Smartphone Application, TDPT-GT 18 November 2023 Chifumi Iseki chi.iseki@gmail.com 0000-0001-9953-7323 Department of Behavioral Neurology and Cognitive Neuroscience Tohoku University Graduate School of Medicine 980-8575SendaiJapan Division of Neurology and Clinical Neuroscience Department of Internal Medicine III Yamagata University School of Medicine 990-2331YamagataJapan Shou Suzuki 0000-0001-9953-7323 Department of Informatics Faculty of Engineering Yamagata University 992-8510YonezawaJapan Tadanori Fukami fukami@yz.yamagata-u.ac.jp Department of Informatics Faculty of Engineering Yamagata University 992-8510YonezawaJapan Shigeki Yamada shigekiyamada393@gmail.com 0000-0001-7158-5569 Department of Neurosurgery Nagoya City University Graduate School of Medical Sciences 467-8601NagoyaJapan Interfaculty Initiative in Information Studies Institute of Industrial Science The University of Tokyo 113-8654TokyoJapan Normal Pressure Hydrocephalus Center Rakuwakai Otowa Hospital 607-8062KyotoJapan Tatsuya Hayasaka hayasakatatsuya1101@gmail.com 0000-0003-1508-6938 Department of Anesthesiology Yamagata University School of Medicine 990-2331YamagataJapan Toshiyuki Kondo 0000-0003-1508-6938 Division of Neurology and Clinical Neuroscience Department of Internal Medicine III Yamagata University School of Medicine 990-2331YamagataJapan Masayuki Hoshi mhoshi@fmu.ac.jp Department of Physical Therapy Fukushima Medical University School of Health Sciences 10-6 Sakaemachi960-8516FukushimaJapan Shigeo Ueda uedashigeo@yahoo.co.jp Shin-Aikai Spine Center Katano Hospital 576-0043KatanoJapan Yoshiyuki Kobayashi kobayashi-yoshiyuki@aist.go.jp Human Augmentation Research Center Kashiwa II Campus National Institute of Advanced Industrial Science and Technology (AIST) University of Tokyo 277-0882KashiwaJapan Masatsune Ishikawa Normal Pressure Hydrocephalus Center Rakuwakai Otowa Hospital 607-8062KyotoJapan Rakuwa Villa Ilios Rakuwakai Healthcare System 607-8062KyotoJapan Shigenori Kanno s-kanno@med.tohoku.ac.jp Department of Behavioral Neurology and Cognitive Neuroscience Tohoku University Graduate School of Medicine 980-8575SendaiJapan Kyoko Suzuki Department of Behavioral Neurology and Cognitive Neuroscience Tohoku University Graduate School of Medicine 980-8575SendaiJapan Yukihiko Aoyagi y.aoyagi@digital-standard.com Digital Standard Co., Ltd 536-0013OsakaJapan Yasuyuki Ohta yasuyuki@med.id.yamagata-u.ac.jp Division of Neurology and Clinical Neuroscience Department of Internal Medicine III Yamagata University School of Medicine 990-2331YamagataJapan Fluctuations in Upper and Lower Body Movement during Walking in Normal Pressure Hydrocephalus and Parkinson's Disease Assessed by Motion Capture with a Smartphone Application, TDPT-GT 18 November 20232865BF84F5E24929C0D1F7F0B8E0DE9A10.3390/s23229263Received: 26 October 2023 Revised: 17 November 2023 Accepted: 17 November 2023fractality1/f noisesmartphone devicegait analysisneurodegenerative diseases We aimed to capture the fluctuations in the dynamics of body positions and find the characteristics of them in patients with idiopathic normal pressure hydrocephalus (iNPH) and Parkinson's disease (PD).With the motion-capture application (TDPT-GT) generating 30 Hz coordinates at 27 points on the body, walking in a circle 1 m in diameter was recorded for 23 of iNPH, 23 of PD, and 92 controls.For 128 frames of calculated distances from the navel to the other points, after the Fourier transforms, the slopes (the representatives of fractality) were obtained from the graph plotting the power spectral density against the frequency in log-log coordinates.Differences in the average slopes were tested by one-way ANOVA and multiple comparisons between every two groups.A decrease in the absolute slope value indicates a departure from the 1/f noise characteristic observed in healthy variations.Significant differences in the patient groups and controls were found in all body positions, where patients always showed smaller absolute values.Our system could measure the whole body's movement and temporal variations during walking.The impaired fluctuations of body movement in the upper and lower body may contribute to gait and balance disorders in patients. Introduction Describing and measuring the gaits of patients have long been clinical challenges.In previous studies, terms describing pathological gait, such as short stepped, shuffling, freezing, wide based, festination, hemiplegic, spastic, ataxic, and instability, have been useful and identifiable characteristics for diagnosing diseases, such as Parkinson's disease (PD) [1][2][3][4], cerebrospinal degeneration [4][5][6], and idiopathic normal pressure hydrocephalus (iNPH) [7][8][9][10].These characteristics are observed in aspects such as the length, speed, timing, and laterality of movement in the feet and legs within the range of human visual perception.Clinicians learn from textbooks and examine patients for many years; however, we are often unable to reach an agreement on descriptions of gait [8].The quantitative dynamic evaluation of gait has also been difficult because of the uncertainty of human vision.Therefore, studies have tried to assess the cycle and variability of gait with various sensors, such as floor sheets [5,6,11], wearable devices [12,13], and motion-capture systems [14,15]. To introduce our method for measuring gait dynamics, let us mention the fluctuations found in nature.Nature hosts some intriguing examples of self-similar structures, such as the Roman cauliflower, in which almost exact copies of the entire flower may be recognized on multiple smaller scale examples, and the correlations between the frequency and size of the copy structure express the 1/f pattern [16].We can also observe the same pattern in many biological phenomena and complex systems in nature [17][18][19].Human motion is diverse and contains inherent variability, yet it is not entirely random.For instance, when standing still in a relaxed state, the swaying motion exhibits fractal, 1/f properties [20], exhibiting a roughly proportional relationship between log power and log frequency in a spectral power analysis.In other words, 1/f relations refer to self-similar fluctuations observed across multiple time scales, indicating fractal patterns.By applying fractality, healthy and pathological gaits, as well as their differences, have been explored in the field of bioengineering [21][22][23][24][25][26][27][28][29].These studies assessed the dynamic aspect of gait, with a focus on inconsistent fluctuation, which was different from classical quantitative analysis centered on calculating averages of key parameters, such as stride length, swing, and speed, to establish stable and consistent patterns in gait.Hausdorff et al. proposed watching the stride-to-stride fluctuations while considering that the fluctuations are not just noise but convey important information [21,30].In healthy adults, the fluctuations from stride to stride are not completely random; instead, each stride time is related to adjacent stride times and to stride times hundreds of strides later [22].The distribution of strides in a healthy walk has a 1/f-like structure [31].This pattern of fluctuation (noise) has also been referred to as pink noise.In contrast, noise that is unhealthy, unstable, and lacks a fractal pattern is referred to as white noise. The advancements in these sensors and methods for capturing the dynamics of human body movements have significantly contributed to the current wealth of knowledge.As a result, our goal is to make these beneficial sensors as easily accessible as possible for patients in need.The system we employed was a smartphone-based markerless motioncapture system using artificial intelligence (AI), named TDPT-GT (Three-Dimensional Pose Tracker for Gait Test), which essentially consisted of taking videos of the patients on an iPhone [32][33][34].A noninvasive motion-capture application allowed us to use this system in the general room for outpatients.The patient setup required only several minutes [32], and, for the analysis, a minimal recording time of a few seconds was sufficient, as seen in the current study.Among recent gait-analysis methods, our system stands out for its superior availability and convenience in clinical applications [34].Hence, we aimed to establish a connection between the application of this measurement tool [32] and its meaningful clinical applications.The initial trial involved extracting two-dimensional characteristics of pathological gait [33], while the second trial delved into the use of AI for distinguishing various gait patterns [34].This manuscript represents our third publication, focusing on sensing the pathology of the temporospatial aspect of patients' gait. The role of the trunk is recognized as one of the key components of the gait system, with many studies having been conducted on this aspect [35][36][37][38][39][40][41].Trunk lean [35], pos-Sensors 2023, 23, 9263 3 of 15 ture [36,37], balance [38][39][40], and muscle power [41] are all associated with maintaining a healthy walking mechanism.Disturbed trunk function results in slowness of gait [37] and falls [38,41].Additionally, the movement of the upper limbs in patients with various diseases has been suggested to be linked to gait disturbance [42][43][44][45][46]. Usually, independent analyses-for example, examinations of writing or hand-to-mouth movements-were conducted, and the connection between the upper and lower limbs was deliberated [42][43][44].Some studies using motion capture evaluated the ranges of motion of hands, elbows, or arms during walking; however, these could not include the temporospatial concepts of the movements of each part [45,46].Hence, for gait analysis, we aim for an inclusive examination of body parts, including the trunk and upper limbs, while also being able to observe the time course during walking. The gaits of patients with PD have been the most common ones subjected to study, mainly using the methods mentioned above to evaluate the fluctuation in motion [22,24,26,28,31].In addition to reduced stride length and gait variability, PD patients showed impaired fractal scaling of the gait [24,26,28], which means that the normal healthy noise (fluctuation) captured during gait cycles was decreased in PD patients.The same tendency was also seen in aged people [29], patients with multiple sclerosis [23], and those with multiple-system atrophy [28].Although the mechanism for changing this fluctuation during gait was not unclear, this approach has an impact on the research on gait dynamics, considering the underlying neural systems [19,22,25,31] and the development of rehabilitation strategies [26].We used this method because it has the potential to assess gait dynamics using data from our system, including up to 27 body positions.This approach enabled us to evaluate the upper body's movements, a previously unexplored area during walking. On the other hand, the gaits of iNPH patients have not been assessed based on their fluctuation in dynamics.The pathological gait specific to iNPH is characterized by freezing gait, wide-based gait, short steps (or senile gait with reduced stride length), shuffling gait (diminished step height), instability (unsteady gait), gait festination, difficulty changing direction, and difficulties in standing up [1,8,47,48].Video-recorded gait performance before and after the spinal tap test and shunt surgery has been recommended for gait assessments in patients with iNPH [8,47].We have prioritized the examination of the iNPH gait [33], primarily due to the fact that the features and diagnosis of gait in iNPH remain unclear, often being referred to as just "gait disturbance".This time, we could focus on the gaits of iNPH patients to find their characteristics and the difference compared to PD patients, and on utilizing a convenient system that requires only a brief recording of a few seconds. The study aimed to identify temporospatial abnormalities during walking-in patients with iNPH and PD, as well as to discern the differences between the two diseases.Additionally, the study sought to explore the measurement and analysis of whole-body movement using a noninvasive iPhone application, making it an easily accessible tool for all. Materials and Methods Ethical Approvals The study design and protocol of this study were approved by the ethics committee for human research at Nagoya City University Graduate School of Medical Science (IRB number: 60-22-0111), Shiga University of Medical Science (R2019-337), and Yamagata University School of Medicine (protocol code: 2021-10).All volunteers and patients participated in this study after providing written informed consent.The study design was prospective and observational.This study was conducted according to the approved guidelines of the Declaration of Helsinki. Study Population We collected information on age, disease history, and gait from May 2021 to November 2022 at Yamagata University Hospital and Takahata Town Hospital.The subjects with PD consisted of 23 patients with clinically established PD diagnosed according to the MDS clinical criteria for Parkinson's disease [49].Patients with iNPH consisted of 23 probable or definite iNPH diagnosed according to the Japanese guidelines and the management of iNPH (3rd edition) [9].The controls were 92 healthy volunteers who participated in local health checkups and who did not have a neurodegenerative disease.Gait trials of patients were examined several times on different days.The gait of a volunteer was recorded at a specific time.To be included in either group, an individual was required to be capable of walking independently and safely for several minutes; using a single-point cane was the only assistance allowed.This criterion leads to the selection of patients with relatively mild motor symptoms. One iNPH and two PD patients had mild resting and postural hand tremors at a rate of 1 on the Unified Parkinson's Disease Rating Scale (UPDRS).No patients expressing dyskinesia during walking were included.Since the study focused solely on the gait capabilities during the recording time, potential influencing factors on mobility, such as medications, the timing of medication intake, shunt treatment, or other examinations, were not controlled for in this study. Data Acquisition of Estimated Three-Dimensional Relative Coordinates during 1 m Circle Walking To fit the frame of the application, which was placed about 3 m away from the gait trail, the gait of each participant was recorded.They walked in a 1 m circle for 1-3 laps, clockwise and counterclockwise (Figure 1 shows a sample of the gait circle; patients were recorded in the daily consultation room). Study Population We collected information on age, disease history, and gait from May 2021 to November 2022 at Yamagata University Hospital and Takahata Town Hospital.The subjects with PD consisted of 23 patients with clinically established PD diagnosed according to the MDS clinical criteria for Parkinson's disease [49].Patients with iNPH consisted of 23 probable or definite iNPH diagnosed according to the Japanese guidelines and the management of iNPH (3rd edition) [9].The controls were 92 healthy volunteers who participated in local health checkups and who did not have a neurodegenerative disease.Gait trials of patients were examined several times on different days.The gait of a volunteer was recorded at a specific time.To be included in either group, an individual was required to be capable of walking independently and safely for several minutes; using a single-point cane was the only assistance allowed.This criterion leads to the selection of patients with relatively mild motor symptoms. One iNPH and two PD patients had mild resting and postural hand tremors at a rate of 1 on the Unified Parkinson's Disease Rating Scale (UPDRS).No patients expressing dyskinesia during walking were included.Since the study focused solely on the gait capabilities during the recording time, potential influencing factors on mobility, such as medications, the timing of medication intake, shunt treatment, or other examinations, were not controlled for in this study. Data Acquisition of Estimated Three-Dimensional Relative Coordinates during 1 m Circle Walking To fit the frame of the application, which was placed about 3 m away from the gait trail, the gait of each participant was recorded.They walked in a 1 m circle for 1-3 laps, clockwise and counterclockwise (Figure 1 shows a sample of the gait circle; patients were recorded in the daily consultation room).The TDPT-GT application could measure the 3D relative coordinates of the human body at 30 frames per second (fps) with 448 × 448 pixels and RGB color using an iPhone camera without any markers for motion capture.Details of the technology of this application and data acquisition were described in our prior publication [32].The TDPT-GT application estimates the 3D relative coordinates of the following 24 body points: the nose, navel, and bilateral points, such as the eyes, ears, shoulders, elbows, wrists, thumbs, middle fingers, hips, knees, heels, and toes-calculated coordinates of 3 body points-the center of the head, neck, and buttocks.The middle fingers and the thumbs were captured around the metacarpophalangeal joint.We extracted the sequential 128 frames (approximately 4 s) at every point during walking in a 1 m circle, when the data is as stable as possible, except at the beginning and the end of walking.In the study, body positions were classified into the trunk positions (Figure 2): the head, neck, nose, navel, eyes, ears, shoulders, hips, buttocks, and knees, and the limb positions: elbows, wrists, thumbs, middle fingers, heels, and toes (Figure 2).The positions of the upper body were marked above the navel, while the positions of the lower body were below the navel (Figure 2). a 1 m circle (line of red dots) at a slow and comfortable speed.The stick figure is constructed with the 24 body points calculated by the deep leaning system of this application.The non-English word at the right side is the option button for changing the skeleton figure to a doll-like animation figure .The TDPT-GT application could measure the 3D relative coordinates of the human body at 30 frames per second (fps) with 448 × 448 pixels and RGB color using an iPhone camera without any markers for motion capture.Details of the technology of this application and data acquisition were described in our prior publication [32].The TDPT-GT application estimates the 3D relative coordinates of the following 24 body points: the nose, navel, and bilateral points, such as the eyes, ears, shoulders, elbows, wrists, thumbs, middle fingers, hips, knees, heels, and toes-calculated coordinates of 3 body points-the center of the head, neck, and buttocks.The middle fingers and the thumbs were captured around the metacarpophalangeal joint.We extracted the sequential 128 frames (approximately 4 s) at every point during walking in a 1 m circle, when the data is as stable as possible, except at the beginning and the end of walking.In the study, body positions were classified into the trunk positions (Figure 2): the head, neck, nose, navel, eyes, ears, shoulders, hips, buttocks, and knees, and the limb positions: elbows, wrists, thumbs, middle fingers, heels, and toes (Figure 2).The positions of the upper body were marked above the navel, while the positions of the lower body were below the navel (Figure 2).In the definition of the study, the trunk positions are included in the parts of gray color, and the limb positions are the others on the extremities.The distance of each position was calculated by the coordinates from the navel (star) to each position (dots).The positions of the upper body indicated the dots above the navel (star) and those of the lower body were below the navel. Fluctuation in Body Positions during Walking From 128 frames of gait cycles, body-position coordinates were calculated as the distances of 26 body positions from the navel, where the Fourier transform done with Python 3.10.0produced body-position data regarding the length from the navel to the other 26 points.A slope () was obtained from the approximate line of the graph plotted by coordinates with the log of the power spectral density against the log of frequency (Figure 3).In the definition of the study, the trunk positions are included in the parts of gray color, and the limb positions are the others on the extremities.The distance of each position was calculated by the coordinates from the navel (star) to each position (dots).The positions of the upper body indicated the dots above the navel (star) and those of the lower body were below the navel. Fluctuation in Body Positions during Walking From 128 frames of gait cycles, body-position coordinates were calculated as the distances of 26 body positions from the navel, where the Fourier transform done with Python 3.10.0produced body-position data regarding the length from the navel to the other 26 points.A slope ( α) was obtained from the approximate line of the graph plotted by coordinates with the log of the power spectral density against the log of frequency (Figure 3).In this study, the slope (α) of each set of body-position data was defined as a "fluctuation index" based on the previous reports where they mentioned gait fractality [13][14][15].We employed the value of the slope (α) to represent the fluctuation.This process was performed by clinically blinded engineers (S.S. and T.F.). Statistical Analysis For the averages of the slopes in every body position, the differences between PD, iNPH, and controls were tested by one-way ANOVA, of which the Tukey test was used to compare each of the two groups.Statistical analysis was performed by Rcmr version 2.8-0 on R version 4.2.2, and the significant level was defined as 5%. Results Clinical Characteristics The total gait trials were 117, 56, and 184 for 23, 23, and 92 patients with iNPH, PD, and the controls, respectively (Table 1).Their average ages and standard deviations were 77.0 ± 6.4, 70.1 ± 6.0, and 72.3 ± 6.3, respectively, and they were not age-matched against both patient groups.Symptoms of iNPH were clinically known to fluctuate with the time In this study, the slope (α) of each set of body-position data was defined as a "fluctuation index" based on the previous reports where they mentioned gait fractality [13][14][15].We employed the value of the slope (α) to represent the fluctuation.This process was performed by clinically blinded engineers (S.S. and T.F.). Statistical Analysis For the averages of the slopes in every body position, the differences between PD, iNPH, and controls were tested by one-way ANOVA, of which the Tukey test was used to compare each of the two groups.Statistical analysis was performed by Rcmr version 2.8-0 on R version 4.2.2, and the significant level was defined as 5%. Results Clinical Characteristics The total gait trials were 117, 56, and 184 for 23, 23, and 92 patients with iNPH, PD, and the controls, respectively (Table 1).Their average ages and standard deviations were 77.0 ± 6.4, 70.1 ± 6.0, and 72.3 ± 6.3, respectively, and they were not age-matched against both patient groups.Symptoms of iNPH were clinically known to fluctuate with the time and day; therefore, we collected several trials from an individual, each with a different date. Fluctuation Index for Each Body Position In the average of the slope (α), as the fluctuation index for each body position, significant differences were found between iNPH and the controls (p < 1 × 10 −7 ) in all positions and those between PD and the controls (at most p < 1 × 10 −3 ) in all positions.All absolute indices in the patient groups were smaller than those of the controls (Table 2).In the comparison between iNPH and PD, significant differences were found in seven upper body positions and eight lower body positions (p < 0.05) (Table 2 and Figure 4), and the differences were spreading to the right-hand area (the wrist, the middle finger, and the thumb) (Figure 4).The order of lows was the ascending averages of absolute fluctuation indices in the controls.The significant differences between iNPH and the controls were with p < 1 × 10 −7 in all positions, and those between PD and the controls were at least with p < 1 × 10 −3 in all positions.In the comparison between iNPH and PD, significant differences were found in 7 upper body positions and 8 lower body positions (* p < 0.05).iNPH, idiopathic normal pressure hydrocephalus; PD, Parkinson's disease. The absolute values in both the upper and lower limbs were smaller than those in the trunk (Figure 5).The absolute values were consistently smaller during circle walking for individuals with iNPH, PD, and the control group in all body positions.Significantly smaller absolute values for iNPH than those for PD were found in seven positions in the upper body and eight positions in the lower body (Figure 5).The order of lows was the ascending averages of absolute fluctuation indices in the controls.The significant differences between iNPH and the controls were with p < 1 × 10 −7 in all positions, and those between PD and the controls were at least with p < 1 × 10 −3 in all positions.In the comparison between iNPH and PD, significant differences were found in 7 upper body positions and 8 lower body positions (* p < 0.05).iNPH, idiopathic normal pressure hydrocephalus; PD, Parkinson's disease. R. toe Discussion In this study, significant random fluctuations during walking were found in all body positions of iNPH and PD patients compared to the controls.Pathological fluctuation patterns, in other words, random fluctuation patterns, were observed even in the limb positions of the upper body.We could measure the pathology of the temporospatial aspect of Discussion In this study, significant random fluctuations during walking were found in all body positions of iNPH and PD patients compared to the controls.Pathological fluctuation patterns, in other words, random fluctuation patterns, were observed even in the limb positions of the upper body.We could measure the pathology of the temporospatial aspect of patients' gaits with a handy motion-capture application on a single iPhone. Analysis of Gait by the System of TDPT-GT For gait analysis, various sensors, such as pedobarography, motion capture, floor sensors, and wearable sensors, have been utilized.Optical motion-capture systems typically require the attachment of several markers to the body and the use of multiple cameras for data collection [1][2][3][4][5][6][7][8].Time-consuming preparation, recording, and the need for large laboratory space limit their use in clinical settings and hospitals.Although wearable devices [7,[16][17][18] are commonly used, they are often incapable of capturing information from multiple body parts simultaneously.Clinical demands have highlighted the necessity of a sensor that is noninvasive, requires short recording times, occupies minimal space for examination, and provides comprehensive systemic information about the body.TDPT-GT, a markerless motion-capture system, meets these requirements by enabling gait recording by capturing videos on an iPhone and instantly generates coordinates for 27 body points. Utilizing TDPT-GT, this study aimed to identify temporospatial abnormalities in the gaits of patients with neurodegenerative diseases.In the realm of time-series analysis, detrended fluctuation analysis (DFA) is a statistical method that evaluates the self-affinity of a signal and was originally rooted in physics.Its application extends across diverse research domains, including gene analysis [50].To quantify how the dynamics fluctuate over time while walking, DFA was used for gait analysis in the 1990s [51] and also heartrate analysis [52].These fluctuations are described as being (1) uncorrelated white noise, (2) long-range correlations with a power-law scaling called pink noise [51] (or 1/f noise), or (3) Brownian noise [52], corresponding to an intentional random walk (generated in a healthy person or composed of artificially shuffled data).In a normal gait pattern, complex fluctuations of an unknown origin appear as (2), which corresponds to a 1/f-like noise [51].Older people or individuals with disease showed (1) white noise; in other words, their variation of cycles was too random [51,52].In the present study, we did not apply the exact DFA, which needed an integrated time series split into equal boxes, where a least squares line was used to fit the data [51,52].The gait analysis in the present study was also referenced from the DFA.Because our gait data initially exhibited significant variability due to the circular walking of many body positions, we processed a whole of 128 frames (less than 5 s) and gathered information from multiple body positions without overlays in each position.Our approach to data capture and analysis was efficient for comparing the gaits of patients and controls. Our recordings were characterized by individuals walking in circles.This walk was necessary within the hospital's consultation room to facilitate a seamless examination for patients with limited mobility.It was also essential to ensure stable recording within one camera frame.This was atypical in earlier gait research, which primarily relied on an analysis conducted in large laboratory rooms.That made it difficult to compare results with those of the previous gait analysis, which typically involved walking in straight lanes.However, circle walking posed a considerable challenge for patients from a medical perspective.This type of walking includes turning movements that require additional balance tasks beyond simple walking.As a result, circle walking likely induced gait disorders more sensitively than straight walking, making it a potentially valuable assessment tool.Until now, circular walking in conjunction with TDPT-GT has been effective in recording pathological gait, as demonstrated in previous studies [32][33][34].The present study further supports this. We were able to detect the dysfunction in the movement of the trunk and upper body including the hands, during walking with this system of TDPT-GT.The mechanism of trunk control and movement of the upper limbs in older individuals or those with various diseases has been suggested to be linked to gait disturbance [37,38,[41][42][43][44][45][46].The present markerless motion-capture technology enabled the simultaneous measurement of trunk, upper limb, and lower limb movements and allowed for a comprehensive analysis over time. Fluctuation during Walking in Patients with iNPH and PD Our study found that the fluctuation system during walking was more impaired in iNPH patients than in PD patients.The difference in gait between iNPH and PD patients has been discussed in some prior research [1,2]; however, no previous reports assessed fluctuation.Both diseases shared reduced gait velocity, due to a diminished and highly variable stride length [1].Specific features of iNPH were broad-based with outwardly rotated feet, diminished stride length [1,2], gait velocity, and disturbed equilibrium [2].A study assessed a series of iNPH patients and found that about 30% of complications of PD (synucleinopathy) [42].Some patients possibly have comorbid iNPH and PD, which are impossible to differentiate; our study did not include comorbid cases.Regarding gait dynamics, we found that the fluctuated body movement of iNPH patients tended to be more impaired than that in the PD patients, meaning that this iPhone application could possibly be used as a tool for differential diagnosis. What made the difference in fluctuation during walking between iNPH and PD patients?The external cues only mildly improved the gait disturbance in iNPH patients, whereas they were highly effective in raising the stride length and cadence in PD patients [2].In PD patients, walking with fixed-tempo rhythmic auditory stimulation can improve many aspects of gait timing; however, it lowers fractal scaling (away from a healthy 1/f structure) [53], which is an unhealthy pattern.The new, interactive rhythmic auditory stimulation could re-establish healthy fluctuational gait dynamics in PD patients [22].The prevalence of dementia in iNPH patients is higher than that in PD patients; therefore, iNPH patients may be distracted from following the instructions of auditory stimulation.Brain networks are supposed to generate, control, and adjust the fractal function [54] captured by signals such as imaging [55], electroencephalography [56], or connectivity [57] as well as the function expressed in gait [21,30,51].In other words, the healthy brain has fluctuations of 1/f.In contrast to these various biological functions of the brain [54][55][56][57], dementia diminishes the brain construction associated with the fractal formation of neurons and networks [21,30,54,58].From the present study, it can be seen that a greater impairment of the entire brain may lead to more pronounced impaired fluctuations in gait, highlighting distinctions between iNPH and PD. We were surprised to observe random fluctuations in the positions of the upper body, including the hands, among the patient groups during walking.The significance of the right hand in the difference between iNPH and PD patients was probably due to the recording condition during the circular walking without controlling the direction of the rounds; therefore, if we controlled that, the difference might disappear between the left and right hands.Although it was possible that the potential differences in laterality within each disease group could have affected the results, we were unable to conduct a subanalysis due to the lack of precise data on laterality.When evaluating the gait of patients, the upper body or the arms are not often the subject of extensive focus.In particular, the characteristics of gait disturbance in iNPH are known to affect their lower body movements.The characteristics of gait disturbance in iNPH are especially known to affect their lower body movements [8].A few studies have evaluated the upper limb function with a peg board in iNPH patients [59,60], where it was improved along with gait after shunt surgery [60].However, the association between dexterity in the upper limbs and the fluctuation of motor control during walking is still unclear.Our previous study, using the same motion-capture system, used AI to differentiate between pathological gait and normal gait [34], and AI emphasized utilizing the trunk positions of the body [34].Thus, we speculated that the trunk-posture adaptation [61] and/or the subjective vertical position [62] play a role in the movement of total or other body positions and contribute to gait or balance (Figure 6).Kinetic problems, including problems with muscle tone, induce posture impairment [63]; this may be associated with the muscle tonus symptoms of PD and iNPH, such as akinesia, dyskinesia, or paratonia (Figure 6).Although the fractality or fluctuation pattern of the upper limb has not been explored previously, using this system, we were able to detect it among patients with PD and iNPH from temporospatial perspectives. Prospects for Using the Technology The future development of TDPT-GT may include the additional automatic function associated with this fluctuation index.Moreover, incorporating the ability to sense correlations between upper and lower limb indices will contribute to advancements.Optional applications working on TDPT-GT, designed to produce seamless recording and display of indices, empower the users to provide feedback on gait information anytime and anywhere.It is likely to be beneficial for determining overall body balance during walking for all patients and older people. The current application has the potential to be employed in diagnosing or assessing the outcomes after therapy or rehabilitation.This is crucial for preventing falls in patients with iNPH [7,8,10], PD [3,4,64,65], other diseases, or old age [66].Fallers tended to present high consistency with power spectral density in the mediolateral axis [67].The unpredictable body movements experienced during walking by patients can lead to imbalance and potential falls.However, patients may not always express or report these symptoms consistently.Hence, integrating the TDPT-GT application to identify bodily fluctuations could serve as a helpful tool for detecting challenging symptoms.Moreover, these less apparent symptoms require thorough assessment before and after rehabilitation.The existing system can be applied effectively in these specific areas. Limitations The limitations of the study stem from the use of the motion-capture system, which generated estimated 3D-relative coordinates.Previously, we demonstrated the correla- Prospects for Using the Technology The future development of TDPT-GT may include the additional automatic function associated with this fluctuation index.Moreover, incorporating the ability to sense correlations between upper and lower limb indices will contribute to advancements.Optional applications working on TDPT-GT, designed to produce seamless recording and display of indices, empower the users to provide feedback on gait information anytime and anywhere.It is likely to be beneficial for determining overall body balance during walking for all patients and older people. The current application has the potential to be employed in diagnosing or assessing the outcomes after therapy or rehabilitation.This is crucial for preventing falls in patients with iNPH [7,8,10], PD [3,4,64,65], other diseases, or old age [66].Fallers tended to present high consistency with power spectral density in the mediolateral axis [67].The unpredictable body movements experienced during walking by patients can lead to imbalance and potential falls.However, patients may not always express or report these symptoms consistently.Hence, integrating the TDPT-GT application to identify bodily fluctuations could serve as a helpful tool for detecting challenging symptoms.Moreover, these less apparent symptoms require thorough assessment before and after rehabilitation.The existing system can be applied effectively in these specific areas. Limitations The limitations of the study stem from the use of the motion-capture system, which generated estimated 3D-relative coordinates.Previously, we demonstrated the correlations of each coordinate against VICON (Oxford, UK) as part of establishing full reliability and validity [32].However, for the current study, it was necessary to analyze each coordinate of each body position independently, specifically focusing on temporospatial changes.This approach was efficient in assessing the observed differences between the disease groups and the control group.The consistent distinct trends between the disease groups and controls might suggest that the coordinates were suitable for this type of analysis. Conclusions By employing TDPT-GT, a user-friendly motion-capture system application on the iPhone, we succeeded in sensing the disrupted fluctuations in the movement of the entire upper and lower body, the trunk, and limbs, during walking in patients with iNPH and PD.The present study may provide new insights into gait analysis, including whole-body movements and their dynamics for patients. Figure 1 . 1 . 11 Figure 1.Sample picture of the gait recording on the iPhone application, TDPT-GT (Three-Dimensional Pose Tracker for Gait Test).Participants were recorded with a smartphone while walking in Figure 1.Sample picture of the gait recording on the iPhone application, TDPT-GT (Three-Dimensional Pose Tracker for Gait Test).Participants were recorded with a smartphone while walking in a 1 m circle (line of red dots) at a slow and comfortable speed.The stick figure is constructed with the 24 body points calculated by the deep leaning system of this application.The non-English word at the right side is the option button for changing the skeleton figure to a doll-like animation figure. Figure 2 . 2 Figure 2. Twenty-six body positions analyzed in the study.White dots are the positions in the study.In the definition of the study, the trunk positions are included in the parts of gray color, and the limb positions are the others on the extremities.The distance of each position was calculated by the coordinates from the navel (star) to each position (dots).The positions of the upper body indicated the dots above the navel (star) and those of the lower body were below the navel. Figure 2 . 2 Figure 2. Twenty-six body positions analyzed in the study.White dots are the positions in the study.In the definition of the study, the trunk positions are included in the parts of gray color, and the limb positions are the others on the extremities.The distance of each position was calculated by the coordinates from the navel (star) to each position (dots).The positions of the upper body indicated the dots above the navel (star) and those of the lower body were below the navel. P( f ) = k f α P : power; f : f requency logP( f ) = αlog f + logk α = fluctuation index, in this study Sensors 2023, 23, 9263 6 of 15 () = + = fluctuation index, in this study Figure 3 . 3 Figure 3.The slope as a fluctuation index.For each set of body-position data, the Fourier transform was completed, and the log of power and the log of frequency correlation were drawn (the black line).The slope (the red line) of the approximate line was a fluctuation index in the study. Figure 3 . 3 Figure 3.The slope as a fluctuation index.For each set of body-position data, the Fourier transform was completed, and the log of power and the log of frequency correlation were drawn (the black line).The slope (the red line) of the approximate line was a fluctuation index in the study. Figure 4 . 4 Figure 4.The body positions of impaired fluctuation in patients with iNPH compared with PD.The black circles (•) represent the body positions with significantly smaller absolute fluctuation indices in iNPH compared with PD.The fluctuation values are shown in Table2.These body positions with different indices between iNPH and PD were spreading to the lower and upper body, including the right-hand area. Figure 4 . 16 Figure 5 . 4165 Figure 4.The body positions of impaired fluctuation in patients with iNPH compared with PD.The black circles (•) represent the body positions with significantly smaller absolute fluctuation indices in iNPH compared with PD.The fluctuation values are shown in Table 2.These body positions with different indices between iNPH and PD were spreading to the lower and upper body, including the right-hand area.Sensors 2023, 23, x FOR PEER REVIEW 9 of 16 Figure 5 . 5 Figure 5.The graph of fluctuation indices.The black line indicates iNPH, the gray line indicates PD and the dotted line indicates the controls.The absolute values were consistently smaller for individuals with iNPH, PD, and the control in this order, across all body positions.The absolute values of iNPH were always smaller than those of PD, with the significances of 7 positions in the upper body and 8 positions in the lower body (*). Sensors 2023 , 16 Figure 6 . 2023166 Figure 6.Schema of the suspected development of gait disturbance and imbalance affected by the random fluctuation of systemic body parts during gait in patients with iNPH and PD.Other symptoms of the diseases affected each other. Figure 6 . 6 Figure 6.Schema of the suspected development of gait disturbance and imbalance affected by the random fluctuation of systemic body parts during gait in patients with iNPH and PD.Other symptoms of the diseases affected each other. Table 1 . 1 Clinical characteristics of three datasets. iNPHPDControlNumber232392Sex (male/female)16/713/1036/56Number of gait trials11756184Age (average ± SD)77.0 ± 6.470.1 ± 6.072.3 ± 6.3iNPH, idiopathic normal pressure hydrocephalus; PD, Parkinson's disease; SD, standard deviation. Table 2 . 2 The averages of fluctuation indices in the three groups of controls, PD, and iNPH, and the p value from the comparison of PD and iNPH. ControlsPDiNPHp Acknowledgments:We appreciate the cooperation of the office staff of Takahata Town, especially Masanori Togashi; the project collaboration of nurses and office staff at Takahata Public Hospital; the cooperation in the gait examination by the rehabilitation staff of Takahata Public Hospital, especially Masashi Shindo and Ikue Nagahashi; and the special assistance provided by Emi Murayama and Takee Kikuchi in contacting the volunteers and patients.Funding: This research was funded by research grants from the Japan Society for the Promotion of Science, KAKENHI (grant number: 21K09098 and 21K07336); the G-7 Scholarship Foundation in 2020; the Taiju Life Social Welfare Foundation in 2020 and 2022; and the Osaka Gas Group Welfare Foundation in 2022.The funders had no effect on or involvement in the writing of this article.The funders played no role in the study design, data collection, and analysis, in the decision to publish the results, or in the preparation of the manuscript.Institutional Review Board Statement:The study design and protocol of this study were approved by the ethics committee for human research at Nagoya City University Graduate School of Medical Science (IRB number: 60-22-0111), Shiga University of Medical Science (R2019-337), and Yamagata University School of Medicine (protocol code: 2021-10).Informed Consent Statement: Written informed consent was obtained from all subjects involved in this study.Data Availability Statement: Data are contained within the article.Conflicts of Interest:Yukihiko Aoyagi was employed by Digital Standard Co., Ltd.The remaining authors declare that this research was conducted in the absence of any commercial or financial relationships that could be interpreted as a potential conflict of interest. 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Prevention of Oncogenic Gammaherpesvirinae (EBV and HHV8) Associated Disease in Solid Organ Transplant Recipients 17 November 2023 Alaa Atamna a.atamna86@gmail.com Infectious Diseases Unit Rabin Medical Center Beilinson Hospital, Petah-Tikva Israel Faculty of Medicine Tel-Aviv University Tel-Aviv, Israel Dafna Yahav Faculty of Medicine Tel-Aviv University Tel-Aviv, Israel Infectious Diseases Unit Sheba Medical Center Ramat-Gan, Israel Cédric Hirzel Department of Infectious Diseases Bern University Hospital University of Bern BernSwitzerland Prevention of Oncogenic Gammaherpesvirinae (EBV and HHV8) Associated Disease in Solid Organ Transplant Recipients 17 November 20232B27A10A3D117771C344F7FE5832DE5210.3389/ti.2023.11856Received: 27 July 2023 Accepted: 07 November 2023human herpes virus 8Epstein-Barr virusKaposi sarcomamulticentic Castleman diseaseprimary effusion lymphomaposttransplant lymphoproliferative disorders Long-term risk for malignancy is higher among solid organ transplant (SOT) recipients compared to the general population.Four non-hepatitis viruses have been recognized as oncogenic in SOT recipients-EBV, cause of EBV-associated lymphoproliferative diseases; human herpes virus 8 (HHV8), cause of Kaposi sarcoma, primary effusion lymphoma and multicentric Castleman disease; human papilloma virus, cause of squamous cell skin cancers, and Merkel cell polyomavirus, cause of Merkel cell carcinoma.Two of these viruses (EBV and HHV8) belong to the human herpes virus family.In this review, we will discuss key aspects regarding the clinical presentation, diagnosis, treatment, and prevention of diseases in SOT recipients associated with the two herpesviruses. Introduction HHV8 is a DNA virus that belongs to the gamma-herpes virus subfamily.It was first discovered in 1994 as the etiologic agent of Kaposi's sarcoma (KS) [1].Four types of KS are distinguished: classic-, endemic-, immunosuppression-associated-, and AIDS-associated KS [2].Other HHV8 associated neoplastic disorders include primary effusion lymphoma and multicentric Castleman disease [3,4]. In SOT recipients, KS is ~200 fold more frequent than the general population, with cumulative incidence of ~3%-5% in endemic areas, and <1% in non-endemic areas [5,6].Post-transplant KS is a consequence of reactivation of latent infection in seropositive recipients, or a primary donor derived infection in seronegative recipients [7]. Non-neoplastic disorders associated with HHV8 are peripheral cytopenias, hemophagocytic syndromes, acute hepatitis, and KS-associated herpesvirus inflammatory cytokine syndrome (KICS) [8,9]. Epidemiology The seroprevalence of HHV8 depends on the geographic region.African countries have the highest seroprevalence rates (>50%), whereas seroprevalence in Europe, North America, South and East Asia is lower [10][11][12][13][14].In low seroprevalence regions, men who have sex with men (MSM) are at increased risk [15]. Both sexual and non-sexual transmission (including blood transfusion and organ transplantation) of HHV8 occurs.SOT recipients may be infected either before transplantation and reactivate the virus post-transplantation, or acquire the virus as a donor-derived infection.Primary HHV8 infection posttransplant increase the risk for HHV8-associated disease [16]. Non-Malignant HHV8 Disorders HHV8 infection in immunocompetent individuals is generally asymptomatic, although occasionally associated with a febrile rash in children [17].In immunocompromised individuals, HHV8 infection has been associated with fever, splenomegaly, maculopapular rash, lymphadenopathy and cytopenia [18] and rarely causes systemic disease with multi-organ failure post-transplantation [6].Bone marrow suppression, with or without hemophagocytosis was linked to donor derived HHV8 infection in the early post-transplant period [19][20][21], and rare cases of sexually transmitted primary HHV8 infections post-transplantation were associated with hemophagocytosis [22]. Malignant HHV8 Disorders Post-Transplant Kaposi Sarcoma (PT-KS) PT-KS is the most commonly encountered HHV8-related neoplastic disease [23].PT-KS mostly develops within the first year post-transplantation [6], and may cause skin lesions involving the extremities, the trunk and the oral cavity [6].Lesions are characterized by red-blue or purple discoloration, representing the vascular nature of the disease [18].Visceral involvement occurs in ~10% of PT-KS cases, with higher rates (up to 50%) in liver transplant recipients and associated with high a mortality [24,25].Disseminated disease without skin lesions exists, and lesions may appear at atypical localizations including the tonsils, urinary bladder and liver [26][27][28].The disease can be rapidly-progressive, especially in donor derived cases [29].In addition to primary infection, risk factors for KS in SOT have been described, with the most prominent factor being residence/ origin in endemic countries.Other risk factors include (from higher to lower risk) older age, male gender, thoracic transplantation, and use of cyclosporine and antilymphocyte antibody [18,30,31]. Multicentric Castleman Diseases (MCD) MCD is characterized by B-cell transformation to plasmablasts, which subsequently infiltrate multiple lymph nodes and distort their architecture.It typically presents with fever, lymphadenopathy, hepatosplenomegaly, and cytopenia [6].MCD and PT-KS may occur concomitantly in SOT patients [32][33][34]. Primary Effusion Lymphoma (PEL) PEL is a non-Hodgkin lymphoma that rarely develops after SOT and affects serous body cavities (pleura, pericardium, and peritoneum) [6].The median time of presentation is 8 years after transplant, with a wide range from 5 months to 28 years [35].It presents as a body cavity effusion in the absence of tumor masses.The prognosis is dismal [35]. Kaposi Sarcoma Herpes Virus (KSHV) Inflammatory Cytokine Syndrome (KICS) KICS is a systemic inflammation that resembles MCD without pathologic findings in lymph nodes.Generally, patients with KICS also have KS and to a lesser extent may have PEL.Patients with KICS have more severe symptoms, and an increased risk of death [36].Two cases of KICS have been reported in SOT recipients (Table 1) [8,37]. Screening and Diagnosis of HHV8 Infection Recent American society of transplant (AST) guidelines on HHV8 provide a weak recommendation for pre-transplant serological screening of donors and recipients in endemic areas in order to stratify the risk for HHV8 associated disease [6].In non-endemic areas it is suggested to consider screening for at-risk donors and recipients only (i.e., MSM, people living with HIV or who inject drugs), or for immigrants from endemic countries [8]. The rationale behind recommending serologic screening in endemic settings is the increased risk for KS among seropositive kidney transplant recipients as compared to seronegative recipients (23%-28% vs. 0.7%) [38].In addition, donor derived post-transplant HHV8 transmission from seropositive donors to seronegative recipients has been described.Nevertheless, HHV8 seropositive individuals are not excluded from organ donation [6].Preventive reduction of immunosuppression has been suggested in D+R− cases [23]. Lack of standardization of serological assays, variable sensitivity and specificity of these tests, and the absence of an algorithm for management according to serologic findings, result in low rates of pre-transplant screening in practice.In a survey including 51 transplant centers, only one-third performed pretransplant HHV8 serology.High HHV8 seroprevalence (>6% seropositivity), Italian centers, available protocols for posttransplant viral load monitoring, and having had a recent case of HHV8 disease were associated with screening. In a study that assessed six different serologic HHV8 assays the Biotrin-DiaSorin IFA and ABI IFA showed the highest agreement with a reference standard of ≥2 concordant positive assays [39]. No AST recommendations are available to direct a schedule of blood viral DNA monitoring in D+R− or R+ SOT recipients, beyond a general recommendation for monitoring in these patients [6].In a recent survey, 41% of centers reported performing HHV8 PCR monitoring post-transplant, with variable indications including symptomatic patients only, riskbased approaches or universal screening [40].In case of detectable HHV8 DNAemia, most centers reduced the immunosuppression or changed from calcineurin inhibitors (CNI) to m-TOR inhibitors, with or without addition of antivirals [40].For viremic patients, guidelines suggest immunosuppression reduction or change to mTOR inhibitors.The rationale for the latter is the antiviral and antiangiogenic effects of sirolimus, though no clinical benefit has been demonstrated in studies [6].Screening for viral DNA in bronchoalveolar fluid has been suggested for lung transplant recipients and is now under investigation (NCT05081141). Immune monitoring by HHV8 ELISPOT test is used by some centers as adjunct to HHV8 PCR monitoring in high-risk patients [40].Absent anti-HHV8 cytotoxic T-cell response has been demonstrated in SOT recipients with KS.It has been suggested that ELISPOT may assist in identifying patients at higher risk for developing KS, and if negative, reducing the immunosuppression may be considered [9]. Table 2 provides a proposed approach for screening or HHV8 pre and post-transplant. Diagnosis of HHV8 Associated Disease The gold standard for diagnosing KS, MCD and PEL is histopathological examination of tissue.Immunohistochemical staining of HHV8 latency-associated nuclear antigen confirms the diagnosis.Tissue PCR for HHV8 may assist in confirming the diagnosis.There is no established role for peripheral blood PCR in diagnosing KS.Positive PCR supports the diagnosis of KS, however, it may be negative in ~20% of KS cases [41].Highest DNAemia levels were reported in MCD, followed by PEL.Hence, PCR may be more sensitive in these cases, and it has been suggested that negative HHV8 PCR may be used to exclude MCD [42].High HHV8 DNAemia (>10,000 copies/mL) supports the diagnosis of MCD over KS [43].In patients with PEL, high viral loads have been demonstrated in effusion fluids [42].Due to limitations of serology discussed above, it is not currently indicated for diagnosis of HHV8 associated disease [44]. Patients with KICS are almost universally DNAemic.The diagnosis of KICS is based on high levels HHV8 DNAemia, exclusion of other possible causes, and possibly detection of HHV8 in involved organs (bone marrow, liver, and others) [9,36].A cutoff value of viral load in plasma ≥1,000 copies/mL or ≥100 copies/10 6 cells in peripheral blood mononuclear cells has been suggested for diagnosis of KICS [36]. Prevention (Val)ganciclovir, cidofovir and foscarnet inhibit the replication of human herpes viruses, including HHV8.Among HIV patients, (val)ganciclovir proved to decrease the incidence of KS [45].However, effectiveness of these drugs as pre-emptive therapy in cases of positive HHV8 PCR has not been demonstrated. The need for a vaccine to prevent HHV8 associated malignancies in susceptible populations has been recently raised by the National Cancer Institute.Since the HHV8 genome is highly conserved, it is possible that a single vaccine would provide protection worldwide [46]. Treatment of HHV8 Related Diseases Treatment of HHV8 associated malignancies and non-malignant conditions in SOT recipients should first include reduction in immunosuppression (RIS) and/or change from CNI to mTOR inhibitors [47,48].Older studies demonstrated between 70% and 100% complete response (CR) of KS following a change from cyclosporin to sirolimus, and 20%-50% CR of KS with RIS [5,38,49]. In a more recent study, including 145 SOT recipients with KS, immunosuppression reduction with/without switch to mTOR inhibitors, resulted in a response in >80% of patients [5]. Systemic chemotherapy with an anthracycline or paclitaxel is usually required for KS patients with visceral involvement, extensive lymph node or mucocutaneous involvement, and for patients not responding to reduction/change in immunosuppression [6,9].Immunomodulatory therapy with interferon-α is avoided in the SOT setting because of the risk for rejection [50].Specific chemotherapy regimens are routinely used for the management of MCD and PEL, in addition to immunosuppression reduction [9,45]. Immunological (ELISPOT) and virological (HHV8 PCR) tests are suggested as part of follow up in the management of KS and other HHV8 related diseases [9]. Several antivirals have in-vitro activity against HHV8, including ganciclovir, foscarnet, and cidofovir, while acyclovir is not highly active [51].Recent NIH guidelines for HIV management do not recommend antivirals as part of KS therapy, based on studies showing limited efficacy [45].For the treatment of PEL, antiviral drugs may be used as a possible adjunctive therapy, with a CIII level of recommendation [45].For MCD, two retrospective studies demonstrated remissions using ganciclovir as part of the treatment regimen in HIV patients [45].This is supported by the rationale of lytic HHV8 infection being present in MCD [52].There is limited data to support the use of anti-IL6 inhibitors for MCD with no recommendation for general use of these drugs for this indication [45].Adoptive immunotherapy with cytotoxic T-lymphocytes specific for HHV8 could have a therapeutic role, though there is currently no commercial product available [9]. EPSTEIN-BARR VIRUS IN SOLID ORGAN TRANSPLANTATION Introduction Epstein-Barr virus (EBV) is a double-stranded DNA virus of the γ-herpesviridae subfamily [53].The virus was discovered in 1964 from cultured lymphoblasts of Burkitt's lymphoma biopsies before being identified as the causative agent of mononucleosis in 1968 [54,55].EBV was the first known human oncogenic virus and it efficiently transforms human B-lymphocytes [56][57][58].Upon infection, EBV establishes lifelong latency in memory B-cells [59,60].The pathogenesis of EBV-associated oncogenesis is complex and it is related to the ability of the virus to transform and immortalize B-cells and to impede apoptosis of infected cells [53,61].EBV is associated with a large spectrum of diseases, including benign diseases (infective mononucleosis, oral hairy leukoplakia), a number of lymphoproliferative disorders (Burkitt's lymphoma, some Hodgkin lymphomas, EBV-positive diffuse large B-cell lymphomas, natural killer/T-cell lymphoma, nasal type angiocentric lymphomas, chronic active EBV), epithelial cancers (nasopharyngeal carcinoma, some forms of gastric cancer), smooth muscle cell tumors, and diseases related to immune dysfunction (multiple sclerosis, EBV-associated hemophagocytic lymphohistiocytosis) [61,62]. In SOT patients, EBV is known to play a major role in the development of EBV-positive post-transplant lymphoproliferative disorders (PTLD), one of the most devastating complications of organ transplantation [53,63,64]. Epidemiology Seroepidemiologic surveys indicate that >90% of adults are infected with EBV [65,66].In developed countries, primary EBV infection tends to occur later nowadays as compared to the past [67][68][69].In the transplant setting, donor transmitted EBV infection is common in EBV mismatched (donor EBV+/ recipient EBV−) patients.Children are more likely to be EBVnegative, and may acquire the virus from the donor organ or by natural infection, putting them at increased risk for posttransplant primary infection. EBV Associated Diseases in SOT Recipients Post-Transplant Lymphoproliferative Disorders (PTLD) Since the first description of five lymphoma cases in kidney transplant recipients (KTR) in 1969, PTLD has been recognized as a serious complication of SOT [70].PTLD encloses a heterogeneous spectrum of conditions characterized by lymphoproliferation after transplantation.These disorders range from uncomplicated infectious mononucleosis-like pathology to true malignancies [71].PTLD is categorized according to the World Health Organization (WHO) 2017 classification, based on its histopathological appearance (Table 3) [77].Additionally, PTLD is classified according to its temporal occurrence: early-onset PTLD arises within the first year post-transplant, whereas late-onset PTLD occurs thereafter [78].In contrast to late-onset PTLD, most cases of early-onset PTLDs are associated with EBV [72,79,80].While the incidence rate for EBV-positive PTLD is highest early after transplant, the incidence rate of EBV-negative PTLD is low immediately after transplantation and increases after 4-5 years, resulting in a biphasic pattern of overall PTLD occurrence [81,82]. A major risk factor for development of EBV-positive PTLD is EBV-seronegativity pre-transplant (hazard rate 5-18 as compared to EBV-seropositive individuals) [80,[83][84][85][86][87].However, in liver transplant recipients the association of EBVseronegativity and PTLD risk is less pronounced [87].As children are more likely to be EBV-seronegative before transplantation, PTLD is more common in pediatric SOT recipients.Further, the risk is affected by the type of transplanted organ with intestinal transplant recipients (~18%) being at highest risk for developing PTLD [88,89], followed by lung (3%-10%), heart (2%-8%), liver (1%-6%), and kidneys (1%-2%) [90].In the current era, there was no conclusive association between the type of induction therapy and PTLD risk [91,92].The contribution of each immunosuppressive agent to PTLD development is unclear, since patients receive multiple agents in different doses at different times [91].However, concerns regarding the use of belatacept in EBV-seronegative transplant recipient have been raised [93].It is for this reason that belatacept is contraindicated in patients who are EBV-seronegative or whose EBV serostatus is unknown prior to transplant [94]. The clinical presentation of PTLD is heterogeneous and depends on the type (non-destructive-, polymorphic-, monomorphic-PTLD) and the localization of disease.Nonspecific constitutional symptoms such as fever, unintended weight loss, night-sweats, and fatigue are common.Lymphadenopathy, tonsillar hypertrophy, dysfunction of involved organs, or compression of surrounding structures may occur.More than half of cases presents with extranodal involvement [72,83,95].PTLD frequently involves the gastrointestinal tract (20%-30%), the allografts (10%-15%), and the central nervous system (CNS, 5%-20%) [72,83,95].Therefore, not only lymphadenopathy but also gastrointestinal bleeding or ulcers, allograft dysfunction in combination with masses, and focal neurological signs should rise suspicion for PTLD. EBV-Associated Smooth Muscle Cell Tumor (EBV-SMT) EBV-SMT is an uncommon neoplasm of immunocompromised individuals [96].The role of EBV in the tumorigenesis is poorly understood.EBV-SMT is thought to be derived from myogenous vascular smooth muscle cells [97].The clinical presentation of EBV-SMT is unspecific and depends on the localization of the tumor [98].Biopsies of smooth muscle tumors in SOT recipients should be evaluated with EBVencoded small nuclear RNA (EBER) stains, to establish the diagnosis of EBV-SMT [98] and the differential diagnosis should include KS and mycobacterial spindle cell pseudotumor [96]. Non-Malignant EBV-Associated Disease After SOT The features of these EBV manifestations may include infective mononucleosis, oral hairy leukoplakia [99], and end-organ infections such as encephalitis/myelitis [100] or hepatitis [101].Some of these manifestations may share clinical features of PTLD (e.g., encephalitis vs. CNS PTLD).Therefore, careful evaluation of these cases is warrant.a There is no uniformly accepted EBV DNAemia uniform cut-off for reduction in immunosuppression.This is related to the different types of samples (whole blood vs. EDTA plasma) used for EDTA monitoring and the considerable inter-laboratory variations in EBV DNAemia measurements (even when using the WHO standard).b There is no established EBV DNAemia cut-off (neither DNAemia level nor duration of persistent DNAemia) for triggering radiologic examinations. Due to the overwhelming clinical importance of PTLD, we will focus on aspects related to PTLD in this review. Diagnosis of Post-Transplant Lymphoproliferative Disorders (PTLD) The diagnosis of PTLD is based on the histopathological examination of appropriate tissue biopsies.Assessing the presence of latent EBV infection of affected cells by (preferably) RNA-in-situ-hybridization targeting EBV-encoded small RNAs (EBER) or by immunohistochemistry targeting latent membrane protein 1 (LMP1) is essential for the diagnosis of EBVassociated PTLD [102].Preceding to tissue sampling, radiographic imaging is a crucial initial step to come to a tentative diagnosis.The radiographic evaluation is similar to that used in the evaluation of suspected lymphoma in the non-transplant population [103].A computed tomography scan (neck to pelvis) is the first step in most centers.MRI may be the preferred modality for suspected cerebral PTLD [104]. Positron emission tomography-computerized tomography has emerged as a useful imaging modality for detecting suspicious lymph nodes and extranodal lesions and may be helpful to identify optimal sites for biopsy [105].Establishing a PTLD diagnosis can be difficult and occasionally multiple attempts for getting conclusive tissue biopsies are necessary (especially for gastrointestinal PTLD).In SOT recipients with persistent gastrointestinal symptoms, PTLD should be part of the differential diagnosis and endoscopy with biopsy of ulcers/lesions should be performed [106]. Studies evaluating the diagnostic test characteristics of EBV DNAemia measurements for diagnosing EBV-positive PTLD are limited.In summary, EBV DNAemia above a specific threshold has good sensitivity (~90%) for detecting EBV-positive PTLD but lacks specificity [107][108][109] and EBV PCR is not useful for detection of EBV-negative PTLD. Prevention of EBV Associated Disease in SOT Recipients Monitoring EBV DNAemia With Reduction of Immunosuppression for Prevention of EBV-Positive PTLD A monitoring strategy of repeated EBV DNAemia measurement with RIS if a certain threshold is reached or if DNAemia is increasing, is applied by many transplant centers [110], especially for high-risk patients (EBV D+/R−) [108,[111][112][113][114][115].However, the optimal way to apply this strategy remains unclear.This is also related to the inter-laboratory variability of EBV DNAemia measurements, despite previous efforts for harmonizing results by introducing an international standard [116,117].In clinical practice, EDTA plasma or whole blood is used for monitoring EBV DNAemia (Table 4).EBV DNAemia levels are higher when determined in whole blood as compared to EDTA plasma [118,119].Therefore, the sensitivity for detection of EBV DNAemia is higher when using whole blood.However, the specificity for detection of EBV-related disease is better when using EDTA plasma samples [120].The controversy with respect to the preferred sample type for monitoring EBV DNAemia is ongoing.In our opinion, it is more relevant to ensure that the same type of sample is used and that DNAemia is determined in the same laboratory when longitudinally assessing EBV DNAemia, instead of focusing on the discussion about the preferred sample type.Even though there is no evidence from randomized-controlled trials supporting the usefulness of EBV DNAemia monitoring and RIS, there is some evidence from cohort studies supporting this approach [75,76,111].However, the results of these studies have to be interpreted with caution because of using historic controls [75,76] (problematic because of decreasing PTLD incidence over time, most likely related to less intense immunosuppression in contemporary versus historic cohorts [72,111,121] and the lack of statistical power to show differences due to the rarity of the disease [111]).Although it seems to be appealing from a pathophysiological point of view, there is no strong evidence supporting EBV DNAemia monitoring with RIS for prevention of EBV-positive PTLD.Furthermore, no specific cut-off value for EBV DNAemia to guide preemptive therapy is available, with some studies using any positive titer [122] while others using increasing loads (>10fold or >1 log10 cp/mL) [122]. Antiviral Prophylaxis for Prevention of EBV-Positive PTLD Several antiviral drugs such as (val)acyclovir, (val)ganciclovir, cidofovir, foscarnet and maribavir inhibit lytic EBV replication [123,124].However, these drugs have no effect on latent EBV infection.Since primary EBV infection after transplantation is a major PTLD risk factor, reducing donor-derived EBV transmission may have an impact on PTLD occurrence.A reduction of primary EBV infection was observed in a cohort of EBV seronegative pediatric KTRs on (val)ganciclovir prophylaxis versus no antiviral prophylaxis [125].In another cohort of EBV mismatched adult KTRs, antiviral prophylaxis for 3-6 months delayed the rate of EBV primary infection at 100 days post-transplant, but the seroconversion rate 12 months posttransplant was identical with and without prophylaxis (72% vs. 74%) [126].Recent cohort studies did not find a protective effect of antiviral prophylaxis on PTLD occurrence [72,127].These findings are consistent with results of a systematic review published in 2017, concluding that antiviral prophylaxis in high-risk EBV-naive patients has no effect on the incidence of PTLD [128]. Rituximab for Prevention of EBV-Positive PTLD The preemptive use of rituximab for prevention of PTLD has become a common strategy in EBV viremic hematologic stem-cell transplant (HSCT) [129,130].B-cell depletion before or directly after HSCT, has shown to reduce EBV replication [131,132] and the incidence of EBV-positive PTLD [133][134][135] in high-risk patients.The potential effect of rituximab on subsequent PTLD development may be attributable to the depletion of CD20 + B-cells, which represent the major reservoir for latent EBV infection.The reduced abundance of these cells at risk for malignant transformation might be linked to a lower PTLD risk [72].Rituximab use is less well established for prevention of EBVpositive PTLD in SOT recipients.A recent multi-center cohort study reported that rituximab given as part of the induction regimen (mostly in ABO-incompatible kidney transplantation) is associated with a decreased risk for PTLD [72].A single-center cohort study reported diminished PTLD rates with rituximab use in heart transplant recipients whose EBV DNAemia did not respond to RIS using a historic control group [75].Similarly, EBV-mismatched KTRs with persistent EBV DNAemia or symptomatic EBV infection given rituximab simultaneously with RIS were less likely to develop PTLD compared to contemporaneous controls [114]. Treatment and Prognosis of PTLD The first therapeutic measure in treatment of PTLD is RIS under close monitoring of the graft function.There are no evidencebased guidelines on how to reduce immunosuppression, but in clinical practice, stopping anti-proliferative agents and dose reduction of the CNI is the common approach [90].Significant RIS may not be feasible in all cases and is especially difficult to achieve in thoracic organ transplant recipients due to the risk of life-threatening graft rejection [136].RIS eradicates the majority of non-destructive PTLD cases.However, for polymorphic and monomorphic PTLD the response to RIS alone is often insufficient [137,138].A radiologic reassessment is performed two to 4 weeks after RIS, and if a CR is achieved no further treatment is needed. In the following section, we summarize the treatment options for polymorphic PTLD and monomorphic diffuse large B-cell lymphoma (DLBCL) PTLD.Treatment of non-DLBCL monomorphic PTLD depends on the histologic classification of the respect lymphoma and follows the same chemotherapy regimens as for immunocompetent patients, and will not be reviewed here.Immunochemotherapy for treatment of DLBCL PTLD is associated with significant toxicity and many SOT recipients are not fit for highly intensive regimens [139].Therefore, sequential and riskstratified treatments are applied for treatment of CD20 + monomorphic DLBCL PTLD.The PTLD-1 [140], the PTLD-1 third amended [141] and PTLD-2 [142] phase 2 trials are landmark studies that established sequential, risk-stratified PTLD treatment modalities.The PTLD-1 study proved the efficacy and safety of a sequential treatment of four cycles rituximab monotherapy followed by four cycles of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) for patients who did not achieve complete remission with RIS [140].The most favorable outcomes were seen in patients who achieved a response to rituximab alone prior to chemotherapy, indicating that these patients may belong to a "good-risk group".In consequence, the PTLD-1 third amended trial assessed a risk-stratified protocol: patients who achieved a CR after four doses of rituximab, received consolidation with rituximab alone while those who did not achieve CR were treated with four cycles of R-CHOP [141].This trial proved that withholding chemotherapy and performing a rituximab consolidation in patients with CR to rituximab alone is safe and associated with less toxicity [141].The PTLD-1 third amended trial identified two subgroups with poor prognosis: thoracic transplant recipients and patients with an International Prognostic Index (IPI) score >2 [141].The PTLD-2 trial, inter alia, assessed treatment escalation (alternating R-CHOP and R-DHOAxrituximab, dexamethasone, cytarabine, oxaliplatin) in these patients with poor prognosis [142].However, the number of patients in this subgroup was low (n = 9), the outcome was poor and the treatment related toxicity was substantial [142]. For further information about novel, less established PTLD treatment options such as infusion of third-party EBV-specific cytotoxic T-lymphocytes, CAR-T cell therapy, proteasome inhibitors, burton-tyrosine kinase inhibitors, and histone deacetylase inhibitors in combination with antiviral nucleoside analogues we refer to the recent review of Atallah-Yunes et al. [143]. The introduction of rituximab, the administration of sequential risk stratified treatment regimens, and optimized supportive care have improved the outcome for patients with PTLD.In the PTLD-1 trial, the median overall survival was 6.6 years [140].Patients with a CR to rituximab alone have better prognosis as compared to rituximab non-responders [141] and thoracic transplant recipients show less favorable outcome as compared to non-thoracic transplant recipients [141,142]. TABLE 1 | 1 Case reports of KSHV Inflammatory Cytokine Syndrome (KICS) in solid organ transplant recipients. RecipientDonorOrganPresentationFindingsKS38 years old female, PSC42 years old, HHV8 IgG positiveLiver andPersistent feverSevere anemia and worsening of renal function,Noand CKD, HHV8 IgGKidney12 months aftersevere splenomegaly and small-sized generalizedNegativetransplantationlymphadenopathy, HHV8 viral load~189,000 copies/mL54 years old male, TOF,39 years old, Eastern Europe, highHeartPersistent feverPancytopenia, ↑creatinine, bilateral pleural effusion,HHV8 IgG Negativerisk sexual behavior, HIV negative,11 months aftergeneralized lymphadenopathy, HHV8 viral loadpositive HHV8 IgGtransplantation~183,000 copies/mL yesCKD, chronic kidney disease; HHV8, human herpesvirus 8; HIV, human immunodeficiency virus; KS, Kaposi sarcoma; KSHV, Kaposi sarcoma Herpes virus; PSC, primary sclerosing cholangitis; TOF, tetralogy of Fallot. TABLE 2 | 2 Proposed approach for HHV8 screening pre-and post-transplant. Serology aPCR bELISPOTPhysical examManagementPreYesNoNoNoNotransplantPostAmong D+R− repeated serologyAmong D+R−, PCR onceAmong D+R− orFor D+R−, skin andIf PCR positive negative → reduction intransplantmay indicate seroconversion-noevery 2 weeks for 3 monthsR+ may assist asmucosal surfacesimmunosuppression or change toschedule suggested [40]followed by once monthly toadjunctive testroutinemTOR inhibitor [6] If ELISPOT negativecomplete 2 years-Among R+,to PCRexaminations [6]→ consider immunosuppressionPCR once monthly forreduction [9]2 years [40] a If a screening approach is not implemented, the following signs and symptoms should merit an investigation for HHV8 if no other cause is found: fever, splenomegaly, maculopapular rash, lymphadenopathy and cytopenia.b There is no gold standard serology assay.c Quantitative cut-offs for PCR tests are missing; optimal testing frequency and duration of surveillance have not been determined.Whole blood may be more sensitive than plasma, because if inclusion of the cellular component.Screening is not routinely used by the authors of this review.Transplant International | Published by Frontiers November 2023 | Volume 36 | Article 11856 TABLE 3 | 3 Overview of post-transplant lymphoproliferative disorders. WHO 2017 categoryEBV-associationClonalityFrequencyClinical featuresNon-destructive PTLD~100% [72, 73]No~5%Early-onset, Benign-Plasmatic hyperplasia-Infectious mononucleosis-like PTLD-Florid follicular hyperplasiaPolymorphic PTLD~90% [72, 73]Variable~10%Early and late-onsetMonomorphic PTLD~50% [72]Yes~80%Early > lateB-cell neoplasm-Diffuse large B-cell lymphoma-Burkitt (like) lymphoma-Plasmablastic lymphoma-Plasmacytoma like lymphoma-OthersT-cell neoplasms~20%Yes<5%Late-onset-Peripheral T-cell lymphoma-OthersHodgkin/Hodgkin-like lymphoma~90% [74]Yes<5%Early and late-onsetEarly-onset, within 1 year post-transplant. Late-onset, >1 year post-transplant. EBV, Epstein-Barr virus; PTLD, post-transplant lymphoproliferative disorder; WHO, world healthorganization. TABLE 4 | 4 Proposed approach for EBV screening pre-and post-transplant. SerologyPCRELISPOTPhysical examManagementPreYesNoNoNoNotransplantPostAmong D+R− repeated serologyAmong D+R−, PCR onceFor researchCheck for lymphadenopathyReduction in immunosuppression iftransplantmay indicate seroconversion-inevery 2-4 weeks forpurposeduring routine clinical controlshigh EBV DNAemia a (author'sclinical practice, measurement of12 months [75, 76]only(author's personal opinion, nopersonal opinion, week evidence)EBV DNA in peripheral blood has(author's personal opinion,evidence)Actively search for PTLD if EBVlargely replaced serology for theweek evidence)DNAemia is persistently high bdiagnosis of primary EBV infection AUTHOR CONTRIBUTIONSAA and DY reviewed the HH8 literature, drafted the HHV8 part and critically reviewed the EBV part of the manuscript.CH reviewed the EBV literature, drafted the EBV part and critically reviewed the HHV8 part of the manuscript.CONFLICT OF INTERESTThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 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The right to science and gender inequalities Peter Larsen Åsa Gunnarsson Yvonne Donders y.m.donders@uva.nl Université de Genève Switzerland Umeå University Sweden Faculty of Law Department of International and European Public Law University of Amsterdam AmsterdamNetherlands The right to science and gender inequalities D55B9EF911F65E0F8BFA1B6B90B15CE9RECEIVED August ACCEPTED October PUBLISHED November CITATION Donders Y ( ) The right to science and gender inequalities. Front. Sociol. : . doi: . /fsoc. .right to sciencegender inequalitieswomen's rightsUNESCOgender bias against womenhuman rightseconomicsocial and cultural rightsright to education Introduction The right to science was included in the Universal Declaration of Human Rights 75 years ago.A very influential person in the drafting of the Universal Declaration was a woman, Eleanor Roosevelt.The position of women in the UDHR is, however, rather secondary.The UDHR speaks of all members of the human family, but its text is rather male oriented.Moreover, despite the fact that equality and non-discrimination are at the heart of the UDHR, women still do not enjoy human rights equally with men.The same is true for the right to science. It is uncontested that deep inequalities between women and men persist in the field of education, sciences and research.Women remain underrepresented and/or disadvantaged in access to scientific education and opportunities to have a career in academia.Furthermore, women suffer from lack of access to scientific applications and scientific applications and technologies may be gender biased and not sensitive to the particularities and needs of women.This inequality is persistent, "[. . .] even in countries with relatively long histories of formal and legal equality".This implies that more subtle and underlying factors play a role, such as gender stereotypes and biases.How ineradicable is the inequality between women and men in sciences and research and what does the right to science as a human right have to offer in response to sex and gender inequalities?This opinion focuses on the right to science as included in Article 15(1)b of the International Covenant on Economic, Social and Cultural Rights and in the UNESCO Recommendation on Science and Scientific Researchers.It argues that the right to science is an important normative tool that should promote and facilitate structural changes that can help to overcome gender-based barriers and ensure that women enjoy the right to science equally with men. Article of the UDHR includes the right of everyone to "share in scientific advancement and its benefits".The term "right to science" is used for reasons of practicality and familiarity in dominant scholarship on this matter.The right to science is however an umbrella term for a cluster of rights. Persistent gender inequalities in sciences and research One of the core principles of all human rights treaties is the obligation of States to ensure equal enjoyment of human rights and to prevent and put an end to all forms of discrimination.There are different forms of discrimination, including direct, indirect and structural discrimination, that should be prevented and eliminated.All three can cause or sustain the disadvantaged position of women in sciences and research. Direct discrimination in relation to sciences occurs when women and girls are formally and thereby explicitly excluded from participating, accessing, or contributing to education, sciences and research.Examples are States that have laws and policies that prohibit women from accessing and participating in education and sciences.The recent measures taken by the Taliban regime in Afghanistan, where girls and women were officially expelled from universities and banned from schools, is a striking and sad example. Indirect discrimination of women as regards their right to science can occur when rules and procedures are seemingly neutral, but in practice have a specific disadvantage for women.For instance, there can be certain requirements for academics to be eligible for promotion or for a leadership position, that may apply to all, but that can be more difficult to meet for women, because of structural inequalities in the right to education and in the right to participate in public life.A concrete example of such a requirement can be that a substantive part of the scientific education and experience has to be acquired in renowned academic institutions abroad.This may constitute a barrier especially for women who want or need to be more confined to their country, region, home or family, or for whom an extensive stay abroad cannot be combined with caretaking tasks (UNESCO Science Report, 2021). Systemic and structural discrimination may overlap with indirect discrimination.Systemic but subtle mechanisms or professional practices may discourage women's participation in sciences and research and contribute to their underrepresentation.For instance, there can be a culture of fierce academic competition for research funding or grants, whereby the competition for such grants is not gender neutral.For example, research has shown that women have less chance of having their work published in peer reviewed international journals (Squazzoni et al., 2021;Kern-Goldberger et al., 2022;Bornmann et al., 2023).A well-known structural barrier for many women is that a successful career in scientific research is only possible if someone invests and devotes large amounts of time to doing research.This may be difficult or impossible for women in periods of their life that are also crucial for founding a family.For men it is easier to postpone becoming a parent until a later age than it is for women. States have clear obligations to promote and to put into effect formal (de jure), substantive (de facto) and transformative (structural) equality of women, responding to direct, indirect and structural discrimination.Achieving formal, substantive and structural equality may also imply taking (temporary) special measures to promote and protect inclusion and participation of women.According to the UN Committee on Economic, Social and Cultural Rights (CESCR), '[. . .] the principle of equality will sometimes require that States parties take measures in favor of women in order to attenuate or suppress conditions that perpetuate discrimination.As long as these measures are necessary to redress de facto discrimination and are terminated when de facto equality is achieved, such differentiation is legitimate.' What can the right to science o er? The right to science covers various aspects and dimensions, including access, participation, contribution and enjoying the benefits, as well as protection against harmful science.All these dimensions have gender dimensions that seem to sustain inequalities between women and men in relation to sciences and A lot has been published on women and their academic careers in relation to motherhood.There is an organization for mothers in science (https://www.mothersinscience.com/) that aims to help an support mothers in STEM research.See, also: research.The Committee on Economic, Social and Cultural Rights, which is the independent body monitoring the implementation of the treaty by the States parties, has elaborated on the normative content of the right to science, including its gender dimensions. In its General Comment on science and economic, social and cultural rights the CESCR explicitly urged States parties to take measures to tackle these gender dimensions.It firstly expressed that States must "[. . .] immediately eliminate barriers that affect girls' and women's access to quality scientific education and careers'.States must also take steps" [. . .] to ensure women's substantive equality in access to scientific education and careers by, for example, raising public awareness in order to eliminate stereotypes that exclude women from science or adopting policies for both men and women to balance domestic life with scientific careers'.It also maintained that "[. . .] temporary special measures, such as quotas for women in scientific education, might be necessary" in order to advance more quickly toward substantive equality in the enjoyment of the right to science. The CESCR furthermore addressed the gender dimensions of scientific research by stating that "[a] gender-sensitive approach is not a luxury for scientific research, but a crucial tool in order to ensure that scientific progress and new technologies adequately take into account the characteristics and needs of women and girls".A gender sensitive approach should be part of all stages of the process of research, from the choice of subjects and the design of methodologies up to the evaluation of its applications and impacts.The CESCR also urges States parties to make decisions concerning funding or general policies in a (more) gender-sensitive manner . The CESCR formulates a wide palette of State obligations to respect, protect and fulfill the right to science, including the equal enjoyment of this right by women and men.Moreover, it named the identification and elimination of all laws, policies, practices, prejudices or stereotypes that undermine women's and girls' participation in science as a so-called core obligation.A core obligation is the minimum essential level of a right that all States parties, irrespective of their political, economic or social situation, should immediately guarantee.The Member States of UNESCO have adopted various instruments on science and education, and for many years "gender equality" is one of the key priorities of UNESCO.The UNESCO Recommendation on Science and Scientific Researchers addresses many of the gender inequality issues related to sciences and research as outlined above. The promotion of equality and non-discrimination is explicitly addressed in relation to education and training of scientific researchers, an area in which States are recommended to take measures to abolish inequalities in opportunities.States are recommended to guarantee equal opportunities in education and training needed to qualify for research careers, as well as for those qualified equal access to available employment in scientific research. States are further recommended, "[. . .] in order to remediate past inequalities and patterns of exclusion", to "[. . .] actively encourage women. . . to consider careers in sciences, and endeavor to eliminate biases against women [. . .] in work environments and appraisal" and they are encouraged to "[. . .] ensure that scientific researchers enjoy equitable conditions of work, recruitment and promotion, appraisal, training and pay without discrimination on the basis of [. . .] sex [and] gender [. . .]". Appraisal is an important area where gender-based differences can play a role.It is therefore recommended that States should "[. . .] design and establish appropriate appraisal systems for independent, transparent, gender-sensitive and tier-based performance evaluation".Such systems should take due account of "[. . .] the difficulty inherent in measuring a performance given the effects of mobility between themes and disciplines [. . .] and the need to appraise all aspects of the individual's performance in context"; and they should "[. . .] transparently account for familycare related interruptions of employment and encourage equitable treatment by means of incentives, so that the careers and research of those who take family related leave, including parental leave, are not negatively impacted as a result".Frontiers in Sociology frontiersin.orgDonders ./fsoc. . In short, the right to science, in combination with the general prohibition of all forms of discrimination, clearly implies that women have the right to equally access, participate in and contribute to sciences and research and that States have positive obligations to ensure this right.There is no lack of applicable norms and no lack of clarity on State obligations.These obligations are not merely obligations of conduct, but also of result.It seems that the continuous inequality between women and men in sciences and research is caused and sustained by a lack of effective implementation and monitoring of these norms. Discussion The right to science includes rights to participate in science, to contribute to science, to have access to science and to enjoy the benefits of science.These rights should be equally enjoyed by all, based on ability and competence.In all these areas, however, women are disadvantaged.The underrepresentation of women in sciences and research also has consequences for the applications of science, which can reflect or sustain problematic gender inequalities. Different forms of discrimination-direct, indirect and structural-negatively affect the enjoyment of the right to science by women.In particular, structural and systematic forms of discrimination caused by persistent gender stereotypes and patterns in societies sustain the subordinate position of women and prevent them from fully participating.Effective participation by women in science education, in sciences and research, as well as in the development of science policies science agendas, therefore requires formal and substantial equality, but mostly transformative equality and structural changes.To overcome institutional and societal gender-based barriers and ensure that women enjoy the right to science equally with men, special policies and measures are required.The elimination of discrimination, including stereotypes that undermine women's participation in science, is a core obligation that States should respect, protect and fulfill under all circumstances. The normative framework of the right to science is wellestablished, with the different human rights treaties and UNESCO instruments.Supported by the general principles of equality and non-discrimination, it is an important right that should promote and protect women's participation in sciences and research, which could also contribute to more gender equality in scientific applications.The international legal instruments on the right to science demonstrate a large degree of consensus among States on the need, at least in theory, to promote gender equality in sciences and research.It does, however, seem to be much more difficult to advance this formal articulation beyond the realm of aspirations.Concrete and effective action is however urgently needed.The right to science exists for 75 years now.It is time to start using it as a normative tool to repair and prevent gender inequalities in sciences and research. International Covenant on Economic, Social and Cultural Rights (ICESCR, ) Articles and ; The Convention on the Elimination of all Forms of Discrimination Against Women (CEDAW, ) contains many relevant norms, including Articles and .This short opinion focuses on the ICESCR, since it contains also the right to science; for elaboration of the CEDAW see the original chapter (Donders, ).Committee on Economic, Social and Cultural Rights, General Comment No. , The equal right of men and women to the enjoyment of all economic, social and cultural rights (art. of the International Covenant on Economic, Social and Cultural Rights) (UN Doc.E/Convention on the Elimination of All Forms of Discrimination against Women, on temporary special measures ( August ); CEDAW Committee, General Recommendation No. on the core obligations of States parties under article of the Convention on the Elimination of All Forms of Discrimination against Women (UN Doc.CEDAW/C/GC/ December ). Committee on Economic, Social and Cultural Rights, General Comment No. : The Nature of States Parties' Obligations (Art., Para., of the Covenant) ( December UN Doc.E/ / ) para. .See, on the work of UNESCO in relation to right to science: ) para.(b).UNESCO Recommendation on Science and Scientific Researchers (Paris November Doc.C/Resolution ) para. .UNESCO Recommendation on Science and Scientific Researchers (Paris November Doc.C/Resolution ) para.(b).UNESCO Recommendation on Science and Scientific Researchers (Paris November Doc.C/Resolution ) para.(d). AcknowledgmentsThis piece is a shortened version of the chapter (accepted for publication): YD, 'The Right to Science: Another Tool to Repair Gender Inequalities in Sciences and Research' in Andrea Broderick and Jennifer Sellin (ed) Socio-economic Rights, Inequalities and Vulnerability in Times of Crises: Building Back Better (Edward Elgar Publishers, forthcoming 2023).Author contributionsYD: Writing -original draft.FundingThe author(s) declare that no financial support was received for the research, authorship, and/or publication of this article.Conflict of interestThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Publisher's noteAll claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. 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Recent Development and Applications of Stretchable SERS Substrates 17 November 2023 Ran Peng rpeng@dlmu.edu.cn 0000-0002-3444-9805 College of Marine Engineering Dalian Maritime University 116026DalianChina Tingting Zhang 0000-0002-3444-9805 College of Marine Engineering Dalian Maritime University 116026DalianChina Sheng Yan shengyan@szu.edu.cn 0000-0002-3463-0786 Institute for Advanced Study Shenzhen University 518060ShenzhenChina Yongxin Song yongxin@dlmu.edu.cn 0000-0002-3463-0786 College of Marine Engineering Dalian Maritime University 116026DalianChina Xinyu Liu xyliu@mie.utoronto.ca 0000-0001-5705-9765 Department of Mechanical and Industrial Engineering University of Toronto M5S 3G8TorontoONCanada Junsheng Wang Department of Information Science and Technology Dalian Maritime University 116026DalianChina Liaoning Key Laboratory of Marine Sensing and Intelligent Detection Dalian Maritime University 116026DalianChina Recent Development and Applications of Stretchable SERS Substrates 17 November 20237AA3EC86BA4A9E5990F660E97BC9EED810.3390/nano13222968Received: 12 October 2023 Revised: 9 November 2023 Accepted: 13 November 2023surface-enhanced Raman scattering (SERS)stretchable SERS substrateflexible SERS substrate Surface-enhanced Raman scattering (SERS) is a cutting-edge technique for highly sensitive analysis of chemicals and molecules.Traditional SERS-active nanostructures are constructed on rigid substrates where the nanogaps providing hot-spots of Raman signals are fixed, and sample loading is unsatisfactory due to the unconformable attachment of substrates on irregular sample surfaces.A flexible SERS substrate enables conformable sample loading and, thus, highly sensitive Raman detection but still with limited detection capabilities.Stretchable SERS substrates with flexible sample loading structures and controllable hot-spot size provide a new strategy for improving the sample loading efficiency and SERS detection sensitivity.This review summarizes and discusses recent development and applications of the newly conceptual stretchable SERS substrates.A roadmap of the development of SERS substrates is reviewed, and fabrication techniques of stretchable SERS substrates are summarized, followed by an exhibition of the applications of these stretchable SERS substrates.Finally, challenges and perspectives of the stretchable SERS substrates are presented.This review provides an overview of the development of SERS substrates and sheds light on the design, fabrication, and application of stretchable SERS systems. Introduction Surface-enhanced Raman scattering (SERS) was discovered in 1977 by Van Duyne [1] and Creighton [2] as an efficient, ultrasensitive, label-free analytical technique.Since its discovery, interest in and use of surface-enhanced Raman spectroscopy has grown exponentially, with attractive advantages in areas such as environmental analysis [3], food safety [4,5], and biomedical analysis [6,7].However, the capability of this technology has not been fully developed due to many unexplored challenges such as the development of highly stable and sensitive SERS substrates [8][9][10][11][12].The intensity of the Raman signal is affected by many factors, such as the size, shape, surface roughness of plasmonic nanostructures, etc., but the key is to locate the target molecules in the gap of the metal structures where the electromagnetic field is highly enhanced (i.e., the "hot spot") [13].The enhancement factor of SERS is proportional to the local electromagnetic field strength; thus, the gap distance of the metal nanoparticle determines the Raman hot-spot intensity, which in turn affects the performance of SERS devices [14]. Traditional SERS substrates are developed on rigid materials such as silicon and glass substrates with fixed hot-spot sizes.The sample loading efficiency for large-size targets into small hot-spots is low, which directly leads to unsatisfactory Raman detection sensitivity [15].Flexible SERS substrates developed based on a flexible matrix, which targets into small hot-spots is low, which directly leads to unsatisfactory Raman detection sensitivity [15].Flexible SERS substrates developed based on a flexible matrix, which enables conformable sampling on arbitrary surfaces, are suitable for in situ and on-site detection of targets [10,16,17].The flexible sampling process can highly improve the detection feasibility, but still with limitations for detecting relatively large-size samples due to the limited hot-spot size [15].Compared to traditional rigid and flexible SERS substrates, stretchable SERS substrates are prepared on stretchable supporting materials which can effectively improve the sample loading efficiency by increasing the distance between metal nanoparticles [18] and then enhance the Raman signal intensity by reducing the distance between metal nanoparticles [19].The integration of stretchable materials with SERS substrates paves the way for the development of high-performance Raman sensors.However, research on the topic of stretchable SERS substrates is still limited. This paper reviews the fabrication and applications of the emerging stretchable SERS substrates (see Figure 1).The working mechanism of SERS and the features of rigid, flexible, and stretchable SERS substrates are briefly overviewed.Stretchable SERS substrates prepared by in situ wet chemical synthesis, physical deposition, physical adsorption, embedding of plasmonic nanostructures in elastomers, and other fabrication techniques including laser ablation and nanocracking are summarized.Subsequently, the applications of stretchable SERS substrates in environmental monitoring, food safety inspection, biomedical analysis, and other aspects are reviewed, followed by discussions on the challenges and prospects of stretchable SERS substrates. A Roadmap to Stretchable SERS Substrates Fundamentals of the SERS Mechanism: A Brief Overview Raman spectroscopy was discovered by the Indian physicist C.V. Raman in 1928 [20], for which he was awarded the Nobel Prize in Physics in 1930.In Raman spectroscopy, a monochromatic laser is directed onto the sample, which causes the molecules in the sample to vibrate.The laser excites the molecule to a higher vibrational energy level, and this excitation energy is then transferred to the surrounding molecules.As the excited molecules return to their original energy state, they emit photons that are scattered in all directions.Most of the scattered photons have the same energy as the incident photons, but a small fraction of the scattered photons have a different energy, which is known as Raman scattering.The energy difference between the incident and scattered photons corresponds A Roadmap to Stretchable SERS Substrates 2.1. Fundamentals of the SERS Mechanism: A Brief Overview Raman spectroscopy was discovered by the Indian physicist C.V. Raman in 1928 [20], for which he was awarded the Nobel Prize in Physics in 1930.In Raman spectroscopy, a monochromatic laser is directed onto the sample, which causes the molecules in the sample to vibrate.The laser excites the molecule to a higher vibrational energy level, and this excitation energy is then transferred to the surrounding molecules.As the excited molecules return to their original energy state, they emit photons that are scattered in all directions.Most of the scattered photons have the same energy as the incident photons, but a small fraction of the scattered photons have a different energy, which is known as Raman scattering.The energy difference between the incident and scattered photons corresponds to the vibrational energy of the molecules which is determined by the symmetricity, bonding, and crystal structure of the molecules.As a consequence, Raman scattering is physically important as a spectroscopic technique that can be used to study the structure, vibrations, and chemical composition of molecules and crystals [9,21,22].However, the intensity of Raman spectroscopy of low-concentration samples is very limited, which inhibits its applications where high sensing sensitivity is required. The discovery of SERS came in the late 1970s, as mentioned above, when Martin Fleischmann and Richard Van Duyne independently discovered that the Raman signal from molecules adsorbed on roughened silver surfaces was dramatically enhanced compared to Raman scattering from the same molecules in solution or on smooth metal surfaces [23].The SERS enhancement mechanism is mainly originated from two aspects.A widely accepted mechanism is called electromagnetic field (EMF) enhancement, in which surface plasmon resonance, the collective oscillation of electrons on a metal surface in response to light, generates a localized electromagnetic field on the metal surface and leads to molecular polarization, which results in a significant increase in the Raman scattering signal [24].The effect of EMF enhancement can be simply described by the following equation: EF = | E loc E 0 | 4 (1) where EF denotes the electromagnetic field enhancement factor, E loc denotes the local electric field excited at the hot-spot, and E 0 denotes the electric field strength of the incident light, and the effect of EMF enhancement is a fourfold relation of the electric field.Another mechanism is chemical enhancement, which refers to the charge transfer or chemical bonding between the metal particles and the molecules, which alter the polarizability and vibrational frequency of the molecules, resulting in the enhancement of the Raman signal.These two mechanisms work together to make SERS an extremely sensitive analytical technique that can potentially be used to analyze trace amounts of molecules even down to individual molecules [25] in various fields such as chemistry, biology, and material sciences.Over the years, researchers have developed a variety of SERS substrates, including roughened metal surfaces, nanoparticles, nanorods, nanoshells, and nanowires made of silver, gold, copper, and other precious metals on various substrates [26].For instance, traditional SERS substrates are developed based on rigid matrices.However, with the development of material sciences, flexible and stretchable materials are applied to assemble SERS substrates for specific applications.Figure 2 illustrates a road map starting from the Raman spectrum to the SERS phenomenon and the development of rigid SERS substrates and flexible SERS substrates and, finally, to the emerging stretchable SERS substrates. Traditional Rigid SERS Substrate Traditional SERS substrates are usually developed based on rigid materials, such as glass, silicon, and porous alumina [27].SERS substrates prepared with rigid materials provide uniform, stable, and reproducible SERS signals.For instance, the SERS substrate prepared by immobilizing silver nanoparticles (AgNPs) on the surface of an organosilanefunctionalized glass showed good mechanical strength and chemical stability and resultant high sensing sensitivity and biocompatibility [28].Silicon wafers were also applied to fabricate the SERS substrate for the detection of DNA, and the result shows that the Si-based SERS substrate has excellent sensitivity and specificity for multiplexed and single-based mismatched DNA detection, which is of great interest in the biological field [29]. However, traditional SERS substrates are rigid and brittle so are not feasible for some specific applications where variable plasmonic nanostructures and conformable sampling are required [30,31].Conventional rigid substrates are usually used to detect molecular samples in liquids by dip-coating or dry samples on object surfaces by contact sampling [32].The sampling processing of dip-coating is relatively complex and the efficiency of contact sampling is limited due to the unconformable contact with non-planar surfaces with the rigid SERS substrates.Similarly, the nanogaps providing the Raman signal are fixed, i.e., the hot-spots cannot be reconfigured once they are formed on the SERS substrates.As a result, large-size samples like viruses and cells can hardly be loaded into the hot-spots, further hindering the usability and sensitivity of traditional SERS substrates in practical applications. Flexible SERS Substrate Flexible SERS substrates are developed based on flexible materials which offer significant advantages over conventional rigid SERS substrates.For example, flexible SERS substrates can highly improve the accessibility of device preparation by cutting the samples into the right size and desired shape, and also enhance the efficiency of sampling by conformable contact scraping or wrapping the SERS substrates around irregular sample surfaces [33].As a consequence, flexible SERS substrates have been researched to compensate for the limitations of traditional SERS substrates.Detection of pesticide residues on the surfaces of fruits [34], in situ detection of trace amount molecules [15], and labelfree detection of phenol-soluble modulins [35] have been achieved by using flexible SERS substrates.In addition, studies have also shown that adjusting the spacing of surface particles can actively change the surface plasmon resonance (SPR), thus optimizing the SPR position and improving the Raman signal [15].However, the hot-spot size of flexible SERS substrates is semi-fixed due to the limited stretchability of the substrate materials, which in turn results in unsatisfactory sampling efficiency and limited detection performance of large-size targets. Stretchable SERS Substrate Stretchable SERS substrates are fabricated by incorporating stretchable materials such as elastomers or polymers with high stretchability into SERS substrates, as mentioned above.Compared to traditional SERS substrates and flexible SERS substates, stretchable SERS substrates can not only retain their original sensing functionality even under the conditions of dynamic mechanical strain or deformation but they also show superb capability in extensively improving sampling efficiency and sensing sensitivity by controlling the layout of nanostructures on the SERS substrates.The enlarging hot-spot size would enable the high-efficiency loading of large-size samples like cells, and the recovering hot-spot size would enhance the magnitude of the electromagnetic field between the hot-spot and, thus, increase the intensity of the SERS signals [36,37].Due to their unique properties, stretchable SERS substrates can be used for the collection and detection of various types of samples (e.g., liquids, solids, and biological samples) in a variety of settings, such as dropping solutions containing organic molecules, inorganics, and biomolecules directly onto the substrate for chemical analysis; swabbing to extract pesticide residues from irregular sample surfaces; and introducing SERS-labeled molecules into biological samples for biomedical research and clinical diagnostics.This review focuses on the fabrication and applications of the emerging stretchable SERS substrates. Fabrication of Stretchable SERS Substrates As mentioned above, methods for preparing stretchable SERS substrates are basically similar to those of preparing traditional and flexible SERS substrates, which involve anchoring plasmonic nanostructures such as nanospheres, nanorods, and nanostars on stretchable materials.However, the preparation of uniform plasmonic nanostructures on flexible and stretchable substrates with high stability is still challenging.Both the size, morphology of plasma nanostructures, and the properties of the stretchable matrix material are important factors that affect the performance of stretchable SERS substrates [38,39].For example, the deciduous nanostructure of the stretchable substrates during dynamic stretching operations would highly affect the stability of the sensors [40].As a result, the material design of the stretchable matrix and the construction of the plasmonic nanostructure on the stretchable supporting material are the key factors for the development of high-performance stretchable SERS substrates. It is noted that most of the fabrication techniques used for preparing stretchable SERS substrates are the same as those of the traditional SERS substrates and flexible SERS substrates.As a consequence, some of the traditional nanofabrication techniques such as e-beam lithography are not presented herein.In this section, the preparation of stretchable SERS substrates is reviewed by six categories including stretchable supporting materials, followed by five typical fabrication techniques: in situ wet chemical synthesis, physical deposition, physical adsorption, and nano-embedding, as well as other unconventional methods such as laser ablation and nanocracking. Stretchable Supporting Materials Materials for the preparation of flexible SERS substrates include nanocellulose [41], cotton fabric [35], graphene/graphene oxide [42], adhesive tapes [43], and so on.All of these materials are flexible and can cover non-planar sample surfaces but with limited stretchability, which prevents their capability to control hot-spot size [44].Stretchable supporting substrates providing the stretchability of the SERS substrates are key to the performance of the SERS device.The materials have to meet the following requirements including good mechanical stretchability for hot-spot size adjustment, high chemical stability to avoid any corrosion and dissolution from the analyte side, high physical stability to ensure the repeatability of the sensing during periodical stretching, and high biocompatibility to meet the biological application needs. A lot of elastomers and polymers with high stretchability have been applied in the development of stretchable SERS substrates.For example, commonly used polymers including polydimethylsiloxane (PDMS) [45], polymethylmethacrylate (PMMA) [46], polyvinyl acetate (PVA) [47], and polyvinylidene fluoride (PVDF) [48] with good stretchability and flexibility have been applied for the development of high-performance flexible/stretchable SERS substrates.To further improve the sampling efficiency of flexible SERS substrates and enhance their capability for hot-spot size control, new elastomers like hydrogels are applied.These materials have been summarized in Table 1, in which the advantages and disadvantages of these materials working as supporting substrates for stretchable SERS sensors are demonstrated. For example, polymers [49], textiles [50], silicone rubber [19], electrical tapes [51], and hydrogels [52] have been widely used to make frames for stretchable SERS substrates.PDMS has the best transparency compared to other types of polymers.High hydrophobicity and Raman enhancement factor at 150% elongation of a micro-nano-structured PDMS substrate were demonstrated by Tan et al. [53].Polycaprolactone (PCL) membrane has ex-cellent flexibility, biodegradability, and biocompatibility, which makes it a suitable material for tape-based stretchable sensor development.The stretchable SERS substrate prepared by decorating Ag nanowire (NW) on the tape showed that an optimized SERS signal can be obtained under the condition of 15% tensile strain [51].Textiles have good toughness, hydrophilicity, and the porous space in the fabric structure allows rapid enrichment of analyte molecules and stabilization of SERS signals in biofluid droplet samples.A SERS substrate prepared using cotton fabrics working under the condition of 30% stretchability was reported by Garg et al. [50].Compared to other materials, silicone rubber and hydrogels with a lower elastic modulus are popular materials for the development of SERS sensors with high stretchability.The gap distance between nanostructures can be tuned easily by applying mechanical stretching to the supporting matrices.For instance, Alexander et al. [19] prepared Au dimers on stretchable silicone rubber membranes that allowed active and reversible tuning of the interparticle gap at levels below 5 nm. Stretchable Substrate Physical Property Advantage Polymer elastomer Stretchable Substrate Physical Property Advantage Polymer elastomer In Situ Wet Chemical Synthesis of Plasmonic Nanostructures In situ wet chemical synthesis refers to the synthesis of plasmonic nanostructures on the surface of stretchable carriers through the reduction of reducing agents, thereby forming nanoparticles or continuous coatings on the carriers [62].In situ wet chemical synthesis is widely used in SERS substrate development because it provides several merits over other methods, including being low cost and easy to scale up where high fabrication yield is required, its capability to tune the size and morphology of nanostructures by controlling the chemical reaction process, and the strong adhesion force between the substrates and the nanostructures enabling stable performance of the sensors. A lot of flexible SERS substrates and stretchable SERS substrates have been developed based on this method [63].For example, a low-cost, easy-to-operate, and stable PDMS-Au nanoparticles (AuNPs) composite membrane formed by dropping gold chloride (HAuCl 4 ) solution directly into a PDMS microchip to construct colorimetric sensors was reported by Wu et al. [64] (Figure 3a).Gold nanostars were also grown on PDMS membranes by using the in situ synthesis method to further improve the flexibility of SERS spectroscopy [65].In this work, AgNPs were synthesized on PDMS and worked as seeds for the growing of Au stars in the following steps.A SERS substrate constructed by Au-coated AgNPs on the PDMS surface can be obtained, as shown in Figure 3b.Chekini et al. [66] reduced metal nanoparticles on a stretchable PDMS substrate in four steps: (i) air plasma treatment, (ii) salinization of the PDMS surface, (iii) deposition of AuNP seeds, and (iv) growing of AuNPs on the PDMS surface, and found that under the condition of mechanical stretching, the particle gap increased in the stretching direction and decreased in the direction perpendicular to the PDMS stretching, resulting in color change of the sample due to the resultant plasma coupling strength change (Figure 3c).The result also indicated that nanoparticles synthesized by the in situ method can adhere to the substrate tightly even when the stretching operation is applied, and the plasma coupling as well as the electromagnetic fields between these nanoparticles can be adjusted by using the stretchable substrate. Due to the reductive nature of 2D nanomaterials like MoS2, metal nanoparticles such as AuNPs can self-assemble directly on the MoS2 surface after reduction, which can effectively adjust the metal nanoparticle gap and improve the performance of photonic devices.As a result, many hybrid nanostructures combined with metals and low-dimensional materials have been developed based on the in situ method [67,68].For example, threedimensional stretchable hybrid substrates have been formed on the surface of graphene and MoS2, and high-sensitive detection of analyte molecules on arbitrary curve structures using these devices was achieved [69,70].Li et al. [71] proposed a stretchable threedimensional AuNPs@MoS2@GF hybrid structure and tested Raman spectra under the condition of different stretching lengths and number of stretches (Figure 3d), showing that the device is highly stable after cycles of 100% stretching and a limit of detection (LOD) as low as 10 −10 M by testing crystal violet (CV) molecules.The authors also demonstrated that the hybrid nanostructure can be used as a cut-paste SERS substrate to cover any shaped surface. In situ wet chemical synthesis of metal nanoparticles is a convenient and simple method for the preparation of SERS active substrates, but this method still has some limitations.Since the plasma metals are randomly arranged on the support substrate, they have poor controllability over size and shape, which in turn leads to the unsatisfactory uniformity and reproducibility of SERS active substrates.Fabrication of highly ordered nanostructures on stretchable substrates while maintaining the dynamic stability of the anchored nanoparticles on various stretchable materials with tunable functionalities is expected. Physical Deposition Method Physical deposition technology refers to the deposition of an ordered array of nanoparticles with specific shapes on supporting substrates [72].This method is usually achieved by physical vapor disposition technology in which condensed phase materials are transited in the vapor phase and then back to the condensed phase and deposited on the supporting substrate under the condition of high vacuum.The depositions often show excellent abrasion resistance and uniform morphology.In addition, this method allows the deposition of a variety of inorganic and organic materials with high controllability and reproducibility, which enables the development of hybrid nanostructures [73,74].The (c) in situ growth of AgNPs on a stretchable PDMS surface in four steps, (i) air plasma treatment, (ii) salinization of the PDMS surface, (iii) deposition of AuNP seeds, (iv) growing of AuNPs on the PDMS surface; (v) a sketch of the experimental setup and (vi) an example of change of color from purple-red to blue-violet during stretching of the sample [66]; (d) fabrication processes of the stretchable hybrid AuNPs@MoS2@GF substrates [71]. Physical Deposition Method Physical deposition technology refers to the deposition of an ordered array of nanoparticles with specific shapes on supporting substrates [72].This method is usually achieved by physical vapor disposition technology in which condensed phase materials are transited in the vapor phase and then back to the condensed phase and deposited on the supporting substrate under the condition of high vacuum.The depositions often show excellent abrasion resistance and uniform morphology.In addition, this method allows the deposition of a variety of inorganic and organic materials with high controllability and reproducibility, which enables the development of hybrid nanostructures [73,74].The surface formed by physical deposition is usually smooth.To enhance the plasma coupling effect, roughened or pre-patterned supporting substrates are usually applied to change the morphology of the metal film. For example, holographic lithography or colloidal lithography [75,76] combined with selective etching [77] on substrates followed by physical deposition of Au are used to pattern uniform plasmonic nanostructures on plane surfaces.Direct coating of Au on stretchable substrates with natural rough surfaces is also a simple method to construct SERS substrates.Alternatively, the rough substrate can be obtained by nanofabrication or soft lithography using templates such as anodic alumina [78,79].For instance, ordered inverse opal array structures composed of PDMS and gold nanoparticles (AuNPs) have been designed for uric acid (UA) detection by using a bottom-up self-assembly method.The periodic distribution of hot-spots was constructed in five steps: assembling of polystyrene (PS) monolayer, casting liquid PDMS, peeling off cured PDMS, sputtering of Au, and transfer of AuNPs film, as sketched in Figure 4a [80]. Interestingly, bioinspired SERS substrates have also been developed for high-performance sensing, in which natural biological materials such as taro leaves [81], reed leaves [49], and cicada wings [82,83] were used as the replication templates.Figure 4b illustrates the procedure of fabricating micro and nanostructured SERS sensors using a natural reed leaf as the template [49].The substrate can be obtained simply in three steps: (i) PDMS template duplication from a reed leaf by soft lithography, (ii) PDMS reed leaf duplication from the PDMS template, and (iii) deposition of Au on the PDMS reed leaf.The detection of metabolites in sweat by using the SERS substrate was achieved. However, most of the substrates prepared by the physical deposition method are prepared on a 2D plane system, which leads to a limited number of hot-spots on the substrates.Recently, Kumar et al. [84] prepared a 3D SERS substrate by using oblique angle deposition (OAD) technology to deposit long silver nanorods (AgNR) onto a PDMS film with a pre-strain of 30%, and bacterial samples were directly added to the substrate (Figure 4c).SERS measurements were carried out on SERS substrates under tensile and released conditions, respectively.The results showed that the Raman signal intensity of the substrate under the condition of the released state was ~10 times higher than that of the pre-stretched condition (Figure 4d).The superb performance of the SERS substrate is due to the 3D cage structure of the nanowrinkled surface with better bacteria and metal nanostructures surface contacts and, thus, more hot-spots.Using nanowrinkling for enhancing the SERS signal was also reported when using shape memory polystyrene substrate [85].This strategy was also reported by Singh et al. [86], in which silver nanorods were deposited on flexible PDMS and polyethylene terephthalate (PET) materials by OAD technology and a silver nanocolumnar film diagram with an inclination angle of >75 • on the surfaces was achieved.In situ SERS measurements of this flexible substrate under tensile strain revealed that after 100 cycles of tensile testing, the nanorods remained undamaged, indicating that the nanostructure fabricated by this method is pretty durable (Figure 4e). Inorganic materials such as graphene are ideal sources for SERS substrate development [87,88].Studies have shown that graphene fold structures can improve the performance of hybrid SERS substrates [89,90].For example, Chen et al. [40] prepared a high-performance hybrid SERS substrate by depositing Au nanoparticles onto a pleated graphene surface (Figure 4f).The height and distribution of the folds in pleated graphene can be tuned by changing the ethanol conditions.The results show that AuNPs are evenly distributed on the pleated graphene surface, and the hybrid substrate is capable of detecting R6G molecules at ultra-low concentrations of 10 −9 M with an enhancement factor (EF) as high as ≈1.19 × 10 7 .The substrate also shows high tensile properties, which can be applied to any curvature surface and can detect multiple analytes at the same time, making it ideal for detecting water contaminants and analyzing crop pesticide residues. SERS substrates prepared by physical deposition technology have good uniformity and reproducibility, offering a powerful tool for creating both flexible and stretchable SERS active substrates.However, this technique still has some limitations.For instance, NPs are directly deposited and assembled onto the surfaces of stretchable polymers.When the substrates are stretched, NPs can easily fall off under the action of tension, resulting in poor stability of the SERS substrates.In addition, physical deposition usually requires special instruments and high vacuum conditions, and special techniques like the above-mentioned OAD are required to obtain patterns with special designs and morphology.SERS substrates prepared by physical deposition technology have good uniformity and reproducibility, offering a powerful tool for creating both flexible and stretchable SERS active substrates.However, this technique still has some limitations.For instance, NPs are directly deposited and assembled onto the surfaces of stretchable polymers.When the substrates are stretched, NPs can easily fall off under the action of tension, resulting in poor stability of the SERS substrates.In addition, physical deposition usually Physical Adsorption Method Physical adsorption is the simplest method to assemble SERS substrates, in which ready-to-use nanomaterials are immobilized onto supporting substrate surfaces through weak bonds such as electrostatic attractions, hydrogen bonding forces, van der Waals forces, or hydrophobic interactions.Before the adsorption operation, the surfaces of the supporting substrates are usually treated by physical or chemical methods, such as changing the surface charge [26] and the hydrophilicity [92] to adjust these interactions.After the modification, ready-to-use plasmonic materials can be transferred onto the substrate, for example, by dip-coating [51], to assemble SERS substrates.The key advantage of the physical adsorption method is that it is easy to operate and cost effective compared to the other fabrication methods. Silver nanowire network film (AgNWNF) has been deposited on PDMS as a substrate to assemble stretchable SERS substrates [26].To firmly adsorb the nanowires, the PDMS substrate surface was treated with ultraviolet ozone (UVO) to generate hydroxyl groups, and the UVO treatment can oxidize and remove grease and organic matter from the substrate surface to achieve a cleaning effect.The excess hydroxyl group can strongly adsorb the deposited AgNWNF on the PDMS surface (Figure 5a).The adhesion strength tests showed that the nanomaterials deposited on the UVO-treated PDMS have better adhesion and stability properties compared to those deposited on the one without UVO treatment.To further enhance the adhesion force between the nanomaterials and the substrates, chemical methods have also been widely applied.The formation of covalent bonds through chemical reactions is often reported on rigid substrates, and many works have also been conducted on flexible or stretchable substrates.Mir-Simon et al. [93] reported the adsorption of AuNPs to the 3-mercaptoproptrimethoxysilane-functionalized PDMS substrate through Au-S chemical bonds and found that the AuNPs array was evenly distributed on the PDMS substrate (Figure 5b).The modification procedures are as follows: the first step is an activation of the PDMS with H 2 O 2 and to salinize the PDMS surface with HCl solution, and then Au nanoparticles were covalently bound to the thiol-terminated PDMS through the salinization groups and Au-S bonds.A tensile stress of 60% was applied to the sample, and the plasmon wavelength decreased linearly from 664 nm to 591 nm, which indicates that the AuNP array fabricated by this method has high stability and uniformity [93]. To further narrow the gap between metal nanoparticles and prepare high-density nanoparticle structures on SERS substrates, a tightly and uniformly arranged AuNP monolayer was autonomously assembled at the water/oil interface and then transferred to a stretchable PDMS substrate by Langmuir-Blodgett transfer technology [94], as shown in Figure 5c [95].This method is very simple and can control the hot-spot size precisely.It is reported that with the increase in the diameter of AuNPs, the average gap size can easily reach below 1 nm.Nanostructure arrays preparing at the organic/water interface were also reported, and by removing the upper organic solution and adding the mixture of PDM on the interface, a stretchable substrate with nanostructure arrays on the cured PDMS surface can be obtained, as shown in Figure 5d [96].By combining the high-density gold nanoparticle monolayer with the stretchable PDMS substrate, the stability, reproducibility, and sensitivity of the SERS substrate can be significantly improved [96]. There are many works related to the physical deposition method; herein, we just listed a few of them.However, immobilization through physical adsorption usually suffers from drawbacks such as desorption of the nanostructures.It is expected that the adsorption of nanostructures can be further improved by pretreatment of the support substrate, which is conducive to improving the stability of SERS detection.Also, it is promising to develop more nanomaterials such as the hybrid nanomaterials to expand the functionality and sensitivity of the SERS sensors.[26]; (b) a scheme of chemical approach to synthesize the derivative substrate [93]; (c) a scheme for fabricating and transferring Au nanoparticle monolayers from the water/hexane interface and an illustration of the SERS experiment [95]; (d) a schematic illustration of as-proposed fabrication process of AgNCs@PDMS SERS substrate [96]. There are many works related to the physical deposition method; herein, we just listed a few of them.However, immobilization through physical adsorption usually suffers from drawbacks such as desorption of the nanostructures.It is expected that the adsorption of nanostructures can be further improved by pretreatment of the support substrate, which is conducive to improving the stability of SERS detection.Also, it is promising to develop more nanomaterials such as the hybrid nanomaterials to expand the functionality and sensitivity of the SERS sensors. Embedding of Nanomaterials Nanomaterials with high surface free energy are easy to agglomerate and oxidize and have poor stability in a free environment.The embedding of plasmonic nanomaterials into host materials such as PDMS can effectively inhibit aggregation of the nanomaterials [26]; (b) a scheme of chemical approach to synthesize the derivative substrate [93]; (c) a scheme for fabricating and transferring Au nanoparticle monolayers from the water/hexane interface and an illustration of the SERS experiment [95]; (d) a schematic illustration of as-proposed fabrication process of AgNCs@PDMS SERS substrate [96]. Embedding of Nanomaterials Nanomaterials with high surface free energy are easy to agglomerate and oxidize and have poor stability in a free environment.The embedding of plasmonic nanomaterials into host materials such as PDMS can effectively inhibit aggregation of the nanomaterials and also create a new type of SERS substrate with enhanced optical properties.The typical embedding method is shown in Figure 6a, in which nanomaterials are adsorbed onto a silica wafer, and a liquid polymer precursor is poured on the solid surface and embeds the particles.After the polymer cures, the nanomaterial-polymer composition is peeled off from the substrate and a transparent and flexible SERS sensor is obtained [97].The optical transparency property of the substrate allows light interaction with the underlying contact surface and the detection of analytes adsorbed on arbitrary surfaces.The sensor has achieved the detection of a trace amount of benzenethiol of 10 −8 M and an EF of ~1.9 × 10 8 .In addition, the SERS sensors can maintain high sensitivity even after 100 cycles of stretching, bending, and torsion deformations (Figure 6b). Other Methods In addition to the above-mentioned fabrication techniques, there are also many unconventional methods by which plasmonic nanostructures can be obtained on stretchable substrates.For example, the laser ablation method has been used to create SERS substrates in an easy, fast, and low-cost manner, in which metal nanoparticles like Ag and Cu can be readily reduced under the condition of high temperature as a laser beam is focused on the sample surface [99,100].Laser-scribed graphene (LSG) technology has been used to develop flexible SERS substrate by generating silver dendrites (AgD) directly on a soft substrate, in which the laser allowed the controlled reduction of graphene oxide for the following growing of AgD by electrodeposition.The SERS signal can be enhanced by the combination of the inherent chemical enhancement of graphene and the electromagnetic enhancement of the Ag nanostructures [90].Figure 7a shows images captured during the fabrication process: (i) a sample of flexible LSG and (ii) GO/LSG, (iii) a zoomed-out view of the LSG surface, and (iv) a zoomed-in view of AgD [90].Similarly, a SERS substrate Most current research has focused on spherical nanostructures, and the tunability of plasma properties of stretchable substrates still needs to be further explored [98].In the work reported by Wen et al. [55], silver-plated gold nanorods were embedded in PDMS to investigate the adjustability of the plasma resonance of the substrate.The preparation process is almost the same as that demonstrated in Figure 6a.First, gold-plated gold nanorods with a silver shell are deposited on a silicon surface, and then the mixture of PDMS is poured onto the silicon and peeled off after the PDMS is cured.Finally, a PDMS device with silver-coated gold nanorods embedded is successfully obtained.Compared with spherical nanorods, silver-coated gold nanorods usually exhibit synthetic tunable plasma resonance over a wider wavelength range and have stronger electric field enhancement, so that the resulting substrate has a high degree of stretchability and stability [55]. The above-mentioned silver-plated gold nanorods are randomly embedded in the elastomer, and the hot-spot structure between the gold nanorods cannot be continuously adjusted.Yan [61] reports an elastic SERS substrate that can simultaneously and continuously tune a large number of AuNP nano-assemblies embedded in the substrate by reversible deformation of the elastic substrate.As shown in Figure 6c, AuNPs are self-assembled into linear nanochains (AuNCs) in an aqueous medium by suitable thiol or phosphine ligands, and then polyvinylpyrrolidone (PVP) powder is added to the AuNCs solution. The obtained solution is dropwise cast on the PDMS substrate for drying, and finally, the blue film is peeled off from the PDMS substrate to form an AuNCs-PVP film, whereby AuNCs are embedded in the PVP matrix.The film is a new type of SERS substrate and the EF can be mechanically modulated in the range of 10 0 to 6.8 × 10 7 , which can be used to directly analyze small molecular analytes in complex biological samples and foods. Nanomaterials are combined with stretchable matrices by embedding, and the surface of the SERS substrate formed is smooth, which makes it easy to cover the sample surface and achieve perfect contact sampling.The unique surface property would also avoid damage to sample surfaces, resulting in higher and more uniform Raman enhancement on the entire With the development of stretchable matrices, a large number of plasmonic nanomaterials can be embedded in the stretchable matrices, which can facilitate the simultaneous and continuous tuning of the SERS enhancement performance via simple mechanical operations. Other Methods In addition to the above-mentioned fabrication techniques, there are also many unconventional methods by which plasmonic nanostructures can be obtained on stretchable substrates.For example, the laser ablation method has been used to create SERS substrates in an easy, fast, and low-cost manner, in which metal nanoparticles like Ag and Cu can be readily reduced under the condition of high temperature as a laser beam is focused on the sample surface [99,100].Laser-scribed graphene (LSG) technology has been used to develop flexible SERS substrate by generating silver dendrites (AgD) directly on a soft substrate, in which the laser allowed the controlled reduction of graphene oxide for the following growing of AgD by electrodeposition.The SERS signal can be enhanced by the combination of the inherent chemical enhancement of graphene and the electromagnetic enhancement of the Ag nanostructures [90].Figure 7a shows images captured during the fabrication process: (i) a sample of flexible LSG and (ii) GO/LSG, (iii) a zoomed-out view of the LSG surface, and (iv) a zoomed-in view of AgD [90].Similarly, a SERS substrate fabricated by femtosecond laser irradiation combined with Au coating and annealing was also reported by Cao et al., and an EF as high as 8.3 × 10 7 was achieved [101].It is easy to achieve large-scale fabrication using the laser ablation method; however, tuning the size and morphology by this method is still challenging.was achieved [101].It is easy to achieve large-scale fabrication using the laser ablation method; however, tuning the size and morphology by this method is still challenging.Breaking metal nanofilm, also called "nanocracking", is also an efficient way to generate plasmonic nanostructures.For example, silver nanopaste (AgNPA) was employed to prepare an ultrasensitive wafer-scale SERS substrate [103].The fabrication process is briefly introduced as follows: AgNPA was spin-coated on Si wafers, and high-density Ag Breaking metal nanofilm, also called "nanocracking", is also an efficient way to generate plasmonic nanostructures.For example, silver nanopaste (AgNPA) was employed to prepare an ultrasensitive wafer-scale SERS substrate [103].The fabrication process is briefly introduced as follows: AgNPA was spin-coated on Si wafers, and high-density Ag nanocracks with small gaps can be spontaneously generated after the evaporation of solvent.The small gaps can provide abundant hot-spots for the ultrasensitive detection of analytes down to single molecules [103].The nanocracking method is a lithography-free method which can also be triggered by machinal stretching.Li et al. reported a large-area pattering of plasmonic nanostructures on ultrathin flexible thermal plastic films through the deposition of Au films, followed by a mechanical stretching process, as shown in Figure 7b.The inset images in Figure 7b illustrate the morphology of the nanocracks under different strains applied.Both the size and arrangement of the nanopatterns can be well controlled by varying the thickness of the nanofilm and the mechanical stretching [60].The authors also demonstrated the transfer of the ultra-flexible sensing films onto curved surfaces for conformable sensing. Another strategy to fabricate high-performance SERS substrates is to combine the listed methods.For example, one can prepare plasmonic nanostructures by in situ synthesis method and then deposit another material onto the nanostructure by a physical deposition method or adsorption process to enhance the functionalities of the substrates.Many devices have been developed based on the hybrid method.For example, a facial method to fabricate a flexible and transparent SERS substrate based on PDMS film modified with a Ag/Au nanowire (NW) forest was reported [102].Au seeds were anchored on the PDMS surface and an in situ chemical synthesis method was applied to grow Au nanowires on the PDMS surface; thereafter, magnetron sputtering coating of Ag nanoparticles was conducted to modify the vertically-aligned Au NWs with Ag nanoparticles (Figure 7c) [15].The Ag/AuNWs/PDMS flexible substrate shows good toughness and a remarkably improved EF value of 6.74 × 10 6 of the Ag/Au NWs/PDMS film was achieved by mechanical stretch.The stretched film is also suitable for the in situ detection of pesticides on the surface of crops, with the advantages of low cost and easy preparation [102].One can believe that the compensation of the fabrication techniques would highly improve the performance of the new-coming SERS substrates. Applications of Stretchable SERS Substrates Stretchable SERS substrates have been developed for a variety of cutting-edge applications, including environmental analysis, food safety and pesticide detection, medical analysis, public safety, and other fields.Here, we briefly describe specific examples of stretchable SERS substrates in the aforementioned fields and both the limitations and the potential of stretchable SERS substrates in these applications are discussed. Environmental Analyses With the rapid development of global industrialization, the problem of environmental pollution is becoming more and more prominent, posing a threat to human existence and the ecological environment, especially from molecular pollutants.Therefore, the development of high-performance SERS substrates for real-time and on-site monitoring of pollutants is urgently needed.Among the current environmental pollution problems, organic pollutants are persistent, difficult to degrade, and highly toxic, posing a significant hazard to the environment.As a result, a lot of stretchable SERS substrates targeting these pollutants have been developed. For example, a stretchable and flexible SERS substrate developed for the detection of organic dyes was reported by Tan et al. [53] The results show that after stretching and shrinking, the PDMS substrate exhibits an irregular wrinkled structure with abundant gaps and grooves that function as hot-spots, thereby improving the testing sensitivity.A detection limit of 1 × 10 −7 M was achieved by using the substrate [53].Benzenethiol (BT), a highly toxic organic pollutant, can contaminate groundwater, watercourses, or sewage systems [104].Kang et al. [18] prepared a stretchable SERS substrate by colloidal lithography followed by electrostatic assembly of gold nanoparticles on the caps.The substrate can be tuned reversibly under external strain and provides a promising mechanism for optimizing the SERS sensitivity (Figure 8a).BT concentrations as low as 10 nM can be detected by using this substrate.The results also show that when detecting BT molecules, the stretchable SERS substrate under the condition of 20% strain is 1000 times more sensitive than that obtained at zero strain (Figure 8b), and the EFs of the sensor under the condition of strains are much higher than those without strain (Figure 8c). detected by using this substrate.The results also show that when detecting BT molecules, the stretchable SERS substrate under the condition of 20% strain is 1000 times more sensitive than that obtained at zero strain (Figure 8b), and the EFs of the sensor under the condition of strains are much higher than those without strain (Figure 8c). Malachite green (MG) is mainly used in aquaculture and industrial production, and its residue is often found in water sources and aquatic products, causing serious harm to human health [105].Based on the stretchable SERS concept, Kumar et al. [81] reported a stretchable biomimetic SERS substrate for the detection of MG (Figure 8d).The substrate was prepared by using taro leaf with microcavities as the template for PDMS replication, followed by the plating of silver nanostructure on the PDMS surface.The Ag-coated microcavity-structured PDMS substrate exhibits high adhesion and hydrophobicity properties, showing a "rose petal effect", which can highly enhance the SERS intensity.A significantly high enhancement factor of ~2.06 × 10 5 and a detection limit of ~10 −11 M for MG with high reproducibility were obtained [81].The applications of stretchable and flexible SERS sensors for monitoring environmental pollutants are still ongoing, and many of them have been extended to fields such as food safety monitoring. Food Safety Monitoring Pesticides, food additives, and other food pollutants can cause food poisoning within a short period and may also accumulate in the human body for a long time.This can lead to fatal harm to humans including but not limited to disrupting body metabolism, damaging genes, and causing cancers [106][107][108].SERS technology has proven to be an effective Malachite green (MG) is mainly used in aquaculture and industrial production, and its residue is often found in water sources and aquatic products, causing serious harm to human health [105].Based on the stretchable SERS concept, Kumar et al. [81] reported a stretchable biomimetic SERS substrate for the detection of MG (Figure 8d).The substrate was prepared by using taro leaf with microcavities as the template for PDMS replication, followed by the plating of silver nanostructure on the PDMS surface.The Ag-coated microcavity-structured PDMS substrate exhibits high adhesion and hydrophobicity properties, showing a "rose petal effect", which can highly enhance the SERS intensity.A significantly high enhancement factor of ~2.06 × 10 5 and a detection limit of ~10 −11 M for MG with high reproducibility were obtained [81].The applications of stretchable and flexible SERS sensors for monitoring environmental pollutants are still ongoing, and many of them have been extended to fields such as food safety monitoring. Food Safety Monitoring Pesticides, food additives, and other food pollutants can cause food poisoning within a short period and may also accumulate in the human body for a long time.This can lead to fatal harm to humans including but not limited to disrupting body metabolism, damaging genes, and causing cancers [106][107][108].SERS technology has proven to be an effective method for the quantitative or qualitative testing of pesticides and other hazards on fruits and vegetables, but the in situ sampling from curved or arbitrary geometry surfaces of fruits and vegetables by traditional rigid SERS substrates is inefficient.Stretchable SERS substrate has better conformable contact with samples and higher loading efficiency than rigid sensors and, thus, higher sensitivity and useability, which can better meet the requirements of food safety analysis.Here, selective application examples of stretchable SERS substrates in the analysis of food contaminants are presented. Pesticides are the most common source of contamination in food and tend to remain on the surface of vegetables and fruits; they can be effectively detected by stretchable SERS substrates via conformable sampling [109].For example, a stretchable SERS substrate for the detection of carbendazim (CBZ) was reported, in which polymorphic silver nanoparticles modified with zinc oxide nanorods were decorated on stretchable substrates by the soft lithography method [109].The 3D hybrid substrate has a high-roughness nanostructure that can enhance the signals of CBZ and achieve a detection limit down to 10 −3 ppm.Similarly, Ma et al. [102] prepared a PDMS membrane-based SERS substrate modified with Ag/Au nanowire forests for trace pesticide detection under various conditions.The SERS activity of the stretchable device can be effectively enhanced by mechanical stretching with an enhancement factor of up to 6.74 × 10 6 .A detection limit of 10 −6 mg/mL for parathion (MP) on tomato surfaces proves that this device is suitable for rapid, on-site detection of pesticides on non-planar surfaces of vegetables and fruits (Figure 9a).Li et al. [60] demonstrate the extension of controlled lithography-free nanofabrication methods for the construction of ultrathin flexible photonic surfaces that can be transferred to curved surfaces of arbitrary topology to form conformal in situ SERS sensors.Thiram residue on apple skin with a detection limit as low as 0.48 ng/cm 2 was realized by using this nanopatterned membrane, as shown in Figure 9b.Liang et al. [110] reported the preparation of Ag-SiO 2 composite nanoparticle structures on stretchable PDMS substrates by co-deposition technique and the detection of thiram on various fruit surfaces.The device showed excellent detection limits of 10 −13 M (Figure 9c).A sandwich-based 3D SERS sensor G@AgNPs@G/PDMS for in situ and label-free detection of malachite green (MG) was reported by Fan et al. [111].The PDMS films can be easily shrunk for 3D structure construction, thus providing advantages in terms of enhancement capabilities and light-matter interactions.Meanwhile, the G@AgNPs@G/PDMS film has a strong adsorption capacity to extract molecules from food surfaces.Detection of MG on fish surfaces as low as 10 −13 M was presented.Another stretchable SERS substrate developed based on a biocompatible poly(ε-caprolactone) membrane embedded with a 3D periodic wavy microstrip array and an array of nanoslots along the stretching direction was also used to detect MG [57].The stretched polymer surface with plasmon resonance nanostructures can produce more than 10-fold signal enhancement compared to the unstretched membranes.The results show that in situ detection of MG molecules as low as 0.1 µM on irregular surfaces of green mussels by using the Ag-deposited stretched substrate can be achieved (Figure 9d).Food additives are chemically synthesized or natural substances that can be added to food to improve the color, aroma, and taste of the food, or to achieve certain preservative effects [113][114][115].However, the misuse of food additives can adversely affect human health.Kitahama et al. [47] reported an ultrathin, stretchable, and adhesive "Place&Play SERS" substrate, which was fabricated by deposition of gold on polyvinyl alcohol (PVA) nanogrid substrate for the detection of the biocide methylchloroisothiazolinone (CMIT) on orange peels (Figure 9e).The gold/PVA nanonet substrate showed high SERS signal enhancement of ~10 8 which enabled the detection of ~10 nM R6G with high reproducibility.Barveen et al. [112] synthesized gold nanostars (AuNSs) directly on polymethylmethacrylate (PMMA) membranes for surface-enhanced Raman spectroscopy (SERS) applications.The transparent and flexible AuNSs/PMMA SERS substrate allowed for the real-time in situ detection of ciprofloxacin (CPX) and chloramphenicol (CAP) on chicken wing samples through the use of a fiber-coupled Raman probe (Figure 9f).The membrane substrate exhibits high sensitivity, high enhancement factor (2.03 × 10 9 ), low detection limit (3.41 × 10 −11 M), and superior multi-detection capabilities including good homogeneity and reproducibility (<7.32%).Tang et al. [116] constructed a flexible dual-plasma SERS substrate for the detection of residuals of triclosan in food samples.Due to the enhanced electromagnetic field from the plasma coupling of the AuNP arrays and aligned AgNWs, the substrate exhibits good sensitivity and reproducibility in a variety of situations, including 100 cycles of stretching, bending, and twisting, with a detection limit for melamine as low as 10 −8 M. From the above examples, one can see that stretchable SERS substrates have been powerful tools in monitoring the misuse of food additives or illegal additions, especially where non-destructive, real-time, and in situ detection is needed. Biomedical Applications In recent years, SERS has gradually become a promising sensing technology in the fields of biomedical research, and many stretchable SERS sensors have been developed to meet the increasing needs in these fields such as healthcare monitoring and diagnosis of diseases. For example, pseudomonas aeruginosa is a conditionally pathogenic bacterium that prefers moist environments and colonizes infection-prone injury sites, making it one of the major pathogens in hospital-acquired infections.Traditional techniques for identifying infectious agents often require prior cultivation in appropriate media, which is time consuming.Stretchable SERS plays an important role in pathogen detection, especially where high-sensitive detection is needed.For example, a stretchable SERS substrate prepared using silver nanorod arrays on PDMS showed that the bent substrate is much more sensitive than the regular substrate when detecting the same concentration of bacterial suspension [84]. In addition to detecting pathogens, stretchable SERS substrates are also used to detect biomarkers for clinical diagnostics, where usually molecular biomarkers in human body fluids are detected [117].Al et al. [61] embedded plasmonic gold nanoclusters (AuNC) in polyvinylpyrrolidone (PVP) substrates for the detection of hypoxanthine in serum.The tunability of the enhancement factor in the range of 100~6.8× 10 7 was achieved by adjusting the nanostructure and the properties of the substrate array through reversible mechanical deformation.At the same time, the PVP substrate allows diffusion of small molecule markers into the hot-spots, which in turn excludes interference from other components.Liu et al. [118] demonstrated a highly scalable, low-cost, ultrathin, stretchable, wearable SERS sensor fabricated based on gold nanonet (Figure 10a).The sensor can be fabricated into any shape and worn on virtually any surface for label-free, large-scale in situ sensing of a wide range of analytes on virtually any arbitrary surface.The wearable SERS sensors have demonstrated excellent utility in detecting human sweat biomarkers.A multifunctional wearable e-skin with hierarchical micro-nanostructures for the detection of metabolites in human sweat was also reported, as shown in the schematic in Figure 10b [49].The Au-coated PDMS reed leaves are thin, pliable, and soft, making them suitable as a wearable sensor for lactic acid and fatty acids detection from sweat samples [49].A novel erasable and reproducible plasmonic hot-spot developed by synthesizing silver nanostructures on liquid metal was reported recently, and the hot-spots can be removed in alkaline solution.The self-adhesive, biocompatible, and stretchable device is able to detect glucose concentrations as low as 1 ng/L [119].Stretchable SERS substrates can also integrate with other technologies such as paperbased microfluidics to achieve desired functions.For example, a paper-based wearable sensor for continuous and simultaneous quantitative analysis of sweat loss, perspiration Stretchable SERS substrates can also integrate with other technologies such as paperbased microfluidics to achieve desired functions.For example, a paper-based wearable sensor for continuous and simultaneous quantitative analysis of sweat loss, perspiration rate, and metabolites in sweat was developed by Mogera et al. [120], in which a serpentine paper layer provides mechanical strain isolation, making the device soft, flexible, and stretchable (Figure 10c).The sensor is capable of sensitively detecting and quantifying uric acid (UA) in sweat at concentrations as low as 1 µM [120].Similarly, Chowdhury et al. [56] constructed heart-shaped nanoparticle dimer arrays on PDMS substrates with high aspect ratios, which can maintain reliable SERS performance even under bending (up to 100 • angle) or stretching (up to 50% stretching) conditions.The device is promising for applications in the field of personalized medicine and remote patient monitoring (Figure 10d).In addition, a SERS substrate for the detection of hydrophobic biomolecules developed by depositing conjugated molecules and Au particles on PDMS was reported by Wang et al. [121].The substrate showed acceptable efficiency due to the tip-focusing effect and high durability even after 100-cycle bending by testing cholecalciferol at ultra-low concentrations (10 −10 M). Other Applications In addition to environmental analysis, food safety monitoring, and biomedical uses, stretchable SERS substrates have also been widely used for public safety inspections.Illicit drugs pose a serious threat to social security and physical health, such as cocaine, a common drug, which can affect the brain and other vital organs when ingested in large quantities and seriously jeopardize lives [122,123].A stretchable SERS substrate with high sensitivity is an excellent candidate for the tests.For instance, Zhang et al. reported a wearable glove-based SERS for rapid sampling and on-site detection of trace amounts of tramadol and midazolam at concentrations of 69.19 ng/mL and 35.03 ng/mL, respectively, as well as for the accurate identification of methamphetamine in complex binary mixtures [124].The efficient analytes extraction was achieved by the design of the substrate with flexible adhesive tape, as shown in the schematic diagram demonstrated in Figure 11a [124].Maddipatla et al. [125] developed a novel pleated-structure-based SERS substrate for the detection of cocaine.The substrate was developed by depositing silver nanoparticle (AgNP) ink onto a pre-stretched thermoplastic polyurethane (TPU) substrate using gravure printing, followed by the release of the TPU (Figure 11b).Dense hot-spots were formed on the pleated structure, and the detection of cocaine under the condition of various stretches was conducted as shown in Figure 11c [125]. The stretchable property of the SERS substrate is also suitable for applications in the biological field.As discussed above, large-size (compared to molecules) cells and proteins can be loaded in hot-spots with high efficiency via elastic stretching and achieve high sensing sensitivity by releasing the stretching.For example, a SERS substrate with active hot-spots constructed on hydrogels for detecting cytochrome and crystal violet was reported by Mitomo et al. [126].The upper sketch in Figure 11d shows the preparation of the tunable plasmonic nanostructure by the self-assembling of AuNPs on glass, followed by the transfer of AuNPs onto a piece of gel membrane, and the lower sketch in Figure 11d demonstrates the working procedure of detecting target molecules by contraction of AuNPs. Figure 11e,f present Raman signals of cytochrome c and crystal violet under three conditions: (a) "open form", where analytes were injected into a gel in an expanded state, (b) "closed form", where analytes were injected into a gel in a contracted state, and (c) active gap control as "open-to-closed form", where target molecules were injected into an expanded state gel and analyzed after contraction.The results indicate that active control of stretchable SERS substates can highly enhance the sensing sensitivity. Stretchable SERS substrates have been proven to be a powerful tool for the identification of molecules.The applications of SERS substrates can be extended to many fields in the future, including but not limited to the detection of trace amounts of explosive substances and illicit drugs in safety inspections and also biomolecule detection in biology research.as "open form"-the approach where analytes were injected onto a gel in an expanded state (in MilliQ water) and analyzed as it is; b) no gap control as "closed form"-the approach where analytes were injected onto a gel in a contracted state (in 1 M NaCl solution) and analyzed as it is; and c) active gap control as "open−to−closed form"-the approach that target molecules were injected onto a gel is an expanded state (in MilliQ water) and analyzed after contraction (in 1 M NaCl solution) [126]. Conclusions and Remarks Stretchable SERS substrates have attracted significant interest in recent years due to their outstanding performance and potential applications in various fields such as environmental monitoring, food safety inspection, and biomedical analysis.In this review, the recent development and applications of the stretchable SERS substrates are summarized, and a roadmap for the development of SERS substrates is introduced.Fabrication techniques for stretchable SERS substrates and the cutting-edge applications of these stretchable SERS-active substrates are introduced.This review provides an overview of the development of SERS substrates and sheds light on the design, fabrication, and application of novel, highly efficient stretchable SERS systems, especially for future applications where tunable hot-spots and Raman spectra are needed. The development of stretchable SERS substrates is still in its early stages, and many challenges need to be addressed, such as reproducibility, scalability, and cost.Addressing as "open form"-the approach where analytes were injected onto a gel in an expanded state (in MilliQ water) and analyzed as it is; b) no gap control as "closed form"-the approach where analytes were injected onto a gel in a contracted state (in 1 M NaCl solution) and analyzed as it is; and c) active gap control as "open−to−closed form"-the approach that target molecules were injected onto a gel is an expanded state (in MilliQ water) and analyzed after contraction (in 1 M NaCl solution) [126]. Conclusions and Remarks Stretchable SERS substrates have attracted significant interest in recent years due to their outstanding performance and potential applications in various fields such as environmental monitoring, food safety inspection, and biomedical analysis.In this review, the recent development and applications of the stretchable SERS substrates are summarized, and a roadmap for the development of SERS substrates is introduced.Fabrication techniques for stretchable SERS substrates and the cutting-edge applications of these stretchable SERS-active substrates are introduced.This review provides an overview of the development of SERS substrates and sheds light on the design, fabrication, and application of novel, highly efficient stretchable SERS systems, especially for future applications where tunable hot-spots and Raman spectra are needed. The development of stretchable SERS substrates is still in its early stages, and many challenges need to be addressed, such as reproducibility, scalability, and cost.Addressing these challenges can lead to the development of more efficient and practical stretchable SERS substrates.The continued exploration of new stretchable supporting material, surface functionalization strategies, and nanofabrication techniques can also lead to discoveries and advancements of new stretchable SERS substrates.For example, the mechanical properties of the substrate must be optimized to ensure that the substrates can be stretched without breaking or losing their SERS activity; the surface functionalization methods should be improved to further enhance the adhesion force between plasmonic nanostructures and the stretchable substrates to avoid any deciduous behaviors; and the plasmonic properties of the nanostructure should also be promoted by optimizing the design of the nanomaterial composite and the shape and size of the nanostructure.Additionally, it is also important to create new SERS substrates with advancements such as renewability, biodegradability, and reducibility to fulfill the needs of applications. Figure 1 . 1 Figure 1.Fabrication and applications of stretchable SERS substrates. Figure 1 . 1 Figure 1.Fabrication and applications of stretchable SERS substrates. Figure 2 . 2 Figure 2. Evolution of stretchable SERS substrates. PDMS ~150% elongationEasy to obtain, low cost, good flexibility and optical transparency (~100%) 13, x FOR PEER REVIEW PDMS ~150% elongationEasy to obtain, low cost, good flexibility and optical transparency (~100%) PCL ~650% elongation Good transparency (~90%) and tempera ture stability (9.62%) 31 Figure 3 . 313 Figure 3. Cont. Figure 3 . 3 Figure 3.In situ wet chemical synthesis of plasmonic nanostructures.(a) In situ synthesis of AuNPs on a PDMS chip (i) with HAuCl4 solution in the reservoir and (ii) AuNPs grown on the PDMS surface [64]; (b) schematic illustration of in situ growth of AgNPs@Au-shell on a piece of PDMS [65];(c) in situ growth of AgNPs on a stretchable PDMS surface in four steps, (i) air plasma treatment, (ii) salinization of the PDMS surface, (iii) deposition of AuNP seeds, (iv) growing of AuNPs on the PDMS surface; (v) a sketch of the experimental setup and (vi) an example of change of color from purple-red to blue-violet during stretching of the sample[66]; (d) fabrication processes of the stretchable hybrid AuNPs@MoS2@GF substrates[71]. Figure 3 . 3 Figure 3.In situ wet chemical synthesis of plasmonic nanostructures.(a) In situ synthesis of AuNPs on a PDMS chip (i) with HAuCl4 solution in the reservoir and (ii) AuNPs grown on the PDMS surface [64]; (b) schematic illustration of in situ growth of AgNPs@Au-shell on a piece of PDMS [65];(c) in situ growth of AgNPs on a stretchable PDMS surface in four steps, (i) air plasma treatment, (ii) salinization of the PDMS surface, (iii) deposition of AuNP seeds, (iv) growing of AuNPs on the PDMS surface; (v) a sketch of the experimental setup and (vi) an example of change of color from purple-red to blue-violet during stretching of the sample[66]; (d) fabrication processes of the stretchable hybrid AuNPs@MoS2@GF substrates[71]. Figure 4 . 4 Figure 4. Physical deposition of SERS substrates.(a) A schematic illustration of SERS substrate preparation by physical deposition method and the detection of uric acid in tears [80]; (b) a schematic illustration of plasmonic nanostructure fabrication using a natural reed leaf as the template [49]; (c) a schematic illustration of the fabrication of the AgNR−PDMS substrate and schematic showing P. aeruginosa on (i) pre−stretched and (ii) wrinkled AgNR−PDMS substrates [84,91]; (d) SERS spectra of P. aeruginosa on pre−stretched and released (wrinkled) AgNR−PDMS substrates; (e) SEM images of a flexible AgNR−PDMS substrate before and after one hundred tensile loading cycles [86]; (f) a schematic illustration of the preparation of WG/AuNPs hybrid SERS platform [40]. Figure 4 . 4 Figure 4. Physical deposition of SERS substrates.(a) A schematic illustration of SERS substrate preparation by physical deposition method and the detection of uric acid in tears [80]; (b) a schematic illustration of plasmonic nanostructure fabrication using a natural reed leaf as the template [49]; (c) a schematic illustration of the fabrication of the AgNR−PDMS substrate and schematic showing P. aeruginosa on (i) pre−stretched and (ii) wrinkled AgNR−PDMS substrates [84,91]; (d) SERS spectra of P. aeruginosa on pre−stretched and released (wrinkled) AgNR−PDMS substrates; (e) SEM images of a flexible AgNR−PDMS substrate before and after one hundred tensile loading cycles [86]; (f) a schematic illustration of the preparation of WG/AuNPs hybrid SERS platform [40]. Figure 5 . 5 Figure 5. Plasmonic nanostructure fabricated by physical adsorption method.(a) Schematic illustration of fabricating AgNWNFs-deposited SERS platform on PDMS nanowrinkles[26]; (b) a scheme of chemical approach to synthesize the derivative substrate[93]; (c) a scheme for fabricating and transferring Au nanoparticle monolayers from the water/hexane interface and an illustration of the SERS experiment[95]; (d) a schematic illustration of as-proposed fabrication process of AgNCs@PDMS SERS substrate[96]. Figure 5 . 5 Figure 5. Plasmonic nanostructure fabricated by physical adsorption method.(a) Schematic illustration of fabricating AgNWNFs-deposited SERS platform on PDMS nanowrinkles[26]; (b) a scheme of chemical approach to synthesize the derivative substrate[93]; (c) a scheme for fabricating and transferring Au nanoparticle monolayers from the water/hexane interface and an illustration of the SERS experiment[95]; (d) a schematic illustration of as-proposed fabrication process of AgNCs@PDMS SERS substrate[96]. Figure 6 . 6 Figure 6.Fabrication of SERS substrates by embedding nanomaterials in elastomers.(a) A general schematic illustration of the fabrication of flexible SERS sensor by embedding nanomaterials in elastomers and (b) the typical working statutes of the stretchable SERS substrate [97]; (c) a schematic of the preparation procedure of AuNCs-PVP film and the detection of small molecule analyte [61]. Figure 6 . 6 Figure 6.Fabrication of SERS substrates by embedding nanomaterials in elastomers.(a) A general schematic illustration of the fabrication of flexible SERS sensor by embedding nanomaterials in elastomers and (b) the typical working statutes of the stretchable SERS substrate [97]; (c) a schematic of the preparation procedure of AuNCs-PVP film and the detection of small molecule analyte [61]. Nanomaterials 2023 , 2023 13, x FOR PEER REVIEW 16 of 31 fabricated by femtosecond laser irradiation combined with Au coating and annealing was also reported by Cao et al., and an EF as high as 8.3 × 10 7 Figure 7 . 7 Figure 7. Cont. Figure 7 . 7 Figure 7.Other methods for stretchable SERS substrate fabrication.(a) Laser ablation method and example images captured during the fabrication process: (i) sample of flexible LSG, (ii) GO/LSG, (iii) zoomed-out view of the LSG surface, (iv) zoomed-in view of AgD; (b) a schematic illustration of the fabrication of the ultra-flexible SERS substrate by nanocracking and the images of the nanocracks [90]; (c) a schematic illustration of the hybrid methods for the preparation of stretched Ag/Au NWs/PDMS film[102]. Figure 7 . 7 Figure 7.Other methods for stretchable SERS substrate fabrication.(a) Laser ablation method and example images captured during the fabrication process: (i) sample of flexible LSG, (ii) GO/LSG, (iii) zoomed-out view of the LSG surface, (iv) zoomed-in view of AgD; (b) a schematic illustration of the fabrication of the ultra-flexible SERS substrate by nanocracking and the images of the nanocracks [90]; (c) a schematic illustration of the hybrid methods for the preparation of stretched Ag/Au NWs/PDMS film [102]. Figure 8 . 8 Figure 8. Stretchable SERS sensors for environmental analysis.(a) AFM images of tunable SERS substrate surface under the condition of stretching; (b) Raman spectra of 1 μM BT−adsorbed plasmonic cap arrays as a function of the applied strain and (c) comparison of the enhancement factor [18]; (d) schematic of detecting MG by using the micro-cavity array PDMS SERS substrate and the Raman signals [81]. Figure 8 . 8 Figure 8. Stretchable SERS sensors for environmental analysis.(a) AFM images of tunable SERS substrate surface under the condition of stretching; (b) Raman spectra of 1 µM BT−adsorbed plasmonic cap arrays as a function of the applied strain and (c) comparison of the enhancement factor [18]; (d) schematic of detecting MG by using the micro-cavity array PDMS SERS substrate and the Raman signals [81]. Nanomaterials 2023 , 31 Figure 9 . 2023319 Figure 9. Detection of pesticides by using stretchable SERS substrates.(a) Schematic of the application of Ag/Au NWs/PDMS films for pesticide testing[102]; (b) photography of basil leaves and apples covered with ultra-flexible gold nano-patterned film and respective surface morphology micrographs[60]; (c) SERS spectra of thiram on various fruits peels[110]; (d) schematic of contacting polymer film onto the green mussel[57]; (e) SERS spectra (solid curves) of an aqueous solution of CMIT at various concentrations on the bottom surface of the gold/PVA nanomesh substrate on an orange peel[47]; (f) in situ detection of CPX and CAP on the chicken wing surface using the flexible Au−NSs/PMMA SERS substrate by the 532 nm excitation[112]. Nanomaterials 2023 , 31 Figure 10 . 20233110 Figure 10.Stretchable SERS substrates for biomedical applications.(a) Concept of wearable SERS on the skin [118]; (b) a schematic of the multifunctional wearable electronic skin for the detecting human sweat [49]; (c) a schematic diagram of paper-based device twisting, deformation, and portable Raman measurement [120]; (d) a schematic diagram of a plasma exciton SERS sensor for detecting wrist sweat [56]. Figure 10 . 10 Figure 10.Stretchable SERS substrates for biomedical applications.(a) Concept of wearable SERS on the skin [118]; (b) a schematic of the multifunctional wearable electronic skin for the detecting human sweat [49]; (c) a schematic diagram of paper-based device twisting, deformation, and portable Raman measurement [120]; (d) a schematic diagram of a plasma exciton SERS sensor for detecting wrist sweat [56]. Figure 11 . 11 Figure 11.Other applications of stretchable SERS substrates.(a) Schematic diagram of the glovebased SERS sensor[124]; (b) a schematic illustration of the fabrication of SERS substrate using gravure printing process and (c) Raman signal of cocaine detected by the wrinkle−structured SERS substrate [125]; (d) preparation of a tunable plasmonic nanostructure and its applications for (e) cytochrome c and (f) crystal violet detection, where a) no gap control as "open form"-the approach where analytes were injected onto a gel in an expanded state (in MilliQ water) and analyzed as it is; b) no gap control as "closed form"-the approach where analytes were injected onto a gel in a contracted state (in 1 M NaCl solution) and analyzed as it is; and c) active gap control as "open−to−closed form"-the approach that target molecules were injected onto a gel is an expanded state (in MilliQ water) and analyzed after contraction (in 1 M NaCl solution)[126]. Figure 11 . 11 Figure 11.Other applications of stretchable SERS substrates.(a) Schematic diagram of the glovebased SERS sensor [124]; (b) a schematic illustration of the fabrication of SERS substrate using gravure printing process and (c) Raman signal of cocaine detected by the wrinkle−structured SERS substrate [125]; (d) preparation of a tunable plasmonic nanostructure and its applications for (e) cytochrome c and (f) crystal violet detection, where a) no gap control as "open form"-the approach where analytes were injected onto a gel in an expanded state (in MilliQ water) and analyzed as it is; b) no gap control as "closed form"-the approach where analytes were injected onto a gel in a contracted state (in 1 M NaCl solution) and analyzed as it is; and c) active gap control as "open−to−closed form"-the approach that target molecules were injected onto a gel is an expanded state (in MilliQ water) and analyzed after contraction (in 1 M NaCl solution)[126]. Table 1 . 1 Stretchable material used as matrix of SERS substrates. Nanomaterials 2023, 13, x FOR PEER REVIEWStretchable SubstratePhysical PropertyAdvantageDisadvantageRef.PDMSPolymer elastomer Table 1 . 1 Stretchable material used as matrix of SERS substrates. Nanomaterials 2023, 13, x FOR PEER REVIEWStretchable SubstratePhysical PropertyAdvantagePDMS~150% elongation ~150% elongation Easy to obtain, low cost, Easy to obtain, low cost, good flexibility and optical transparency (~100%) good flexibility, and optical transparency (~100%) Not resistant to high temperature [53-56]PCLPCL~650% elongationGood transparency (~90%) and tempera ture stability (9.62%)Polymer elastomerPMMA~3160 MPa elastic modulusExcellent optical transparency (~92%) an good flexibilityPVA4400~5400 MPa elastic modulusUltrathin, flexible, stretchable, adhesive and bio-integrablePC~2200 MPa elastic modulusHigh tensile, flexural, and compressive strengthsPVP-Biocompatible, highly plastic, and adhesivTextiles-~30% elongationWashable and reusableSilicone rubber-200~900 MPa elastic modulusReversible gap adjustmentElectrical tape-~150% elongationPaste and peel off and cost-effectiveHydrogelsPoly (acrylic acid)1~100 kPa elastic modulusStimulus responsiveness, large volume change, and biocompatibility Table 1 . 1 Stretchable material used as matrix of SERS substrates. Nanomaterials 2023, 13, x FOR PEER REVIEWStretchable SubstratePhysical PropertyAdvantagePDMS~150% elongationEasy to obtain, low cost, good flexibility and optical transparency (~100%)PCL~650% elongation ~650% elongation Good transparency (~90%) Good transparency (~90%) and tempera ture stability (9.62%) and temperature stability (9.62%) Insufficient mechanical strength [57]Polymer elastomerPMMA PMMAelastic modulus ~3160 MPagood flexibility Excellent optical transparency (~92%) anPVA4400~5400 MPa elastic modulusUltrathin, flexible, stretchable, adhesive and bio-integrablePC~2200 MPa elastic modulusHigh tensile, flexural, and compressive strengthsPVP-Biocompatible, highly plastic, and adhesiTextiles-~30% elongationWashable and reusableSilicone rubber-200~900 MPa elastic modulusReversible gap adjustmentElectrical tape-~150% elongationPaste and peel off and cost-effectiveHydrogelsPoly (acrylic acid)1~100 kPa elastic modulusStimulus responsiveness, large volume change, and biocompatibility Table 1 . 1 Stretchable material used as matrix of SERS substrates. Stretchable SubstratePhysical PropertyAdvantagePDMS~150% elongationEasy to obtain, low cost, good flexibility and optical transparency (~100%)PCL~650% elongationGood transparency (~90%) and tempera ture stability (9.62%)Polymer elastomerPMMA~3160 MPa elastic modulus Excellent optical ~3160 MPa good flexibility elastic modulus transparency (~92%) and Excellent optical transparency (~92%) an good flexibility Low surface to scratch hardness, easy [58,59]PVA4400~5400 MPa elastic modulusUltrathin, flexible, stretchable, adhesive and bio-integrablePC~2200 MPa elastic modulusHigh tensile, flexural, and compressive strengthsPVP-Biocompatible, highly plastic, and adhesiTextiles-~30% elongationWashable and reusableSilicone rubber-200~900 MPa elastic modulusReversible gap adjustmentElectrical tape-~150% elongationPaste and peel off and cost-effectiveHydrogelsPoly (acrylic acid)1~100 kPa elastic modulusStimulus responsiveness, large volume change, and biocompatibility PVANanomaterials 2023, 13, x FOR PEER REVIEW Table 1 . 1 Stretchable material used as matrix of SERS substrates. 4400~5400 MPa elastic modulusUltrathin, flexible, stretchable, adhesive, and bio-integrablePoor water resistance[47]Textiles-~30% elongationWashable and reusableSilicone rubber-200~900 MPa elastic modulusReversible gap adjustmentElectrical tape-~150% elongationPaste and peel off and cost-effectiveHydrogelsPoly (acrylic acid)1~100 kPa elastic modulusStimulus responsiveness, large volume change, and biocompatibility Stretchable Substrate Physical Property Advantage Polymer elastomer PDMS ~150% elongation Easy to obtain, low cost, good flexibility, and optical transparency (~100%) PCL ~650% elongation Good transparency (~90%) and temperature stability (9.62%) PMMA ~3160 MPa elastic modulus Excellent optical transparency (~92%) and good flexibility PVA 4400~5400 MPa elastic modulus Ultrathin, flexible, stretchable, adhesive, and bio-integrable PC ~2200 MPa elastic modulus High tensile, flexural, and compressive strengths PVP -Biocompatible, highly plastic, and adhesiv PC Nanomaterials 2023, 13, x FOR PEER REVIEW Table 1 . 1 Stretchable material used as matrix of SERS substrates. Table 1 . 1 Stretchable material used as matrix of SERS substrates. -Biocompatible, highly plastic, and adhesive-[61]Textiles Textiles Silicone rubber Silicone rubber Electrical tape-----~30% elongation ~30% elongation Washable and reusable 200~900 MPa Reversible gap adjustment elastic modulus 200~900 MPa elastic modulus ~150% elongation Paste and peel off and cost-effectiveNot resistant to high temperature Washable and reusable [50] Low tensile strength [19] Reversible gap adjustment Non-biodegradable [51]Electrical tape-~150% elongationPaste and peel off and cost-effectiveHydrogelsPoly (acrylic acid)1~100 kPa elastic modulusStimulus responsiveness, large volume change, and biocompatibility Stretchable Substrate Physical Property Advantage Polymer elastomer PDMS ~150% elongation Easy to obtain, low cost, good flexibility, and optical transparency (~100%) PCL ~650% elongation Good transparency (~90%) and temperature stability (9.62%) PMMA ~3160 MPa elastic modulus Excellent optical transparency (~92%) and good flexibility PVA 4400~5400 MPa elastic modulus Ultrathin, flexible, stretchable, adhesive, and bio-integrable PC ~2200 MPa elastic modulus High tensile, flexural, and compressive strengths PVP -Biocompatible, highly plastic, and adhesiv Hydrogels Poly (acrylic acid) Nanomaterials 2023, 13, x FOR PEER REVIEW Table 1 . 1 Stretchable material used as matrix of SERS substrates. Nanomaterials 2023, 13, x FOR PEER REVIEW Funding: This research was funded by the National Natural Science Foundation of China (83121063, 83122015), the Fundamental Research Funds for the Central Universities of China (3132023210, 3132023506) and Natural Science Foundation of Liaoning Province (2022-MS-156), and China Postdoctoral Science Foundation (2022MS720621), and the APC was funded by the National Natural Science Foundation of China (83122015).Author Contributions: R.P., T.Z. and S.Y. wrote the review, Y.S., X.L. and J.W. edited the manuscript, R.P., X.L. and J.W. coordinated and supervised this project.All authors have read and agreed to the published version of the manuscript.Conflicts of Interest:The authors declare no conflict of interest. Surface Raman Spectroelectrochemistry Part I. Heterocyclic, Aromatic, and Aliphatic Amines Adsorbed on the Anodized Silver Electrode. D Jeanmaire, R P Van Duyne, 10.1016/S0022-0728(77)80224-6J. Electroanal. 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Daytime napping, biological aging and cognitive function among middle-aged and older Chinese: insights from the China health and retirement longitudinal study 17 November 2023 Huiyi Wu Department of Epidemiology and Biostatistics School of Public Health and West China Fourth Hospital West China Sichuan University ChengduChina Lei Huang School of Medicine West China Hospital West China Sichuan University ChengduChina Shushan Zhang Department of Neurology Affiliated Hospital of North Sichuan Medical College NanchongChina Yang Zhang yangzhang0729@hotmail.com Department of Periodical Press and National Clinical Research Center for Geriatrics West China Hospital Sichuan University ChengduChina Chinese Evidence-Based Medicine Center West China Hospital Sichuan University ChengduChina Yajia Lan lanyajia@sina.com Department of Environmental Health and Occupational Medicine School of Public Health and West China Fourth Hospital Sichuan University ChengduWest ChinaChina Stevo Popovic Yongjie Chen Sup Amornpinyo Adriana Ladeira De Araújo University of Montenegro Montenegro Tianjin Medical University China Morteza Taher University of Tehran Iran Khon Kaen University Thailand University College Dublin Ireland Daytime napping, biological aging and cognitive function among middle-aged and older Chinese: insights from the China health and retirement longitudinal study 17 November 202394FC830C3ADE39CC74C4D3D0DC3A112610.3389/fpubh.2023.1294948RECEIVED 15 September 2023 ACCEPTED 06 November 2023cognitive agingAlzheimer's diseaseneurodegenerative diseaselongevitygerosciencebiological aging Objective: The complicated association of daytime napping, biological aging and cognitive function remains inconclusive.We aimed to evaluate the cross-sectional and longitudinal associations of daytime napping and two aging measures with cognition and to examine whether napping affects cognition through a more advanced state of aging.Methods: Data was collected from the China Health and Retirement Longitudinal Study.Napping was self-reported.We calculated two published biological aging measures: Klemera and Doubal biological age (KDM-BA) and physiological dysregulation (PD), which derived information from clinical biomarkers.Cognitive z-scores were calculated at each wave.Linear mixed models were used to explore the longitudinal association between napping, two aging measures, and cognitive decline.Mediation analyses were performed to assess the mediating effects of biological age acceleration on the association between napping and cognition.Results: Participants aged over 45 years were included in the analyses.Nonnappers had greater KDM-BA and PD [LS means (LSM) = 0.255, p = 0.007; LSM = 0.085, p = 0.011] and faster cognitive decline (LSM = −0.061,p = 0.005) compared to moderate nappers (30-90 min/nap).KDM-BA (β = −0.007,p = 0.018) and PD (β = −0.034,p < 0.001) showed a negative association with overall cognitive z scores.KDM-BA and PD partially mediated the effect of napping on cognition.Conclusion:In middle-aged and older Chinese, compared to moderate nappers, non-nappers seem to experience a more advanced state of aging and increased rates of cognitive decline.The aging status possibly mediates the association between napping and cognition.Moderate napping shows promise in promoting healthy aging and reducing the burden of cognitive decline in Chinese middleaged and older adults. Introduction Aging is commonly characterized as a progressive, generalized impairment in system integrity, manifested as increased vulnerability to disease and death (1).With the projected trends in global population aging, the prevalence of age-related diseases has become a major concern.Currently, dementia has emerged as the greatest challenge in global health and social care (2).In 2019, 55 million people worldwide were estimated to be living with dementia, a number that is projected to rise to 139 million by 2050 (3).Previous research has shown that moderate napping duration (e.g., 30-90 min) is associated with better cognitive performance (4).On the other hand, no naps or overly long naps (e.g., >90 min) may be detrimental (5).However, most of the evidence comes from older adults, especially those aged over 65 years, many of whom are living with chronic illness or experiencing cognitive impairment.Since there is increasing evidence that active interventions for risk factors in midlife may reduce the incidence of dementia, especially Alzheimer's disease (AD) (2,6,7), it is necessary to know whether these associations still exist in the middle-aged and older population, which is essential for early intervention or delaying the dysfunction and AD (8). As the greatest risk factor, age is strongly associated with an increased risk of dementia, with incidence rates increasing exponentially after the age of 65 (2).No effective medical treatment is currently available for dementia, and as a result, researchers have sought to prevent and intervene in cognitive impairment by understanding its pathophysiological mechanisms and modifiable risk factors.The geroscience hypothesis suggests that interventions to delay the biological processes of aging could prevent age-related diseases and extend healthy lifespan (9,10).Prior studies found that people who experienced an accelerated aging process tended to have poorer cognitive or memory function (8,(11)(12)(13).On the other hand, laboratory studies indicated that sleep disorders can affect the biological aging process through various pathways, such as mitochondrial metabolism, DNA damage, telomeric shortening, and chronic inflammation (14).A study in the United Kingdom Biobank cohort found the acceleration of biological aging would be the consequence of sleep quality (including nighttime sleep duration) (15).A new study has revealed that a higher frequency of napping was causally associated with epigenetic age acceleration based on DNA methylation level (16).Given the potential connections, it seems warranted to evaluate the association of napping duration, BA, with cognitive function, however, the current evidence is limited. In this study, we used two newly validated composite biological age (BA) measures in the Chinese population, Klemera and Doubal biological age (KDM-BA) and physiological dysregulation (PD).We hypothesized individuals who take no naps or excessive naps exhibit worse cognitive function and greater BA compared to those who take moderate naps.We examined the associations among napping during, BA and cognition in the China Longitudinal Study of Health and Retirement (CHARLS), a national cohort study.Then, we investigated the longitudinal effect of baseline napping and BA on cognitive decline during follow-up.Additionally, we examined the mediating role of BA in the relationship between daytime napping and cognitive function to test whether the acceleration of BA is an underlying pathway linking daytime napping to cognitive function. Materials and methods Study design and participants CHARLS is a nationally representative longitudinal survey of the middle-aged and older population in mainland China.The baseline survey comprising individuals aged 45 years and older was conducted between June 2011 and March 2012, and three follow-up visits were conducted in 2013 (wave 2), 2015 (wave 3), and 2018 (wave 4).A multistage probability sampling method was used to select interviewees.Data on demographic characteristics, health circumstances, socioeconomic status, and other health-related information was obtained from individual interviews and physical measurements.Whole blood specimens were collected during the baseline and wave 3 surveys.More details on the data have been previously described (17).All participants in the CHARLS study were required to provide signed informed consent.The Ethics Committee of Peking University approved this study (IRB00001052-11015). In brief, we included participants who provided blood samples at baseline (N = 11,847) and restricted to those aged 45-99 years to ensure that individuals were sufficiently old to detect age-related variants in biomarkers but not too old as to represent a chosen population with above-average health (N = 1,1,416) (18,19).Next, we excluded individuals whose information on one or more biomarkers was missing (N = 2,205).Participants diagnosed with memory-related diseases and those with missing data on napping, cognition, and other covariates were excluded (N = 2,564).Finally, 6,647 respondents were enrolled in the cross-sectional analyses.A total of 6,031 respondents who had at least one remeasurement of cognitive function at follow-up were enrolled in the longitudinal analyses (Supplementary Figure S1). Exposure Baseline daytime napping, irrespective of napping frequency, was assessed by asking, "During the past month, how long did you take a nap after lunch?"We obtained information on the duration of one napping session at a certain time of the day (after lunch) for all participants.According to the previous evidence, participants in our study were divided into four groups according to their napping duration at baseline: non-nappers (0 min), short nappers (≤30 min), moderate nappers (30-90 min), and extended nappers (≥90 min).The moderate napping group was set as the reference in our analysis, as moderate nappers showed better cognitive function in previous studies (4,5,20). Biological age measures We calculated composite BA using two published algorithms: the KDM (18) and PD (21).Based on these two algorithms, KDM-BA and PD have been developed and used in research on senescence and age-related diseases (22,23).In a recent study, the two BA measures were validated in a Chinese population and demonstrated robust prediction of mortality and disease counts (19).The KDM algorithm is derived from a series of regressions of each individual's biomarkers on chronological age (CA) in a reference population (24).PD was derived from the Mahalanobis distance by extracting information on multiple biomarkers with respect to a reference or mean baseline population.Unlike KDM-BA, PD does not directly estimate BA and its value represents how anomalous each individual's physiological profile is compared with a reference sample.PD increases over time within individuals, with higher values indicating an advanced state of aging. Considering the availability and correlations with CA, a total of eight biomarkers used to estimate KDM-BA and PD for individuals were obtained from blood and physical examinations at the baseline survey and were similar to the biomarker set employed by Liu for the validation analysis in the CHARLS (19).The biomarker list included creatinine, high-sensitivity C-reactive protein, total cholesterol, triglycerides, glycosylated hemoglobin, urea nitrogen, platelets, and systolic blood pressure.The KDM-BA algorithm parameters were trained using data from the China Nutrition and Health Survey (CHNS) cohort and then projected onto CHARLS.For PD, we selected a sample aged 20-39 years from the CHNS as the reference population to parameterize the algorithms, as in previous studies (19,25).More details on the CHNS have been presented elsewhere (26).The BA measures mentioned above were calculated using the BioAge R package (27).For KDM-BA, we calculated the residuals, referred to as KDM-BA acceleration (KDM-BAacc), resulting from a linear regression model when regressing KDM-BA on the CA for each participant.A value greater than 0 indicates advanced biological age beyond that expected in terms of CA, and values less than 0 are reversed.For PD, the primary statistical distance was log-transformed for approximate normalization and standardized in subsequent analyses. Outcome According to previous publications, cognitive function comprising the three aspects of memory, orientation, and executive function was assessed in all CHARLS waves through a battery of tests, including some components of the Telephone Interview of Cognition Status, word recall, and picture drawing (28)(29)(30)(31).In brief, the memory function was measured by immediate and delayed word recall for 10 unrelated words, and the memory score was the sum of two individual test scores, ranging from 0 to 20.For the orientation function, respondents were asked to tell investigators the day of the week, date of the month, month of the year, and year.One point was awarded for each correct answer.The executive function test contained two items of the serial sevens test, that is, serial subtraction of 7 from 100 (up to five times, ranging from 0 to 5 points), and a picture drawing test, in which respondents were required to redraw a picture of two intersecting pentagons shown to them, three points were given for a successful drawing and 0 points for a failed drawing.The total scores for orientation and executive function ranged from 0 to 4 and from 0 to 8, respectively.These tests have been demonstrated to be valid and have been widely used in previous studies on cognition in the CHARLS population and others (28)(29)(30)(31)(32)(33). To compare across cognition tests, z scores of each domain at each wave standardized to baseline were generated by subtracting the mean score at baseline from each participant's scores at any wave and dividing by the standard deviation (SD) of the baseline scores.We calculated the global cognitive z scores for an individual at each wave by averaging the z scores of the three domains and re-standardizing them to the baseline global z scores.This approach has been widely adopted (29,(31)(32)(33)(34). Covariates Potential confounders related to napping, cognition and biological aging were selected for analysis (4,5,19,25,29,35).All covariates were collected during the first study visit, including age, sex, residence, education level, body mass index (BMI), marital status, current smoking status, alcohol consumption, night sleep duration, depressive symptoms, and self-reported chronic diseases such as hypertension, diabetes, heart diseases and stroke.Residents were divided into those living in urban and rural areas.Education was divided into three levels: low (elementary school or below), middle (middle school, high school, and vocational school), and high (associate's degree or above).Weight and height for BMI calculation were measured by objected physical examination.Marital status was classified as married (living with spouse present) or others (including separated, divorced, widowed, and never married).All participants were divided into current smokers and nonsmokers.Alcohol consumption was divided into non-drinkers, drinking less than once a month, and drinking more than once a month.Night sleep duration was assessed using the question, "During the past month, how many hours of actual sleep did you get at night (average hours for one night)?"Participants were divided into three groups: short (<7 h), moderate (7-8 h), and long (>8 h).The ten-item Center for Epidemiologic Studies Depression Scale was used to evaluate depressive symptoms.Each item was scored from 0 to 3, with the sum ranging from 0 to 30, and a score of 12 or higher was defined as depressive symptoms (36).Chronic disease history was measured by self-reporting at baseline. Statistical analyses The characteristics of the participants at baseline were described with mean and SD for continuous variables and count and percentages for categorical variables, respectively.The Kruskal-Wallis test or analysis of variance was used for continuous variables, and the chi-square test was used for categorical variables to examine differences across napping duration groups. We used analyses of covariance to calculate the mean difference across groups to evaluate the association between napping duration and cognitive z scores, as well as napping duration and BAs at baseline after adjusting for potential confounders, respectively.The results were expressed as least-square means (LSM) of cognitive z scores or the values of two BA measures compared with the reference group (moderate nappers).Multivariable linear model was used to evaluate the association between KDM-BAacc/PD and cognitive z scores at the baseline.Model 1 was adjusted for age and sex, and Model 2 was additionally adjusted for the aforementioned covariates. To further examine the associations of baseline napping and the two BA measures with cognitive decline, we fitted a set of linear mixed models in which the cognitive z scores were included as a longitudinal outcome.The models incorporated all available follow-up data and treated missing data as missing at random.A random intercept and slope for each individual were considered to account for the remeasurement of cognitive function and to allow for individual differences at baseline and varying rates of cognitive decline over time.Specifically, the primary models included baseline napping duration, time (years from baseline to each subsequent longitudinal cognitive measure), and the interaction term between napping duration and time.The coefficient of the interaction term manifested the mean difference in the rate of change in cognitive z scores (SD/year) compared with the reference group.Model 1 was adjusted for age and sex, and Model 2 was further adjusted for the covariates mentioned above.We hypothesized that greater BA would be associated with faster rates of decline in both global cognitive and three cognitive domains.Additional models (Models 3a and 3b) were further adjusted for two BA measures and the interactions between BA measures and time, based on Model 2, respectively. Mediation analyses were conducted to assess the effects of BA measures on the association between napping duration and cognitive function.The estimation was performed using the medication R package with bootstrapping (1,000 simulations).The parameter estimates and proportion mediated (the proportion of napping duration effect on cognitive function medicated through BA) were documented. We also conducted some additional analyses.Firstly, given that the statistically significant association of napping duration with BA was only observed between the non-nappers and the reference group, all respondents were dichotomized into two subgroups, non-nappers and nappers, to assess the effects of napping on cognitive function and BA acceleration, and to set non-nappers as the reference group.Secondly, as the potential non-linear association between daytime napping and cognition, the data were smoothed and then a restricted cubic spline model was used.We selected the model with the smallest AIC to determine the number of knots, and there are 4 knots in the final model.Thirdly, considering that family clustering existed in the CHARLS participants, we performed a sensitivity analysis to further adjust for family effect in our models.Statistical significance was considered to be p < 0.05.All statistical analyses were 2-sided and conducted using R version 4.1.1. Results Baseline characteristics and sample size Among the 6,647 participants included in our study, 3,363 individuals (50.6%) were male.The mean chronological age (SD) of the sample was 58.3 (8.8) years with an average KDM-BA of 57.3 (9.2) years and an average PD of 0.71 (0.38) (Table 1).The Pearson correlation coefficients of KDM-BA and PD with CA were 0.95 and 0.15, respectively (Supplementary Figure S2).The detailed distribution of BA and the correlation of BA with CA in each napping group can be found in Supplementary Figure S3.A total of 6,031 respondents were enrolled in the longitudinal analyses over a median of 7.0 years of follow-up (Supplementary Table S1).The original scores of cognitive domains during follow-up were presented in Supplementary Table S2. Cross-sectional associations of baseline daytime napping and biological age measures with cognitive function After adjusting for possible confounders, the LSM of the global cognitive z scores were lower in the non-napper and extended napper subgroups than in the moderate napper subgroup (Table 2).These associations were also observed in orientation function but not memory function and executive function (Supplementary Table S3).The associations of two BA measures with global cognition and z scores of the three domains are presented in Table 3.After adjusting for age and sex (Model 1), the baseline KDM-BAacc was negatively associated with global cognitive and memory z scores (β = −0.008,95% CI: −0.014, −0.002; β = −0.010,95% CI: −0.018,-0.002),and PD was negatively associated with global cognition and all specific domains.These associations persisted after controlling for more covariates, except for the association of PD with memory function (Model 2). Longitudinal associations of baseline daytime napping and biological age with cognitive decline The cross-sectional analysis only assessed the status of cognition as measured at baseline.We further investigated how the duration of napping and biological age at baseline relate to cognition decline during the follow-up.The mean difference in the global cognitive z scores and the mean difference in the rate of change in global cognition are presented in Table 4. Non-nappers and extended nappers had an increased decline rate in global cognitive z scores compared with moderate nappers, and the multivariable-adjusted rates by −0.013 SD/year (95% CI: −0.024, −0.003) and − 0.020 SD/ year (95% CI: −0.036, −0.005), respectively (Table 4).However, no difference in the rate of decline in z scores of the three cognitive domains was observed between the napping duration groups (Supplementary Tables S4-S6).We further adjusted for two BA measures and the interaction term of BAs with time in our models (Models 3a and 3b), indicating that higher PD was associated with baseline global cognitive z scores and the cognitive decline rate at follow-up (β = −0.011,p< 0.001) (Table 4). The mediating effect of biological age measures After adjusting for covariates, non-nappers tended to have higher LSM for both BA measures, indicating an advanced biological senescence state compared with moderate nappers.However, the association was not significant for the short and extended nappers (Table 2).Based on the results, non-nappers, but not extended nappers, were associated with greater BA compared with moderate nappers.Hence, we set the non-nappers as the treatment group and the moderate nappers as the control group for comparison in mediation analyses.Considering both greater BA measures were associated with cognitive function at baseline, though it was the PD, not the KDM-BAacc, that predicted cognitive decline during follow-up.This remained relevant for examining the effect of current KDM-BAacc on the association between napping and cognition.Mediation models were performed separately for the two BA measures.KDM-BAacc and PD mediated 2.9 and 4.7% of the napping duration effect on global cognition, respectively, compared to those in the non-napper subgroup and those who reported taking moderate naps (Figures 1A,B). Additional analysis All respondents were dichotomized into two subgroups, non-nappers and nappers, cross-sectional and longitudinal analyses suggested that nappers were associated with higher global cognitive z scores at baseline (LSM =0.056, 95% CI: 0.022, 0.083) and a decreased rate of change in global cognitive decline (β = 0.009, 95% CI: 0.001, 0.017), compared with those who did not take naps (Supplementary Tables S7, S8).Furthermore, non-nappers were associated with higher LSM for both BA measures, similar to the main analysis (Supplementary Table S5).Similarly, mediation models were constructed, and 4.3 and 4.2% of the napping effect on global cognitive function was mediated by KDM-BAacc and PD, respectively (Figures 1C,D).In addition, we exploratory analyzed the nonlinear relationship between daytime napping and cognitive function scores, with the highest scores occurring at 60-70-min naps (Supplementary Figure S4).The effect of family clustering was further examined in sensitivity analysis, which did not alter the significance of the results, confirming the robustness of our findings (Supplementary Table S9). Discussion In this community-based study, we observed that non-napping and excessive daytime napping duration (≥90 min) were associated with a lower cognitive performance level and faster cognitive decline among middle-aged and older Chinese.More importantly, we found that non-napping may be associated with BA acceleration and that acceleration is associated with cognitive function.Our findings suggest that two BA measures, the KDM-BAacc and PD, partially mediate the association between daytime napping and cognitive function. The results of the cross-sectional and longitudinal analyses on the association of daytime napping with cognitive function were generally consistent, similar to the results of previous studies (4, 37-39).Our results revealed that no napping and excessive napping duration (≥90 min) were associated with worse cognitive function and increased rate of cognitive decline than moderate napping (30-90 min).Interestingly, irrespective of the napping duration, the overall cognitive function of nappers was better than that of non-nappers, and the parameter estimates were much larger than those grouped by napping duration in our main analyses.Sleep is essential for memory consolidation and normal brain function (40).Sleep disorders or sleep deprivation are linked to β-amyloid (Aβ) deposition, increased Tau, and interference with the function of neuronal pathways, especially those of GABA and cAMP, leading to AD (40,41).Sleep is considered a crucial restorative process for the brain and body, contributing to the recovery of energy and attention, as well as cellular restoration (14).Napping may help compensate for nighttime sleep loss, compared to non-nappers, moderate napping may provide extra resistance to cognitive decline in people who sleep poorly at night (38).On the other hand, excessive daytime sleepiness may be detrimental to maintaining cognitive function.Nevertheless, it is worth noting that the excessive duration cutoff value varied in previous studies, which may have contributed to the inconsistent results (4,(37)(38)(39). As far as we know, our study is the first to evaluate the association between daytime napping, biological aging, and cognitive function in a large prospective cohort of community-dwelling individuals.Our results demonstrated that non-napping was significantly associated with two BA measures established by drawing information on clinical biomarkers from multiple systems in the human body, indicating that non-napping could be a potential risk factor for BA acceleration.A study reported that abnormal (too short or excessive) sleep is implicated in bigger BA, and normal night sleep duration (7-8 h) was associated with 0.245 years decreases in KDM-BAacc (15).Although a new study reported the evidence for causal effects of daytime napping frequency on epigenetic age, and a higher frequency of daytime napping was associated with epigenetic age acceleration (16), more evidence is required on the effects of other napping habits such as duration and timing.A significant association was observed between non-napping and increased biological age in our analyses, it seemingly shed light on the possibility of napping in the resistance to accelerated aging.However, due to the limited evidence, we believe that the results should be interpreted cautiously and that more objective studies are warranted to replicate our results. People with higher values of KDM-BAacc or PD are more likely to experience worsening cognitive performance.These findings are consistent with those of previous studies (8,10,11).Biologically older individuals had poorer cognitive performance at midlife, and this difference mirrored a real decrease in cognitive function over time (8).Notably, our results suggested that the two measures revealed inconsistent associations with specific domains.We observed that PD was negatively correlated with all cognitive domains, whereas KDM-BA was associated only with memory function.PD appeared to be more predictive and sensitive to the unique aspects of cognitive function.One possible reason is that KDM-BA and PD were calculated by two different algorithms that could capture disparate characteristics of biomarkers and quantify the aging process differently (10, 19), although we selected the same set of biomarkers in our calculations. This study provides initial evidence for using two accepted measures in quantifying biological age as an underlying pathway linking daytime napping to global cognitive function.Sleep disturbances and sleep deprivation could play a direct role in aging mechanisms (14). Current biological mechanisms linking sleep to biological aging, such as inflammation, mitochondrial dysregulation and epigenetic changes, were also thought to be the underlying aging process for cognitive decline and neurological diseases, including ADRD (14,42).Our results suggest that if we could intervene in changeable risk factors that contribute to an advanced state of biological aging, we might be able to improve cognitive decline even in the preclinical stage of AD.Furthermore, our results highlight the critical role of moderate daytime napping in slowing down the rate of cognitive decline and biological age acceleration, although the magnitude of the effect we observed was limited.The mediation effect sizes tended to be small and insignificant in the three cognitive domains of our study; however, we believe that this effect should not be completely ignored.Previous studies have suggested that inflammatory factors mediate the relationship between sleep-related behaviors and cognitive function (35); however, KDM-BA and PD, have captured clinical profiles of multiple organs and systems, not just inflammation systems.This is promising to understand further the pathways involved in the daytime napping effect on cognition, and our results provide statistical epidemiological evidence for subsequent mechanistic studies.A strength of our study was the large population size of the Chinese community-based population, which allowed us to obtain sufficient blood data for biological age estimation.In addition, we used parallel analyses of two conceptually diverse composite biological age measures that had been previously trained and validated in the same sample (19), which enhanced the robustness of our conclusions.However, our study also had some limitations.Firstly, the measure of cognition used in this study is rudimentary when compared to a complete neuropsychological evaluation, although the abbreviated nature of the study's cognitive evaluation made it practical to assess such a large sample size.Secondly, this study is only limited to the investigations of naps taken after lunch.Other dimensions like frequency and complete range of nap timing throughout the day were lacking.Third, self-reported information may not accurately capture unintentional naps or forgotten napping instances, which led to more credible information not being available for our study.Future studies would benefit from including more reliable and objective measures.Furthermore, while we adjusted for several covariates in our models, there may still be some potential confounders, such as sleep quality, night shifts (which may lead to longer and more frequent daytime naps), work status and ApoE genotypes.A genome-wide association study revealed strong associations between two well-established approaches to BA estimation and ApoE (43).In addition, participants might alter their napping behavior at follow-up, which deserves to be followed in the future.Finally, we could not completely rule out reverse causality, a published study found a bidirectional relationship between daytime napping and Alzheimer's dementia, and the complicated mechanisms involved need to be further investigated (44). In summary, we discovered the association between napping, aging, and cognitive function among a nationally representative sample of middle-aged and older Chinese individuals.The significant mediation effect revealed a pathway from napping to cognitive decline through the acceleration of biological age.Our study underscores that daytime napping could work as an early sign or risk factor for biological aging and clinically significant cognitive impairment.The findings highlight that daytime napping behavior, as a lifestyle, is a cost-effective intervention that can be easily promoted to middle-aged and older populations.Future research with more objective measures of napping behaviors and more methods to quantify biological age is required to validate our findings. TABLE 1 1 Baseline characteristics of overall participants and napping duration subgroups.Low education level was defined as elementary school or below; middle was defined as middle school, high school, and vocational school; and high was defined as an associate degree or above.Depressive symptoms were defined as a ten-item Center for Epidemiologic Studies Depression (CES-D 10) scale score of 12 or above. CharacteristicsAllNon-ShortModerateExtendedF/I 2p valuenappersnappersnappersnappersvalue(N = 6,647)(N = 3,017)(N = 1,159)(N = 1707)(N = 764)Age58.3 ± 8.857.8 ± 8.558.6 ± 8.958.6 ± 9.159.1 ± 9.314.20.003Gender (male)3,363 (50.6)1,337 (44.3)586 (50.6)996 (58.3)444 (58.1)105.9<0.001Residence (urban)2,489 (37.4)1,065 (35.2)473 (40.8)664 (38.9)287 (37.6)13.10.004Education level aLow4,327 (65.1)2062 (68.3)698 (60.2)1,075 (63.0)492 (64.4)Middle1,681 (25.3)699 (23.2)335 (28.9)441 (25.8)206 (27.0)33.8<0.001High639 (9.6)256 (8.5)126 (10.9)191 (11.2)66 (8.6)Marital status (married)5,675 (85.4)2,579 (85.5)976 (84.2)1,484 (86.9)636 (83.2)7.40.060BMI (kg/m 2 ) b<18.5378 (5.7)194 (6.4)53 (4.6)97 (5.7)34 (4.5)18.5-23.93,446 (51.8)1,650 (54.7)573 (49.4)847 (49.6)376 (49.2)37.3<0.00124-282017 (30.3)850 (28.2)365 (31.5)554 (32.5)248 (32.5)≥28806 (12.1)323 (10.7)168 (14.5)209 (12.2)106 (13.9)Current smoking2,811 (42.3)1,168 (38.7)463 (39.9)804 (47.1)376 (49.2)49.6<0.001Drinking statusNon-drinker4,321 (65.0)2086 (69.1)756 (65.2)1,024 (60.0)455 (59.6)Less than once a month543 (8.2)207 (6.9)112 (9.7)163 (9.5)61 (8.0)59.3<0.001More than once a month1783 (26.8)724 (24.0)291 (25.1)520 (30.5)248 (32.5)Night sleep duration<7 h3,335 (50.2)1,618 (53.6)601 (51.9)794 (46.5)322 (46.9)7-8 h2,802 (42.2)1,176 (39.0)482 (41.6)786 (46.0)358 (42.1)52.9<0.001>8 h510 (7.7)223 (7.4)76 (6.6)127 (7.4)84 (11.0)Depressive symptoms c1740 (26.2)886 (29.3)283 (24.4)398 (23.3)173 (22.6)29.9<0.001Chronic diseasesHypertension1,657 (25.0)636 (21.1)342 (29.5)471 (27.6)208 (27.2)45.5<0.001Diabetes398 (6.0)140 (4.6)92 (7.9)120 (7.0)46 (6.0)20.9<0.001Heart disease776 (11.7)303 (10.0)180 (15.5)213 (12.5)80 (10.5)26.6<0.001Stoke129 (1.9)50 (1.7)25 (2.2)33 (1.9)21 (2.7)4.150.245Memory score7.4 ± 3.37.2 ± 3.37.7 ± 3.37.5 ± 3.37.1 ± 3.28.1<0.001Orientation score3.1 ± 1.03.1 ± 1.03.2 ± 1.03.2 ± 0.93.1 ± 1.034.3<0.001Executive score5.8 ± 2.45.6 ± 2.55.9 ± 2.36.0 ± 2.35.8 ± 2.338.3<0.001KDM-BA57.3 ± 9.256.8 ± 8.857.4 ± 9.357.6 ± 9.558.2 ± 9.514.50.002PD0.71 ± 0.380.73 ± 0.370.71 ± 0.380.68 ± 0.380.71 ± 0.388.4<0.001BMI, body mass index; ADL, activities of daily living; IADL, instrumental activities of daily living; KDM-BA, Klemera and Doubal method-biological age; PD, physiological dysregulation.Data are expressed as mean ± standard deviation (SD) for continuous variables and as count(percentage) for categorical variables. a b BMI was calculated as weight in kilograms divided by height in meters squared.c TABLE 2 2 Cross-sectional associations of baseline napping duration with global cognitive function or biological age measures.After adjusting for age, sex, education level, residence, BMI, marital status, current smoking and drinking status, night sleep duration, depressive symptoms, and self-reported chronic diseases. Napping groupGlobal z scoresKDM-BAaccPDLSM a (95%CI)p valueLSM a (95%CI)p valueLSM a (95%CI)p valueNon-nappers (0 min)-0.061 (−0.106, −0.015)0.0050.255 (0.058,0.451)0.0070.085 (0.016,0.154)0.011Short nappers (≤ 30 min)0.012 (−0.045,0.068)0.899−0.179 (−0.424,0.064)0.2020.031 (−0.056,0.115)0.701Moderate nappers (30-90 min)Reference-Reference-Reference-Extended nappers (≥ 90 min)−0.078 (−0.143, −0.011)0.0130.053 (−0.226,0.333)0.9160.067 (−0.031,0.165)0.257LSM, least-squares means; KDM-BAacc, Klemera and Doubal method-biological age acceleration; PD, physiological dysregulation; CI, confidence interval. a TABLE 3 3 Cross-sectional associations of biological age measures with cognitive function.Klemera and Doubal method, biological age acceleration; PD, physiological dysregulation; CI, confidence interval.Model 1 was adjusted for age and sex.Model 2 was additionally adjusted for education level, residence, BMI, marital status, current smoking and drinking status, night sleep and napping duration, depressive symptoms, and self-reported chronic diseases. CognitiveKDM-BAaccPDfunction (z scores)Model 1Model 2Model 1Model 2β (95%CI)p valueβ (95%CI)p valueβ (95%CI)p valueβ (95%CI)p valueGlobal−0.008 (−0.014, −0.002)0.008−0.007 (−0.012, −0.001)0.018−0.045 (−0.062, −0.028)<0.001−0.034 (−0.050, −0.018)<0.001Memory−0.010 (−0.018, −0.002)0.013−0.008 (−0.017, −0.000)0.043−0.034 (−0.058, −0.010)0.005−0.021 (−0.045,0.001)0.065Orientation−0.002 (−0.011,0.005)0.480−0.007 (−0.015,0.001)0.112−0.050 (−0.074, −0.026)<0.001−0.042 (−0.065, −0.018)<0.001Executive−0.007 (−0.015,0.000)0.067−0.007 (−0.015,0.001)0.102−0.052 (−0.075, −0.028)<0.001−0.041 (−0.063, −0.018)<0.001KDM-BAacc, TABLE 4 4 Longitudinal associations of baseline napping duration and biological age measures with global cognitive decline.Klemera and Doubal method, biological age acceleration; PD, physiological dysregulation; CI, confidence interval.Model 1 was adjusted for age and sex.Model 2 was additionally adjusted for education level, residence, BMI, marital status, current smoking and drinking status, night sleep duration, depressive symptoms, and self-reported chronic diseases based on Model 1. Model 3a was additionally adjusted for KDM-BAacc, based on Model 2. Model 3b was additionally adjusted for PD, based on Model 2. VariableModel 1Model 2Model 3aModel 3bβ (95%CI)p valueβ (95%CI)p valueβ (95%CI)p valueβ (95%CI)p valueNapping duration, min0−0.108 (−0.152, −0.063)<0.001−0.051 (−0.095, −0.014)0.009−0.053 (−0.093, −0.012)0.011−0.051 (−0.092, −0.010)0.013≤ 300.016 (−0.040,0.072)0.566−0.009 (−0.060,0.041)0.700−0.011 (−0.061,0.040)0.675−0.009 (−0.059,0.042)0.73430-90Reference-Reference-Reference-Reference-≥ 90−0.084 (−0.147, −0.019)0.011−0.081 (−0.140, −0.027)0.007−0.080 (−0.141, −0.026)0.008−0.080 (−0.140, −0.025)0.005Time since baseline (years)−0.060 (−0.068, −0.052)<0.001−0.058 (−0.066, −0.050)<0.001−0.058 (−0.066, −0.050)<0.001−0.059 (−0.068, −0.051)<0.001Napping duration x Time since baseline0−0.014 (−0.024, −0.004)0.008−0.013 (−0.024, −0.003)0.012−0.013 (−0.024, −0.003)0.012−0.013 (−0.023, −0.002)0.016≤ 30−0.001 (−0.014, 0.012)0.9360.000 (−0.013, 0.013)0.9900.000 (−0.013, 0.013)0.9890.001 (−0.012, 0.014)0.92330-90Reference-Reference-Reference-Reference-≥ 90−0.021 (−0.036, −0.006)0.006−0.020 (0.034, −0.005)0.007−0.020 (−0.035, −0.005)0.008−0.019 (−0.034, −0.004)0.012KDM-BAacc----−0.007 (−0.013, −0.001)0.024--KDM-BAacc x Time since baseline----(−0.003,0.000) −0.0010.082--PD------−0.027 (−0.044, −0.010)0.002PD x Time since baseline------−0.011 (−0.016, −0.007)<0.001KDM-BAacc, Wu et al. 10.3389/fpubh.2023.1294948Frontiers in Public Health 09 frontiersin.orgAcknowledgmentsWe thank all project staff and participants in the China Health and Retirement Longitudinal Study (CHARLS).Data availability statementThe datasets presented in this study can be found in online repositories.The names of the repository/repositories and accession number(s) can be found at: http://charls.pku.edu.cn/.FundingThe author(s) declare financial support was received for the research, authorship, and/or publication of this article.This study was supported by the Sichuan Science and Technology Program of the Science and Technology Department of the Sichuan Province (Nos.2022YFS0422 and 2023NSFSC1736).Ethics statementThe studies involving humans were approved by the Ethics Committee of Peking University.The studies were conducted in accordance with the local legislation and institutional requirements.The participants provided their written informed consent to participate in this study.Conflict of interestThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Publisher's noteAll claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.Supplementary materialThe Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpubh.2023.1294948/full#supplementary-material Understanding the odd science of aging. 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Exploring potent aldose reductase inhibitors for anti-diabetic (anti-hyperglycemic) therapy: integrating structure-based drug design, and MMGBSA approaches 20 November 2023 Muhammad Shahab State Key Laboratories of Chemical Resources Engineering Beijing University of Chemical Technology BeijingChina Guojun Zheng zhenggj@mail.buct.edu.cn State Key Laboratories of Chemical Resources Engineering Beijing University of Chemical Technology BeijingChina Fahad M Alshabrmi Department of Medical Laboratories College of Applied Medical Sciences Qassim University BuraydahSaudi Arabia Mohammed Bourhia bourhiamohammed@gmail.com Department of Chemistry and Biochemistry Faculty of Medicine and Pharmacy Ibn Zohr University AgadirMorocco Fentahun Gezahign Wondmie Department of Biology College of Science Bahir Dar University Bahir DarEthiopia Ahmad Mohammad Salamatullah Department of Food Science and Nutrition College of Food and Agricultural Sciences King Saud University RiyadhSaudi Arabia Ali H Al-Marzoqi Jianzhong Chen Opeyemi Iwaloye University of Babylon Iraq Shandong Jiaotong University China Federal University of Technology Nigeria Exploring potent aldose reductase inhibitors for anti-diabetic (anti-hyperglycemic) therapy: integrating structure-based drug design, and MMGBSA approaches 20 November 20232A8492CF54D1563D4335E9B939E561C310.3389/fmolb.2023.1271569RECEIVED 02 August 2023 ACCEPTED 20 October 2023aldose reductasediabetes melitusSBVSmolecular docking simulationMMGBSA Aldose reductase (AR) is an important target in the development of therapeutics against hyper-glycemia-induced health complications such as retinopathy, etc.In this study, we employed a combination of structure-based drug design, molecular simulation, and free energy calculation approaches to identify potential hit molecules against anti-diabetic (anti-hyperglycemic)-induced health complications.The 3D structure of aldoreductase was screened for multiple compound libraries (1,00,000 compounds) and identified as ZINC35671852, ZINC78774792 from the ZINC database, Diamino-di nitro-methyl dioctyl phthalate, and Penta-o-galloyl-glucose from the South African natural compounds database, and Bisindolylmethane thiosemi-carbazides and Bisindolylme-thane-hydrazone from the Inhouse database for this study.The mode of binding interactions of the selected compounds later predicted their aldose reductase inhibitory potential.These com-pounds interact with the key active site residues through hydrogen bonds, salt bridges, and π-π interactions.The structural dynamics and binding free energy results further revealed that these compounds possess stable dynamics with excellent binding free energy scores.The structures of the lead inhibitors can serve as templates for developing novel inhibitors, and in vitro testing to confirm their anti-diabetic potential is warranted.The current study is the first to design small molecule inhibitors for the aldoreductase protein that can be used in the development of therapeutic agents to treat diabetes. Introduction Aldo-Keto-Reductases (AKRs) are versatile enzymes involved in metabolizing carbonyl-containing substrates like sugars, lipid aldehydes, ketosteroids, and keto prostaglandins (Bohren et al., 1989;Komoto et al., 2004).This superfamily comprises 16 families, ranging from AKR1 to AKR16 (Komoto et al., 2004), that share a high degree of sequence similarity and a common protein folding structure.The AKR website (http://www.med.upenn.edu/akr/)provides information about the AKR superfamily.AKR enzymes exhibit similar catalytic and structural properties and are NAD(P)H-dependent oxidoreductases expressed as 34-37 kDa polypeptides (Jez et al., 1997).The AKR1 is further categorised into the A to E subfamilies; among them, AKRB1 is comprehensively studied and plays a major role in the emergence of diabetic complications (Blakeley et al., 2008).These enzymes make up the "polyol pathway," an alternative glucose metabolism process that runs concurrently with glycolysis and causes hyperglycemia in diabetic patients (Singh et al., 2021).Hyperglycemia-induced pathways drive oxidative stress in diabetic organs (heart, kidney, and eye) through AGEs, the polyol pathway, the mitochondrial electron transport system, and PKC activation (Srivastava et al., 2005;Bhatnagar and Srivastava, 1992).In metabolic processes like the glutathione reductase/glutathione peroxidase system's detoxification of reactive oxygen species (ROS), NADPH plays reductive roles (Paul et al., 2020).An increased cytosolic NADH/NAD + ratio causes mitochondrial NADH-dependent pathways, which induce ROS (Vedantham et al., 2012) Increased NADH may also ameliorate the production of diacylglycerol (DAG), which activates PKC and causes oxidative stress by activating NAD(P)H oxidase by regulating PKC.The prevalence of diabetes has been rising alarmingly worldwide.According to the World Health Organization (https://www.who.int/health-topics/diabetes),more than 400 million people worldwide are currently suffering from diabetes.As a result, diabetes complications have risen in tandem with the rise in the number of people with the disease (Taslimi et al., 2018;Demir et al., 2020).Cardiovascular disease (CVD) is the main cause of morbidity and mortality in people with diabetes mellitus, among the different diabetic complications (Jandeleit-Dahm and Cooper, 2002).Diabetic cardiomyopathy is a unique cardiovascular condition characterized by impaired cardiac function in individuals with diabetes that is unrelated to coronary artery disease (Sower et al., 2001;Lopaschuk, 2002), Diabetes increases myocardial sensitivity to ischemia and raises the risk of cardiovascular disease and myocardial infarction (Lehto et al., 1994;Stone et al., 1995).Cardiovascular dysfunction in diabetics has been attributed to increased sorbitol buildup and a decline in NADPH due to an AR flux (Flores et al., 2023).Diabetic patients demonstrate a heightened incidence of cardiovascular disease and myocardial infarction (Jhuo et al., 2022).Cardiac dysfunction in diabetic patients is attributed to increased sorbitol accumulation and reduced NADPH levels resulting from aldose reductase (AR) flux (De Geest and Mishra, 2022;Garg and Gupta, 2022).According to studies, AR activation causes oxidative stress, (Ramana et al., 2006b;Ramana and Srivastava, 2006), which in turn can activate the NF-κB pathway (Ramana et al., 2006a;Ramana et al., 2006b;Ramana and Srivastava, 2006).Additionally, by lowering oxidative stress, AR inhibition can prevent acute hyperglycemia-induced cardiac contractile dysfunction (Aly et al., 2023).The blocking of the NF-κB pathway and oxidative stress by AR suppression is a new strategy for avoiding cardiovascular diseases. Materials and methods Preparations of protein structure The 3D structure of an enzyme aldose reductase with (PDB ID; 3S3G) was extracted by utilizing the PDB database (Zheng et al., 2012).The three-dimensional structure was further checked for chain breaks, and missing atoms and water molecules were extracted.Furthermore, the retrieved protein structure was subjected to preparatory procedures using the Dock prep module of UCSF Chimera v1.10.2 software program (Goddard et al., 2007).The partial charges were used, which properly set the protein model's protonation phase at a neutral pH.The GBVI/WSA rescoring approach and London dG scoring function were also utilised in combination with the Triangle matcher docking algorithm.Finally, a protein-ligand interaction fingerprint was used to figure out hydrophobic bonds, ionic bonds, and hydrogen bonds (Méndez-Álvarez et al., 2023).In addition, the three-dimensional structure of the standard drug (tolmetin) was retrieved from the Pub Chem database.The structure was energyminimised and prepared by AutoDock Vina. Preparation of commercial and in house databases Virtual screening was conducted using three databases: the ZINC database (Ghufran et al., 2022) (1 million compounds), the South African Natural Compounds database (available at http:// african-compounds.org/about/afrodb/), and with the help of the research of our collaborators, the three-dimensional structures of the compounds were put into a database called an In-house containing 1,600 compounds.These databases were utilized to identify highly active and potent inhibitors against aldose reductase.LogP, LogS, Lipinski's, Pfizer, GSK, and the Golden Triangle rules were among the properties predicted for the finally selected hits.Subsequently, aldose reductase screening was performed on the compounds passing the RO5 criteria from each database, utilizing scoring and Minimization with AutoDock Vina (smina) (Ding et al., 2023).Following Smina screening, the top hits were subjected to further evaluation using ADFR.ADFR employed flexible docking with high accuracy for each compound against aldose reductase, utilizing the AutoDock four scoring function (Aly et al., 2023).Finally, from in-house and commercial databases (ZINC and the South African Natural Compounds Database), the top two hits were finalized from each database on the basis of docking score and binding interaction using Schrodinger Maestro and Pymol software and carried out for MD simulation (Maya Díaz, 2023;Taherkhani et al., 2023). Screening of libraries The structure base Virtual screening uses the three-dimensional structure of ligands and proteins in the database.In order to determine potent ligands, we carried out molecular docking to assess the methods by which ligands and proteins bind.This approach predicts the beneficial and enhanced interactions between proteins and ligands.In addition, SANCDB, an in-house database, and ZINC databases The drug libraries, including the ZINC database, the in-house database, and SANCDB, were screened by the SBVS using AutoDock 4. AutoDock 4, a docking suite, was utilised to screen the compounds obtained from SANCDB, ZINC, and the in-house databases, employing a homology model.To ensure the robustness and reliability of our molecular docking analysis, we employed three distinct servers: Glide module software (Schrödinger Maestro v12.1) (Schrödinger, 2011;Kaushik et al., 2018).These servers are renowned for their userfriendly interfaces and accuracy in delivering reliable docking results.This multipronged approach enhances the reliability of our findings and strengthens the scientific rigor of our study also it allowed us to assess the consistency and reliability of our docking scores.The model's protonation phase was appropriately adjusted for neutral pH, and partial charges were included.The London dG scoring function, Triangle Matcher Docking algorithm, and GBVI/ WSA rescoring method were employed.Additionally, a force fieldbased scoring function was utilized for post-docking refinement.Thereafter, the interaction of each ligand with the active site was determined by utilizing the protein-ligand interaction fingerprint in AutoDock 4. PLIF determines how hydrogen atoms, water molecules, and ions interact with each other (Adessi et al., 2023).Finally, MD simulation was performed in order to verify the molecular docking approaches. Molecular dynamic simulation A molecular dynamic (MD) simulation was carried out in order to evaluate the dynamic behavior of receptors with inhibitors at the atomic level.On the basis of binding interactions and docking scores, the best hits were used to perform MD simulation, and free energy calculation-based validation was carried out by using AMBER22 (Case et al., 2005;Shahab et al., 2023a).Initially, the drug topology was created by utilizing the parmchk2 and antechamber (Khan et al., 2023).Thereafter, all complexes obtained were constructed using the Tleap preparation programme.An octahedral box was used, and by introducing the Na + or Clions, all complexes were neutralised.To prepare the complexes, topology and coordinate files were used for a two-stage minimization process: 1) 12,000 steps for the first round and 2) 6,000 steps for the second round.Subsequently, each complex underwent heating and equilibration for 20 ns In the production stage, a 100 ns simulation was conducted.For accelerated MD simulation, the GPU version of PMEMD.cuda was employed.Trajectories obtained were processed using the CPPTRAJ and PTRAJ tools (Jama et al., 2023;Wang et al., 2023). Binding free energy evaluation The use of the MMPBSA.pyscript in the MD simulation run, trajectories were identified, which were further used in calculating the binding free energy (Shahab et al., 2023b).For calculating the binding free energy of any complexes, including protein-ligand, nucleic acid-protein, and protein-protein, this type of approach was used (Khan et al., 2023).Hence, we also applied this approach here to accurately compute the total binding free energy of the proteinligand complexes.Mathematically the binding free energy can be estimated as: ″ΔG bind G complex − Greceptor + Gligand ″ Different contributing components of total binding energy were calculated by the following equation: ″G Gbond + Gele + GvdW + Gpol + Gnpol″ G bond , G electrostatic , and G vdW refer to interactions involving bonded, electrostatic, and van der Waals states, respectively.On the other hand, G polar and G npolar describe polar and non-polar interactions, respectively, which are calculated based on the assumed free energy through precise Generalized Born (GB) methods (Chen et al., 2019;Chen et al., 2022). Prediction of bio activity and dissociation constant (K D ) The bio-activity and dissociation constant (KD) were computationally predicted for top hits by utilizing an online web server, Molinspiration (https://www.molinspiration.com/cgi-bin/properties) and PRODIGY-Ligand (https://wenmr.science.uu.nl/ prodigy/lig) respectively (Vangone et al., 2019).These online tools were reported previously to demonstrate the bioactivity and KD of various molecules against diseases (Khan et al., 2021). Results and discussion Aldose reductase, also known as AKR1B1, is an enzyme belonging to the aldo-keto reductase family.It relies on NADPH as a cofactor and is responsible for catalyzing the reduction of both hydrophilic and hydrophobic aldehydes.It serves as the initial enzyme in the polyol pathway, which converts glucose into sorbitol.Sorbitol is then further metabolized to fructose by the action of sorbitol dehydrogenase.The activation of the polyol pathway, particularly in hyperglycemic conditions, is widely accepted as the key event leading to various long-term complications associated with diabetes.Due to the significant role of AKR1B1 in the development of diabetic complications, researchers have targeted this enzyme for the development of molecules that can inhibit its activity (Balestri et al., 2022).In our study, the computational analysis revealed that the identified compounds form specific interactions with key amino acids within the active site of aldose reductase, such as Ser302, Phe122, Trp219, Cys298, Ala299, Val297, and Trp20 and Leu300 integrating structure-based drug design (SBDD) and molecular mechanics/ generalized born surface area (MMGBSA) approaches (Kinoshita, 1990;Ramachandran et al., 2023).For instance, these residues are also reported to act as inhibitor hotspots for other drug targets (Fang et al., 2023).The topmost active ligands from each database (South African, ZINC, and in-house) based on docking results and interaction analysis were further subjected to validation through molecular dynamics (MD) simulations.Post-MD analyses were performed to assess the ligands' behaviour and proper-ties.Therefore, the present study used structure-based virtual screening, molecular dynamic simulation, and binding free energy approaches to determine potent inhibitors against aldose reductase.The combination of these computational techniques facilitated the rational design and prioritization of compounds based on their predicted binding energies and structural characteristics.In various computational approaches, including structure-based virtual screening and MD simulation, great concern is given to accelerating the cycle of drug development.The identified inhibitors hold promise for further development and experimental evaluation, potentially leading to novel treatments for hyper-glycemic patients and addressing the complications associated with diabetes.I believe this research offers promising insights into potential aldose reductase inhibitors for managing hyperglycemia and diabetes-related complications, it is important to acknowledge its limitations.The findings are primarily based on computational methods and simulations, specifically structurebased drug design (SBDD) and molecular mechanics/generalized born surface area (MMGBSA) approaches.These predictions, although promising, necessitate rigorous experimental validation to assess the efficacy, safety, and specificity of the identified compounds in biological systems.Additionally, the study does not address critical aspects such as pharmacokinetics, potential off-target effects, and the variability among diverse patient populations.Recognizing these limitations is crucial to offer a balanced and realistic perspective on the study's implications and the potential for clinical translation of the findings. Molecular docking Analyzing binding modes of top hits retrieved from african medicines database The South African Natural Compounds Database (SANCDB) is a valuable resource for various disease treatments.Screening SANCDB identified diamine-dinitro-methyl dioctyl phthalate and penta-o-galloyl-glucose as the top hits among the 570 compounds.These compounds demonstrated docking scores of -12.24 kcal/mol and -11.34 kcal/mol, respectively.Then we validated molecular docking score by Schrödinger Maestro v12.1 (−12.265,−11.532 kcal/mol).In terms of interaction, Diamino-di nitro-methyl dioctyl phthalate established five hydrogen bonds with Ser302, Phe122, Trp219, Cys298, and Leu300 residues Figures 1A, B. The interacting residues observed in the South African Natural Compounds Database (SANCDB) align with the compounds reported in the ZINC database.The binding residues, including Trp20, Cys298, Tyr309, and Asn260, were found to be involved in the interaction with the compounds.Additionally, a π-π interaction was established with the Tyr209 residue.These findings indicate consistent and specific interactions between these small molecules and the critical residues of aldoreductase.The compound penta-ogalloyl-glucose established four hydrogen bonds involving Ala299, Val297, Ser302, and Trp20 (Figures 1C, D).Diamino-di nitromethyl dioctyl phthalate was found to interact with aldose reductase through hydrogen bonds, exhibiting a similar interaction pattern to the compounds reported in the ZINC and Inhouse databases in this study.This suggests potential pharmacological activity against aldose reductase.The interaction patterns of each compound and their respective docking scores are presented in Table 1. Binding modes of top hits from the inhouse database The in-house database, containing over 1,300 derivatives of bisindolylmethane, proves to be a valuable resource for designing natural product-based remedies for various diseases.Based on docking conformations, the ligands Bisindolylmethane thiosemicarbazides and Bisindolylmethane-hydrazone hybrids were selected as top hits, demonstrating binding scores of -10.25 kcal/mol and -9.51 kcal/mol, respectively.Bisindolylmethane thiosemi-carbazides formed two hydrogen bonds with Trp48 and His110, while Bisindolylmethane-hydrazone hybrids formed three hydrogen bonds with Phe122, Tyr49, and Asn160.The docking score for Bisindolylmethane against the aldoreductase protein was reported as -10.25 kcal/mol, with key interactions involving Phe122, Tyr49, and Asn160.To validate the molecular docking score, we used Schrödinger Maestro v12.1, which yielded scores of -10.422 and -10.827 kcal/mol.The compounds identified in this particular database have smaller molecular structures and show promising pharmacological activity against aldoreductase.The interaction patterns of each compound are depicted in Figures 2A-D, and their respective docking scores are presented in Table 1. Binding modes of top hits from the ZINC database The ZINC database, comprising 100,000 druggable compounds, was screened, and 6,575 compounds were found to comply with the LogP, LogS, Lipinski's, Pfizer, GSK, and Golden Triangle rules, which were among the properties predicted for the top two hits.Among these, 1,145 compounds were identified as the best hits.From these, two compounds, namely ZINC35671852 and ZINC78774792, were selected as the top hits based on their docking scores of -7.49 kcal/mol and -6.71 kcal/mol, respectively.To validate the molecular docking score, we used Schrödinger Maestro v12.1, which yielded scores of −8.753and -7.827 kcal/ mol.ZINC35671852 formed three hydrogen bonds with the residues Trp20, Tyr309, and Asn260, while ZINC78774792 exhibited a similar hydrogen bond interaction pattern.These shortlisted compounds from the ZINC database displayed excellent docking scores and exhibited an interaction pattern that covered the entire active site, effectively blocking key residues and highlighting the pharmacological potential of these small molecules.The interaction patterns of each compound are depicted in Figures 3A-D, and their respective docking scores are presented in Table 1. Molecular dynamic simulation To properly understand the structure's dynamic features, the binding of proteins with ligands is an essential parameter to reveal.Therefore, in the molecular dynamic simulation technique, we performed RMSD (structure stability), RMSF (residual flexibility), RoG, and hydrogen bond analysis to understand each ligand's stability.The structure stability, which is calculated through RMSD as a function of time, shows that all ligands stably bind to the target protein except for some minor deviations. Root mean square deviation The dynamic stability of a protein-ligand complex plays a crucial role in determining the pharmacological efficacy of a compound.A stable binding between the ligand and the protein's active site indicates a higher potential for pharmacological activity.To evaluate the stability of the simulation trajectory, the Root Mean Square Deviation (RMSD) function is utilized in combination with simulation tools.In this study, the RMSD values for the trajectories of each complex were computed over time to assess their stability.The RMSD values for each complex are illustrated in Figures 4A-F.For this purpose, we carried out RMSd analysis to calculate the top two ligands from each database and the standard drug in the active site of aldose reductase.The RMSd analysis demonstrates that the top ligands exhibit stable behaviour but have minor deviations.The RMSd graph of the standard drug exhibits a small deviation within 0.5-1 Å up to 60 ns; thereafter, it increases from 1.2 to 1.7 Å up to 100 ns., ns and shows unstable behaviour (Figure 4).In the South African database, the SA1 complex exhibits significant stability throughout MD simulation from 0.6 to 0.7 Å till end of simulation (Figure 4A).Interestingly, the SA2 complex, according to the RMSd analysis presented, exhibits highly stable behaviour throughout the MD simulation period (average RMSD analysis 0.9Å (Figure 4B).In addition, for the compound Bisindolylmethane thiosemi-carbazides from the in-house database, RMSd graphs reveal stable behaviour at 1.0 to 1.3 Å at 65 ns; after that, the RMSd curve increased to 1.7 Å till 85 ns, thereafter reaching a stable state till the end of simulation (Figure 4C).Furthermore, Bisindolylmethane-hydrazonehybrids complex, the initial RMSd curve increases at 1.8 until 55 ns, then gradually decreases to 1.2, as presented in Figure 4D.In the Zinc database, initially the ZINC35671852 complex mediates an increase in the RMSd graph, reaching 1.9 Å up to 35 ns; thereafter, it gradually decreases to 1.5 Å and shows minor fluctuation till 100 ns (Figure 4E).Furthermore, the ZINC78774792 complex initially presents a decrease in the RMSd curve at 1.4 Å after reaching 55 ns, and the system shows minor fluctuations within 1.5-1.8Å till end of simulation (Figure 4F).The control complex exhibited stable RMSD values, but the complexes formed with the novel compounds showed lower RMSD values, indicating more stable dynamics.This suggests that the novel compounds have a stronger and more stable interaction with aldoreductase.These findings highlight the potential therapeutic value of these compounds for the treatment of anti-diabetic (antihyperglycemic) conditions based on their interaction with aldoreductase. Root mean square fluctuation (RMSF) The calculation of residue flexibility has provided crucial information regarding molecular interaction patterns, interresidue communication, protein coupling, inhibition potential, Biocatalysis, and enzyme engineering.The flexibility of each residue was evaluated and visually represented in Figures 5A-C.The fluctuations for each amino acid of aldose reductase in complex with ligands were evaluated through the RMSF curve, which accesses the stability of the active site towards compounds during the 100-ns MD simulation period.A lower number or reduced fluctuation signifies well-structured and less distorted regions within the complex.Interestingly, the flexibility of residues in the control complex exhibited similarities to the complexes of the top hits. Similarly, in the African database, all the compounds displayed a nearly identical pattern of residue flexibility.The region between 30 and 280 presented higher flexibility in the penta-o-galloyl-glucose complex only, while the regions between 10 and 120, 130-160, and 200-230 demonstrated higher fluctuation in Diamino-di nitromethyl dioctyl phthalate complex in all the complexes (Figure 5A).Additionally, the compound Bisindolylmethane thiosemi carbazides exhibited significantly higher flexibility in the regions 75-180, while Bisindolylmethane-hydrazone hybrids showed higher flexibility in the regions 0-75, 80-110, and 230-280, as shown in Figure 5B.Moreover, a notable fluctuation was observed in the zinc database, where almost all regions displayed higher fluctuations except for 110-300.The RMSF graphs depicting the fluctuations in the zinc database complex are displayed in Figure 5B.Overall, these findings indicate that the binding of each ligand affects the internal dynamics in a different manner. It is noteworthy that the regions 160-190 encompass the active site residues, suggesting that the movement of this loop assists the drug in optimizing its position within the cavity by increasing the volume of the pocket.This ultimately facilitates the stable binding of ligands to the active site of aldose reductase. Radius of gyration The compactness of the system was evaluated by plotting the correlation between RoG (radius of gyration) and time.In comparison to conformational entropy, lower RoG values indicate a highly stable and compact structure, while higher RoG values indicate a lower degree of compactness in the structure.RoG is used to explore the folding and compactness of proteins; lower RoG values present strong compactness and strong structural rigidity, whereas high RoG values present less compactness and an unfolded state.The study of MD simulation presents the effects of inhibitors upon binding with proteins.As presented in Figure 6, RoG analysis shows that these obtained compounds from databases bound to aldose reductase, having lower RoG values as compared with standard drugs, which shows that after binding with ligands, aldose reductase stability and compactness increase.In the control complex, a consistent RoG value of 19.1 Å was observed, indicating a uniform and stable structure. Similarly, the compounds from the African database showed a comparable RoG pattern to the RMSD results.Specifically, the Diamino-di nitro-methyl dioctyl phthalate-AR complex exhibited a higher RoG pattern, with an average RoG of 19.3 Å.In contrast, the Bisindolylmethane-hydrazone hybrids displayed a lower RoG pattern, consistent with the RMSD results, with an average RoG of 19.0 Å Figures 6A, B. Similarly, the structural compactness of each complex from the in-house database was assessed to examine the variations observed during the simulation.Interestingly, the RoG patterns for the top complexes from the in-house database align strongly with the RMSD results.For instance, the RoG of Bisindolylmethane thiosemicarbazides displayed a uniform RoG value with an average of 10.0 Å.The RoG exhibited a consistent graph with no significant changes in protein size, except for an abrupt decline observed at 40-50 ns.The average RoG for this complex was estimated to be 19.As for the ZINC78774792-AR complex, a consistent and uniform RoG was observed throughout the first 100 ns of the simulation.The average RoG for this complex was calculated to be 19.1 Å Figures 5E, F. In conclusion, the results indicate that the identified hits exhibit stable protein compactness, as reflected by similar average RoG values.Additionally, minimal unbinding events were observed.These findings suggest that these hits have promising pharmacological potential and could potentially serve as effective therapeutics against aldoreductase for the treatment of diabetes. Analysis of hydrogen bond Hydrogen bond analysis plays an essential role to understand any protein-ligand complex stability.Herein, we studied strength of hydrogen bonds during MD simulation.In current study, we carried out analysis of hydrogen bonds of top two hits from each database for entire 100 ns MD simulation period.As illustrated in Figure 7, the number of hydrogen bonds were increase in all inhibitors from Zinc, South African and in-house databases by comparing with standard drug.The SA1 exist four hydrogen bond where as SA2 mediate FIGURE 4 RMSD analysis was conducted to assess the dynamic stability of the hits from four databases in complex with aldoreductase.Figures (A-F) show the RMSD profiles for the hits from the African, Inhouse, and ZINC databases, respectively, compared to the control (Tolmetin-Aldoreductase).The results indicate that the hits exhibit lower RMSD values, suggesting more stable dynamics and promising interactions with aldoreductase. FIGURE 5 RMSf analysis of the finally selected hits (A) RMSf analysis Diamino-di nitro-methyl dioctyl phthalate/aldoreductase complex (red), Penta-O-galloyl glucose/aldoreductase complex (Blue) (B) Bisindolylmethane thiosemi-carbazides/aldoreductase complex (pink), Bisindolylmethane-hydrazone hybrids/Aldoreductase Complex (green) (C) ZINC35671852/Aldoreductase Complex (Purple), ZINC78774792/Aldoreductase Complex (Blue).three, IH1, IH2, ZN2, and standard drug have number of two hydrogen bonds during MD simulation. Binding free energy calculation The MMPBSA is one of the commonly employed approaches that is used to access the ligands binding energy with protein molecules.This calculation is an imperative assessment that properly re-evaluates the accuracy and binding conformation of the interacting partner.This approach is the least expensive and has higher accuracy as compared with other techniques.By considering the precious applicability of this approach, we computed the binding free energy for the top inhibitors from each database by utilising MD Frontiers in Molecular Biosciences frontiersin.org10 trajectories.The details of binding free energy are illustrated in Table 2. Dissociation constant and bioactivity analysis PRODIGY-LIG generates the KD results as ΔG upon submission of the complex.For the top six hits, the KD values were calculated to be Diamino-di nitro-methyl dioctyl phthalate (−12.44),penta-Ogalloyl-glucose (−11.63),Bisindolylmethane thiosemi carbazides (−9.87),Bisindolylmethane-hydrazone hybrids (−10.25),ZINC35671852 (−10.14), and ZINC78774792 (−10.87)respectively.The dissociation constant for the control complex was −8.21, indicating its strong activity against aldoreductase.In terms of bioactivity, a score between −0.5 and 0.5 is considered.A molecule with a bioactivity score greater than 0.00 is likely to possess significant biological activity, while scores between 0.50 and 0.00 indicate moderate activity.Scores below 0.50 are indicative of inactivity.The bioactivity for each of the top hit molecules was estimated to be Diamino-dinitro-methyl dioctyl phthalate (0.15), penta-o-galloylglucose (0.27), Bisindolylmethane thiosemi carbazides (0.24), Bisindolylmethane-hydrazone hybrids (0.41), ZINC35671852 (0.23), and ZINC78774792, respectively.The control complex (aldoreductase-tolmetin) was predicted to have a bioactivity score of 0.42, indicating significant biological activity.This suggests that the compounds have a strong potential to inhibit aldoreductase under in vitro conditions. Conclusion Different attempts have been made to identify potent drugs against aldose reductase.But none of the drugs show strong efficiency.Therefore, the present study was designed to apply structure-based virtual screening, MD simulation, and the MMPBSA approach to identify a potent drug against aldose reductase.By performing virtual screening, we identified the top six inhibitors from each database, including the South African natural database, the in-house database, and the zinc database, respectively.All these inhibitors were validated and compared with standard drugs through various computational approaches.In these obtained inhibitors, compounds from South Africa and IH1 were found to be the best inhibitors, which presented less carbon alpha deviation, less fluctuation, binding interaction, and a good binding score.However, in vivo and in vitro analysis were required, which will help validate the activity of compounds for clinical usage. Publisher's note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. FIGURE 1 (A) The binding pocket of diamine-dinitro-methyl dioctyl phthalate with the aldoreductase receptor (B) the binding interaction pattern of the diamine-dinitro-methyl dioctyl phthalate demonstrating the key interacting residues.(C) The binding pocket of penta-O-galloyl-glucose with the aldoreductase receptor (D) the binding interaction pattern of the penta-O-galloyl-glucose, demonstrating the key interacting residues. FIGURE 2 (A) The binding pocket of Bisindolylmethane thiosemi-carbazides with the aldoreductase receptor (B) the binding interaction pattern of the Bisindolylmethane thiosemi-carbazides, demonstrating the key interacting residues.(C) The binding pocket of Bisindolylmethane-hydrazone hybrids with the aldoreductase receptor (D) the binding interaction pattern of the Bisindolylmethane-hydrazone hybrids, demonstrating the key interacting residues. 3 Å.In line with the RMSD results for Bisindolylmethane-hydrazone hybrids, the RoG pattern also demonstrated a gradual increase in the RoG Trajectory Figures6C, D. The top hits from the ZINC database displayed slightly lower but relatively stable RoG values for all the complexes.In the case of the ZINC35671852-AR complex, the RoG initially decreased and reached 19.1 Å at 20 ns It then continued to decrease and stabilised at 19.0 Å for the remaining 100 ns of the simulation. FIGURE 3 (A) The binding pocket of ZINC35671852 with the aldoreductase receptor (B) the binding interaction pattern of ZINC35671852, demonstrating the key interacting residues.(C) The binding pocket of ZINC78774792 with the aldoreductase receptor (D) the binding interaction pattern of the ZINC78774792 demonstrating the key interacting residues. FIGURE 6 ( 6 FIGURE 6 (A-F) Illustrating the analysis of hydrogen bond of standard drug (black) and the final hits. FIGURE 7 7 FIGURE 7RoG of each hit from four databases in complex with aldoreductase (A,B) represent the RMSDs for the tolmetin-aldoreductase and top hits from the African database with aldoreductase.(C,D) represent the RoG for the tolmetin-AR and top hits from the in-house database with aldoreductase.(E,F) represent the RoG for the tolmetin-aldoreductase and top hits from the ZINC database with aldoreductase. TABLE 1 1 Protein-ligand interaction details and their docking of the standard drug and final hits score. CompoundDockingInteraction detailsscoreLigandReceptorInteractionDistanceE(kcal/mol)African database (A)Diamino-di nitro-methyl−12.24C1SGCYS298H-donor3.80−0.2dioctyl phthalateO3CD1LEU300H-acceptor3.87−1.5O17CD1LEU300H-acceptor3.52−0.2O22OGSER302H-acceptor3.09−6.4O17CD1PHE122H-acceptor pi-H3.76−3.3C76-ringTRP219pi-H3.65−1.3C56-ringTRP204.33−2.1Penta-O-galloyl-glucose−11.34O6SGCYS122H-donor3.16−1.4O28OVAL297H-donor2.86−1.6C316-ringPHE122H-pi pi-H4.13−0.76-ringCBPHE1223.67−0.8In-house database (B)Bisindolylmethane−10.25N7OVAL47H-donor3.06−0.5thiosemi-carbazidesS16OASP216H-donor4.06−0.1C19OASP216H-donor3.55−0.2C19OD2ASP216H-donor3.77−0.2C45OCYS298H-donor4.27−0.1S16CDPRO LEU218H-acceptor4.48−0.1O19CD1300H-acceptor3.61−0.1Bisindolylmethane-−9.51S16SGCYS298H-donor3.77−0.7hydrazone hybridsN39OVL47H-donor3.39−0.8O13NLEU300H-acceptor3.09−1.6S16NE1TRP111H-acceptor4.22−0.06-ring6-ringTRP219Pi-Pi3.78−0.7Zinc database (C)ZINC35671852−7.90N522CELYS77H-acceptor3.38−0.7C56-ringTRP20H-pi3.50−0.55-ring86-ringTYR209Pi-pi3.19−0.06-ring6-ringTRP219Pi-pi3.60−0.0ZINC78774792−7.49N318CELYS77H-acceptor3.45−0.2O3ND2ASN160H-acceptor3.03−0.26-ringCALEU300H-pi4.37−0.26-ring19CD1LEU300Pi-pi3.48−0.16-ring6-ringTYR209Pi-pi3.32−0.06-ringTRP2193.82−0.0Control drug (Tolmetin)−6.71O218NE1TRP11H-acceptor3.16−2.4(Continued on following page)Frontiers in Molecular Biosciencesfrontiersin.org TABLE 1 ( 1 Continued) Protein-ligand interaction details and their docking of the standard drug and final hits score. CompoundDockingInteraction detailsscoreLigandReceptorInteractionDistanceE(kcal/mol)O218ND2ASN160H-acceptor2.69−6.5O319OHTYR48H-acceptor2.75−0.5 TABLE 2 2 Illustration of binding free energy of control drug and finally selected hits. ComplexesVDWEELESURFEGBΔG TotalStd. Err. of MeanDiamino-di nitro-methyl dioctyl phthalate−67.8424−2.9115−6.857816.9381−60.67360.1813Penta-o-galloyl-glucose−56.22161.2134−6.239410.5192−50.72830.1592Bisindolylmethane thiosemi-carbazides−52.3106−0.5548−6.589112.3153−47.13920.1433Bisindolylmethane-hydrazone hybrids−61.1410−4.3809−7.035031.2747−41.28220.2546ZINC35671852−43.8532−0.2644−5.00528.9494−40.17340.1046ZINC78774792−42.0320−0.8339−4.84438.8884−38.82180.1819Control drug (Tolmetin)−32.0807−52.8459−3.538064.1539−24.31080.1200 Frontiers in Molecular Biosciences frontiersin.org Data availability statementThe raw data supporting the conclusion of this article will be made available by the authors, without undue reservation.Author contributionsMS: Investigation, Writing-review and editing.GZ: Project administration, Writing-original draft.MB: Investigation, Writing-original draft.WG: Investigation, Writing-review and editing.AM: Investigation, Validation, Writing-original draft.FundingThe author(s) declare that financial support was received for the research, authorship, and/or publication of this article.This research was funded by the National Key R&D Program of China Nos 2021YFC2102900 and 2021YFC2101503, and Beijing Natural Science Foundation No. L212001.This work was supported by the State Key Laboratories of Chemical Re-Sources Engineering, Beijing University of Chemical Technology, Beijing, China.The authors would like to extend their sincere appreciation to the Researchers Supporting Project, King Saud University, Riyadh, Saudi Arabia for funding this work through the project number (RSP-2023R437).Conflict of interestThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 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Neuropsychological assessment methodology revisited: metatheoretical reflections 17 November 2023 Xerxes D Arsiwalla Shimon Marom Josh Joseph Ramminger Department of Psychology University of Marburg Marburg, Germany Department of Psychology Humboldt University of Berlin Berlin, Germany Martin Peper Department of Psychology University of Marburg Marburg, Germany Alexander Nicolai Wendt alexander.wendt@psychologie.uni-heidelberg.de Faculty of Psychology Sigmund Freud University ViennaAustria Department of Psychology Heidelberg University HeidelbergGermany Pompeu Fabra University Spain Technion -Israel Institute of Technology Israel Neuropsychological assessment methodology revisited: metatheoretical reflections 17 November 2023B7AF3CB838DB64F54738AC4FC6648D0F10.3389/fpsyg.2023.1170283RECEIVED 20 February 2023 ACCEPTED 23 October 2023 The contemporary discourse surrounding the issue of low replicability rates in psychology (Open Science Collaboration, 2015) posits that such rates can be attributed, at least in part, to deficiencies in theory building(Muthukrishna and Henrich, 2019;Oberauer and Lewandowsky, 2019;Witte, 2022).Therefore, the validity of empirical research is contingent upon the soundness of scientific theory.Scientific theories encompass convictions pertaining to the subject-matter under investigation, as well as the interrelationships between the various entities or attributes being examined Theory building in neuropsychology, similar to other disciplines, rests on metatheoretical assumptions of philosophical origin.Such assumptions regarding the relation of psychological and physiological variables influence research methodologies as well as assessment strategies in fields of application.Here, we revisit the classic procedure of Double Dissociation (DD) to illustrate the connection of metatheory and methodology.In a seemingly unbridgeable opposition, the classical neuropsychological procedure of DD can be understood as either presupposing localizationism and a modular view of the brain, or as a special case of the generalized neuro-lens model for neuropsychological assessment.In the latter case, it is more easily compatible with a perspective that emphasizes the systemic-network, rather than the modular, nature of the brain, which as part of the organism, proportionately mediates the situatedness of the human being in the world.This perspective not only makes it possible to structure ecological validation processes and give them a metatheoretical foundation, but also to interlace it with the phenomenological insight that the laboratory as one context of empirical research may be analyzed in terms of situated experience.We conclude with showing that both the localizationist and the system science approach can agree on a view of the brain as a dynamical network, and that metatheory may thus offer important new perspectives of reconciliation. KEYWORDS neuropsychological assessment, lense model paradigm in neuropsychology, philosophy of science, phenomenological psychology, metatheory, ecological validity, modularity, neural networks The problem of consciousness and neuropsychological methodology 10.3389/fpsyg.2023.1170283(Borgstede and Eggert, 2022).To the extent that these are metatheoretical or ontological, they also belong to the scope of philosophy (cf.Hastings et al., 2020).However, it should be noted that a metatheoretical framework differs from a specific scientific theory in that it can structure competing concrete individual theories (Muthukrishna and Henrich, 2019).Metatheoretical frameworks that belong to the relation of psychological and physiological variables are coherently also found in psychology, physiology, and cognitive neuroscience (cf.Marom, 2020;Pauen, 2021).These disciplines thus take a stance toward a problem which philosophers call the mind-body problem or problem of consciousness (see Pauen, 2021;Schleim, 2022).Yet, throughout history, philosophers could not achieve a consensus on the solvability of this problem.In 1872, Du Bois-Reymond gave a series of lectures on the limits of scientific explanation, one of these limits being the problem of consciousness (Du Bois-Reymond, 1872; see also Schleim, 2022).In 2013 Kügler has regarded the "evershifting problem" of consciousness as an unsolvable riddle (Kügler, 2013;but see Pauen, 2021).Regardless of matters concerning the solvability of the problem, recent work in theoretical psychology and neuroscience has emphasized that philosophical positions or metatheoretical frameworks, such as the postulate of neuropsychological reducibility or the postulate of psychophysical causality may influence theory-building, research methodology, as well as diagnostics or even therapeutic interventions (see Fahrenberg, 2013, 404;Fuchs, 2017;Marom, 2020).Explicit metatheoretical frameworks for the subject-matter of these sciences are, for instance, biological naturalism, which regards mental phenomena as properties of the brain (Searle, 1992), or enactivism, which holds them as emergent properties of an organism in a dynamic-reciprocal interplay with its environment (see Lee, 2023).Krakauer et al. (2017) have claimed that cognitive neuroscientists and psychologists, while guided by philosophical beliefs, implicitly adumbrate the lack of an explicit metatheoretical or conceptual framework when they use filler terms.Without such a framework, statements like "The circuit X is involved in behavior Y" (ibid., 485) would be a mere restatement of the correlative or causal relation and would not (further) contribute to any explanation.The lack of explicit metatheoretical frameworks coincides with the notion of a neglect of (formal) explanatory theory in psychology (Teigen, 2002;Oberauer and Lewandowsky, 2019;McPhetres et al., 2021;Borgstede, 2022;Wendt and Wolfradt, 2022).We wish to call attention to the influence of different metatheoretical frameworks, as it may be the case, that a single empirical finding can be accounted for by multiple explanatory frameworks.The recourse to parsimony to justify the primacy of framework x over framework y is only logically permitted if it is not made unreflectively based on framework x, otherwise one would be committing the fallacy of a petitio principii. In light of the broad array of philosophical views concerning the problem of consciousness, we do not commit ourself to any particular one.This article investigates the metatheoretical beliefs regarding the relation of physiological and psychological variables, which beliefs inherent to different neuropsychological assessment procedures, such as double dissociation and the concept of reverse experimentation (see Kadlec and van Rooij, 2003). Our intention is to assert that metatheoretical stances may stimulate improved approaches for addressing specific methodological requirements in neuropsychological research, such as internal and ecological validity.To achieve this objective, we draw upon a phenomenological orientation which can be found in 20th century psychology (Lewin, 1936;Herzog, 1992; but also see Wendt, 2022), philosophy (Gurwitsch, 2010), as well as neuropsychology (Goldstein, 1995;Frisch, 2014a). The entanglement of metatheory and methodology in neuropsychological assessment Our endeavor commences with an analysis of a widely used neuropsychological practice known as double dissociation (DD).The rationale of DD holds that, if a brain lesion A leads to the impairment of the psychological function 1 but not of function 2 and a brain lesion B leads to the impairment of function 2 but not of function 1, a relative functional independence of the two brain areas can be assumed (see, e.g., Stone and Davies, 1993, 594).A prototypical example is the dissociation of speech production, impaired in patients with lesions in Broca's area, and the impairment of speech comprehension, impaired in patients with lesions in Wernicke's area (see Gazzaniga et al., 2014, 472-474). One classical presupposition regarding DD is that its validity rests on the metatheoretetical assumption of modularity, eventhough this assumption was subject to extensive critique (Shallice, 1988;Plaut, 1995).It should be emphasized that multiple accounts of modularity exist (cf.Gottschling, 2020).For instance, Shallice (1988, 20) discusses Fodor (1983), whose account of modularity defines a module as a subsystem exhibiting specific characteristics, including domain specificity, innate specification, indecomposability into basic elements, hardwiredness, computational autonomy, information encapsulation, and a distinctive pattern of development.Fodor argues that modules are "computationally autonomous" in the sense that they operate independently without relying on general-purpose processes from other modules."Informational encapsulation" refers to the limited access of a module to a specific subset of information within the overall system (Shallice, 1988, p. 20).Shallice critically contends that this conceptualization of modularity may be excessively rigid, considering the subjectmatter of neuropsychology.Because of these concerns Shallice adheres to the concept of functional differentiation in regard to subsystems.In accordance with Tulving (1983), Shallice asserts that two subsystems exhibit functional dissimilarity when one system functions independently but potentially less efficiently without the support of the other intact system.In the case of functional dissimilarity, enhancements or suppressions in the operations of one system do not necessarily impact the other system in a similar manner.Accordingly, this functional disparity indicates that the systems operate differently and are governed by distinct principles, at least partially (Tulving, 1983, p. 66).However, it is still common to interpret double dissociation as methodological correlate of the metatheoretical assumption of modulartiy [see for a critique (Shallice, 1988;Plaut, 1995)].Still, it must be noted, that the concepts of modularity and functional dissimilarity bear relevant similarities.When we speak of 'modular' we will adress this wide sense of modularity. Reflections on double dissociation The explanatory paradigm of DD may be subjected to critique, for example, from the phenomenological standpoint of enactivism which has been advanced by Thomas Fuchs.In our view, DD is also consistent with a metatheoretical position Fuchs termed "biological epiphenomenalism".This approach regards consciousness as a "dispensable varnish" (2017, 227), i.e., views conscious experience as a causally ineffective byproduct of brain processes.DD's primary focus lies in investigating the influence of brain lesions on behavior or experience, specifically examining how physiological variables affect psychological aspects.However, it does not typically investigate the reverse relationship, where psychological factors affect physiological variables.Fuchs rejects the notion of a dualism between mind and brain that is implied by such perspectives.In his view, psychological variables are not separate from bodily processes.He regards psychological variables as abstractions used to describe properties of an embodied mind.For Fuchs, it is the conscious, living organism, which possesses causal power, not the abstraction (2017).Marom (2015) largely agrees with Fuchses perspective (2015, pp.49-68).For Marom, psychological and physiological variables are viewed as categorically, but not necessarily ontologically distinct (see Fahrenberg, 2013Fahrenberg, , 2015)).It may be argued that DD does not adequately consider this categorical distinction.Furthermore, if DD is approached from a biological epiphenomenalist standpoint, it becomes challenging to reconcile certain empirical findings.Examples of such findings are that subjectively experienced stress is predictive of somatic health outcomes (Tsukerman et al., 2020), that meditation enhances hippocampal connectivity (Lardone et al., 2018), or that psychotherapy improves the linkage between the amygdala and the cognitive control network (Shou et al., 2017).The reason for this explanatory difficulty is that the conceptual framework of biological epiphenomenalism does not accommodate the effects of psychological variables on physiological variables.Enactivism, on the other hand, argues that through downward causality, psychological variables, as emergent properties of the embodied mind, can influence "simpler" biological variables (Fuchs, 2020).However, the potential for circular causality remains a subject of debate (see, for example, Lee, 2023), and for the purpose of our discussion, we remain true to the metatheoretical perspective by bracketing the decision for one or the other standpoint. One of has summarized further arguments against DD in a previous article.On the one hand, the aforementioned concept of dissociation of function seems problematic due to a lack of factor independence.Additionally, DD has been subject to criticism for relying on non-experimental ex post facto data.Consequently, DD faces limitations in establishing causal relationships between neurobiological and mental phenomena.Moreover, it fails to demonstrate necessary identity between psychological and physiological phenomena on the ontological level due to the existence of an indefinite number of potential neural networks that can implement the same psychological function (Peper, 2018). 1ouble dissociation but also its critical adversaries, are substantially influenced by their underlying metatheoretical pre-suppositions.This highlights the importance of methodological reflection, as it has the potential to facilitate metatheoretical reconciliation and potential improvement.In the following discussion, we will illustrate how a meta-model for neuropsychological assessment (Peper, 2018), as well as the phenomenological orientation in psychology (Wendt, 2018) and neuropsychology (Goldstein, 1995;Frisch, 2014a,b) might contribute to addressing the limitations of DD and potentially overcome its shortcomings. A lens type meta-model for neuropsychological assessment Within neuropsychological assessment theory, one of us has put forth the neuro-lens model (NLM) which is a neuropsychological generalization of DD since the latter can be regarded as a special case of the former (cf.Peper, 2018).NLM's epistemological approach to relate distal and proximal entities draws on the metaphor of the lens (cf.Brunswik, 1952). The NLM framework poses the following pre-conditions for inferring causal relations between psychological ( ) and physiological ( ) domains incorporates the following three preconditions: (a) the ability to experimentally manipulate the psychological and physiological variables of interest, (b) the identification of convergent and discriminatory correlations, which are indicators of validity, and (c) the investigation of both causal directions between psychological and physiological variables, that is, examining the influence of on ( → ) as well as the influence of on ( → ). According to the logic of this so-called reverse experimentation approach, a psychological function of interest could be stimulated to show that a specific biophysical activation depends on that function, and not on another activation.For instance, a visual stimulus could be presented in an fMRI experiment to capture the neural correlates associated with visual perception.2In contrast, neural system manipulation could be utilized to demonstrate the modification of a specific psychological function while leaving others unaffected (Peper, 2018): transcranial magnetic stimulation (rTMS) could be applied, for example, to induce a temporary disturbance in the motor cortex (M1), selectively impacting 10.3389/fpsyg.2023.1170283hand movement in one region and arm movement in another (Peper, 2018). Methodologically speaking DD can be seen as a specific application of the NLM.The NLM offers methodological advantages, such as its hierarchical multilevel structure, which addresses the issue of factor independence in both mental and physiological variables.In addition to these methodological considerations, the NLM brings about a shift in the metatheoretical assumptions of neuropsychological assessment strategies.According to this, experimental manipulation can be applied to both categorical domains of neural and psychological phenomena.It thus captures the range of possibilities that have been developed within the field of neuropsychological assessment and research and offers a more comprehensive approach to exploring the complexities of brain-mind relationships. Double dissociation and the NLM both describe methodological procedures, while e.g., epiphenomenalism or the system science/network view are metatheories.Yet, metatheory and methodology are not independent.Because DD (merely formally) can be seen as a special case of the NLM, one could employ DD's methodology while adhering to a metatheoretical network perspective.However, it is not possible to be a metatheoretical epiphenomenalist and simultaneously employ the NLM as methodological framework. The generalization by the NLM encompasses methodological and metatheoretical perspectives concerning the contextdependency of psychophysiological variables.This context-dependency, however, may not be adequately addressed within the framework of classical discriminant diagnosis of which DD is an instance.This is particularly the case when this framework is approached from a modularist perspective, which according to Frisch (2014b) often assumes that knowledge acquisition occurs solely within standardized environments.However, methodologically there is no inherent reason why (experimental) research cannot be conducted beyond the confines of the laboratory (Fahrenberg et al., 2007).We therefore see that the metatheory associated with the NLM is preferable to one that does not consider the context and context-dependency of psychological, as well as physiological variables.The NLM emphasizes the context dependency of psychological and physiological attributes with regard to methodology and metatheory. Concerning the issue of ecological validity, Peper follows Brunswik, in stating that "the conditions and materials of assessment should be representative of the environment of the person.Multiple interacting environments, for instance, shared or non-shared contexts of personal life events can be identified.Thus, different types of lens models are needed to improve ecological validity" (Peper, 2018, 272).This assertion seems especially important since the ability of some neuropsychological tests to predict the impairment of patients in their daily living environment appears to be limited (e.g., Peper and Loeffler, 2014;Suchy et al., 2022). The concept of "ecological validity" has been criticized recently for conceptual vagueness and risk of antagonizing the "real world" and the "neutral lab" (Holleman et al., 2020).Consistent with Peper's assumptions of differences in contexts, phenomenological psychology's paradigm of situation analysis can shed light on the fact that the laboratory is but one context of experience, as one of us has argued (Wendt, 2018). 3It is crucial to understand, however, that complementary to an understanding of the context of the "physical" environment of an organism, it is also necessary to assume a subjective experienced environment (in the sense of Umwelt the works of theoretical biologist Jakob Johann Von Uexküll, 1921).Among other reasons, because it is possible, that people situated within the same physical environment experience a different Umwelt (Gurwitsch, 1976), a descriptive approach to the assessment of the situation of an individual may contribute to neuropsychological procedures (cf.Frisch and Métraux, 2021).This perspective thus helps both to avoid the justified criticism by Holleman et al. concerning the antagonization of the "real world" and a supposedly neutral laboratory and to take different types of experienced situations into account (Wendt, 2018, 4).Striving for ecological validity makes it necessary to reflect on metatheoretical stances regarding the contextual nature of the human condition. Contextuality and metatheoretical dialogue Metatheoretical reflections regarding the contextual nature of the human condition can be found in phenomenological psychology, which has a long tradition of emphasizing that human experience is situated (Lewin, 1936;Merleau-Ponty, 1962;Gurwitsch, 2010;Wendt, 2018).The observation that the laboratory, unlike many other contexts of human experience and behavior, is characterized by an elimination of many everyday stimuli does not contradict the observation that contexts outside the laboratory are heterogeneous.In shared work one of us has argued that [n]atural situations differ from lab situations in multiple ways as they require more complex planning, organizational and monitoring processes.In contrast, lab environments are typically void of distractors that divert the subjects attention from the task.Moreover, the test administrator, who structures the test session and supports the subject throughout the procedure, is not present in real life; thus, a crucial social agent that compensates for deficits and provides extrinsic motivation is absent (Peper and Loeffler, 2014, 233-234). According to Eling (2015), the phenomenologically oriented physician Kurt Goldstein (1878Goldstein ( -1964) ) spoke of some test situations as being "lebensfremd, " (not true to everyday life) and of others as being "lebenswahr" (true to everyday life).Goldstein, together with the gestalt psychologist Adhémar Gelb (1887Gelb ( -1936)), played a central role in the advent of contemporary neuropsychology.Goldstein's phenomenologically inspired positions can be understood as a metatheoretical or 3 The acknowledgment of the laboratory as a meaningful situation, governed at least partially by, among other experiential factors, social rules, and individual expectations, may create an opportunity to analyze, for instance, the Milgram Experiment as an investigation into the authoritative role of science in Western societies (see Haslam et al., 2014).Overlooking the fact that the act of entering a laboratory stimulates a distinct experience may result not only in an overestimation of the generalizability of experimental results but eventually also leads to impaired interpretations of empirical findings. Frontiers in Psychology 04 frontiersin.orgmetascientific attempt at structuring the various schools of theory and methods presented here.Accordingly, Goldstein was an early critic of modularity, stressing that psychological functions can only be understood if the whole organism is taken into consideration (Gelb and Goldstein, 1920;Rimpau, 2009).This position possesses at least some similarity with enactivism which commonly regards psychological variables as properties of the entire organism (cf.Fuchs, 2017).Frisch (2014a) notes that Goldstein viewed practices contingent on some versions of modularity, as DD according to some authors (Warrington, 1981), as insufficient, because they do not consider that patients can partially regain psychological functions after brain lesion.The possibility of such recovery indicates that extended networks can realize the realization of a psychological function.Furthermore, the realization of a psychological function via a complex system can be disrupted if one damages a part of the system. 4This does not imply that one can infer a localization of the function within the lesioned part. Frisch argues, that the loss of a psychological function may be dependent on a situation.For instance, the recall of the same words may be disturbed in the symbolic context (naming) but not in the concrete-emotional context (scolding).5 Lastly, Goldstein's clinical work indicated that brain lesions usually do not affect only a single function.Likewise, it would rarely be the case, that a psychological function is fully absent after lesion, with other psychological functions being completely intact (Frisch, 2014a,b).It seems reasonable to assume that these sophisticated aspects can be better addressed by the generalized NLM than by DD.Goldstein regarded the brain as a network (Netzwerk) situated within the organism which he again viewed as situated within its life and within a concrete situation (Goldstein, 1927(Goldstein, , 1995;;Frisch, 2014a,b;Frisch and Métraux, 2021). The metatheoretical potential of Goldstein's position lies in the fact that it does not imply that we need to abandon any assumption of local specification at a particular time t.Equally, in our opinion, a view of the brain as a dynamic network nested in an organism which is nested in a world is also largely consistent with some versions of modularity.As we have noted, metatheoretical beliefs structure scientific theories; yet, they are not easily falsifiable.Since a lesion rarely leads to a complete loss of psychological function (cf.Frisch, 2014a), one can either argue that the case is not "pure" enough and therefore in favor of modularity or interpret the findings as evidence against modularity.6However, it obviously makes a difference whether the hippocampus or the PFC is affected by a lesion, whether this is due to the modular structure of the brain or to the fact that a part of a circuit has been damaged.Given that modularists must acknowledge the plasticity of the brain, the branch of modularity that seems largely consistent with a system science neuropsychological assessment strategy can be regarded as dynamic modularity.Furthermore, Frisch (2014a) emphasizes that Goldstein did not subscribe to equipotentialism, the idea that solely the size of the lesion was of functional importance.Moreover, some authors argue that a network approach to the brain is compatible with versions of modularity (Alexander-Bloch et al., 2010). We need not settle the question of whether the modularity assumption holds, since, our aim is only to demonstrate that philosophical assumptions have the potential to shape both research and assessment in neuropsychology.In this context, Goldstein's belief that the brain is a dynamic and adaptable network, and that lesions have a comprehensive impact on the entire organism, which in turn adapts its Umwelt to cope with the new situation, aligns with various metatheoretical frameworks in neuropsychology.The adaptation of the organism encompasses not only physical aspects of the environment, but also subjective experiences structured by demand characteristics and affordances (cf.Lewin, 1936;Dings, 2020).By considering the contextual aspects of individuals and patients, both in terms of their distal environment (physical surroundings) and proximal environment (Umwelt), generalized lens models might help to effectively examine the relationship between proximal and distal aspects of the subject matter of neuropsychology. Conclusion Our aim was to revisit the metatheoretical or philosophical beliefs that accompany neuropsychological research and assessment.Despite appearing to be in opposition, the classical neuropsychological approach of DD can be understood either as assuming localizationism and a modular view of the brain, or as a specific case of lens-type modeling approaches (NLM) to neuropsychological assessment.The latter interpretation more readily aligns DD with a comprehensive systemic view of the human brain as a network that, as part of the whole organism, mediates the situatedness of human beings in the world.These perspectives closely intersect with the empirical and theoretical work of early neuropsychologist Kurt Goldstein, who emphasized the situatedness of the organism within its Umwelt (subjectively experienced environment).Thus, both modularity and the system science approach sketched here, converge in Goldstein's claim that the brain is a dynamic and adaptable network, and that lesions impact the entire organism, which then adapts its Umwelt to cope with the new overall situation.This perspective not only enables the structuring of ecological validation processes through a metatheoretical foundation, but also aligns with the idea from phenomenological psychology, that the laboratory is only one of many situations.Lens-type models may provide a methodological framework to better adapt neuropsychological assessment strategies, that accommodate a minimal consensus among the different metatheories of neuropsychology.The analysis therefore shows that metatheory in neuropsychology is not in opposition to therapeutic practice and research.All three levels are in epistemic continuity and can complement each other in a substantial manner. Data availability statement The original contributions presented in this study are included in the article/supplementary material, Frontiers in Psychology A note for the philosophically inclined: The argument of ex post facto data is especially relevant to non-identity theorists. The argument concering necessary relation is especially relevant to identity theorists. Pepers critique thus remains forceful from different metatheoretical standpoints. It should be noted, however, that identifying the substrate, i.e., the correlating brain state of a psychological function, is a difficult undertaking. Every state of consciousness is accompanied by its neural enabling conditions, its neural substrate, and its neural consequences.de Graaf et al. (2012) argue that only enabling conditions and consequences can be separated from each other, while the assumed substrate of mental function always remains intertwined with one of the two and thus eludes identification in empirical analysis.Frontiers in Psychology It has been argued that this was demonstrated by von Monakow (cf.Frisch, 2014a). According to Frisch (2014a), this was demonstrated by Hougling Jackson. Van Orden et al. claim that the first interpretation leads to the iterative introduction of new modules, as there are no criteria for the acceptance or rejection of modules (cf. 2001). frontiersin.orgRamminger et al. 10.3389/fpsyg.2023.1170283further inquiries can be directed to the corresponding authors.FundingOpen Access funding provided by the Open Access Publishing Fund of Philipps-Universität Marburg.Author contributionsJR developed the initial idea and wrote the first manuscript.AW and MP made substantial contributions to the text.JR, AW, and MP jointly finalized the manuscript.All authors contributed to the article and approved the submitted version.Conflict of interestThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Publisher's noteAll claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. 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Aligning climate scenarios to emissions inventories shifts global benchmarks Matthew J Gidden gidden@iiasa.ac.at International Institute for Applied Systems Analysis LaxenburgAustria Climate Analytics BerlinGermany Thomas Gasser International Institute for Applied Systems Analysis LaxenburgAustria Giacomo Grassi Joint Research Centre European Commission IspraItaly Nicklas Forsell International Institute for Applied Systems Analysis LaxenburgAustria Iris Janssens International Institute for Applied Systems Analysis LaxenburgAustria Department of Computer Science University of Antwerp AntwerpBelgium William F Lamb Mercator Research Institute on Global Commons and Climate Change BerlinGermany School of Earth and Environment Priestley International Centre of Climate University of Leeds LeedsUK Jan Minx Mercator Research Institute on Global Commons and Climate Change BerlinGermany School of Earth and Environment Priestley International Centre of Climate University of Leeds LeedsUK Zebedee Nicholls International Institute for Applied Systems Analysis LaxenburgAustria Melbourne Climate Future's Doctoral Academy School of Geography, Earth and Atmospheric Sciences University of Melbourne ParkvilleVictoriaAustralia Climate Resource NorthcoteVictoriaAustralia Jan Steinhauser International Institute for Applied Systems Analysis LaxenburgAustria Potsdam Institute for Climate Impact Research PotsdamGermany Keywan Riahi International Institute for Applied Systems Analysis LaxenburgAustria Aligning climate scenarios to emissions inventories shifts global benchmarks 4A3AE7AD8C0E26959F58E03E89B8E17A10.1038/s41586-023-06724-yReceived: 13 February 2023 Accepted: 6 October 2023 Taking stock of global progress towards achieving the Paris Agreement requires consistently measuring aggregate national actions and pledges against modelled mitigation pathways 1 .However, national greenhouse gas inventories (NGHGIs) and scientific assessments of anthropogenic emissions follow different accounting conventions for land-based carbon fluxes resulting in a large difference in the present emission estimates 2,3 , a gap that will evolve over time.Using state-of-the-art methodologies 4 and a land carbon-cycle emulator 5 , we align the Intergovernmental Panel on Climate Change (IPCC)-assessed mitigation pathways with the NGHGIs to make a comparison.We find that the key global mitigation benchmarks become harder to achieve when calculated using the NGHGI conventions, requiring both earlier net-zero CO 2 timing and lower cumulative emissions.Furthermore, weakening natural carbon removal processes such as carbon fertilization can mask anthropogenic land-based removal efforts, with the result that land-based carbon fluxes in NGHGIs may ultimately become sources of emissions by 2100.Our results are important for the Global Stocktake 6 , suggesting that nations will need to increase the collective ambition of their climate targets to remain consistent with the global temperature goals. The 2021 UN Climate Change Conference (COP26) marked a shift in the focus of climate policy from pledge-making to implementation towards the long-term temperature goal of the Paris Agreement, the collective progress towards which is assessed through periodic Global Stocktakes (GSTs).In spring 2022, the first GST was launched 7 and continues through the 2023 United Nations Climate Change Conference (COP28) to establish evaluation mechanisms among parties.Comparing present emission trends from the NGHGIs and future targets in a collective benchmarking effort rooted in the best available science will be key for a rigorous, precedent-setting first GST and the overall success of the Paris Agreement 6 . Countries have gradually increased the ambition of their national targets in response to the latest IPCC report findings 1,8 .Notably, several nations made long-term net-zero emission commitments in the run-up to COP26 (ref.9), which brought the long-term temperature goal of the Paris Agreement within striking distance, although much of the assessed temperature reductions arose from long-term and non-binding promises rather than immediate climate action [10][11][12] .Global climate scenarios show that both deep reductions of near-term emissions as well as enhancement of anthropogenic land-based carbon sinks are needed to achieve net-zero emissions and limit global warming to achieve the temperature goal of the Paris Agreement 13,14 . A key discrepancy exists, however, in how model-based scientific studies and NGHGIs account for the role of anthropogenic land-based carbon fluxes 4, 15,16 , with national inventories incorporating a broader scope of removals 2 , resulting in lower emission estimates in NGHGIs.Previous studies [2][3][4] have quantified the magnitude of this difference to be approximately 5.5-6.7 Gt CO 2 yr −1 .This conceptual difference hinders the comparability of the aggregate targets set by countries and future mitigation benchmarks.Although this problem has been acknowledged in the most recent IPCC assessment 17 and raised by parties during the GST 18 , the impact of this discrepancy on national and global mitigation benchmarks is still not well understood.Aligning mitigation pathways assessed by the IPCC with NGHGI conventions is therefore needed to support the science-based formulation of nationally determined contributions (NDCs) and to measure collective global action against emission levels necessary to achieve the Paris Agreement goal. Aligning climate pathways and inventories The IPCC-assessed mitigation pathways are typically generated by integrated assessment models (IAMs) that capture transitions in anthropogenic energy and land-use systems consistent with stated global climate policy objectives.The reporting conventions for land-use, land-use change and forestry (LULUCF) carbon fluxes of these models follow that of detailed global carbon-cycle models (that is, 'bookkeeping' models).These models simulate and account for direct anthropogenic fluxes (due to human-induced land-use changes, forest harvest and regrowth) separately from indirect fluxes that are the natural response https://doi.org/10.1038/s41586-023-06724-y of land to environmental changes (for example, CO 2 fertilization or response to climate change) 5,15,19,20 and define anthropogenic emissions as those owing to the direct effect.Because it is practically not possible to separate direct and indirect fluxes through observations, the NGHGIs submitted by parties to the United Nations Framework Convention on Climate Change (UNFCCC) follow reporting conventions 21 that define anthropogenic fluxes using an area-based approach 22 in which all fluxes occurring on managed land are considered anthropogenic, with few exceptions to isolate natural disturbences 16,23,24 .The NGHGIs include a wider definition of managed land compared with models, which includes any forested area that 'perform[s] production, ecological, or social functions' 25 (Fig. 1).As a result, present-day LULUCF fluxes estimated with scientific modelling conventions indicate that the land sector is a net source of emissions 3 , whereas the NGHGIs collectively report it as a net sink 26 , resulting in fundamentally different present and future perspectives of the role of land-based fluxes. To estimate the direct and indirect components of land-based carbon fluxes necessary to align mitigation pathways with conventions used in the NGHGIs, we use a reduced-complexity model with an explicit treatment of the land-use sector, OSCAR 5 , one of the models used for the annual Global Carbon Budget 3 , applied in a probabilistic setup and at a resolution of five global regions used in the IPCC assessments (Methods).We calculate a difference of 4.4 ± 1.0 Gt CO 2 yr −1 in LULUCF emissions globally averaged over 2000-2020 between model-based (higher) and NGHGI-based (lower) accounting conventions, which is in line with the existing estimates 2,5 .We then assess the pathways with OSCAR to quantify how the difference between conventions evolves over time.A total of 914 of the 1,202 IPCC-assessed pathways provided sufficient land-use change data to enable this alignment (Extended Data Table 1; data are available at https://data.ece.iiasa.ac.at/genie/). Across both the 1.5 °C and 2.0 °C scenarios (Fig. 2a,b; see definitions in the Methods), LULUCF emissions estimated using the NGHGI conventions show a strong increase in the total land sink until around mid-century.However, the NGHGI alignment factor (that is, the difference between the two accounting conventions; Fig. 2c) decreases over this period, nearing zero in the 2050s to 2060s for the 1.5 °C scenarios and the 2070s to 2080s for 2.0 °C scenarios.This convergence is primarily a result of the simulated stabilization and then decrease of the CO 2 -fertilization effect as well as background climate warming reducing the overall effectiveness of the land sink 27,28 , which in turn reduces the indirect removals included in NGHGIs.These dynamics lead to land-based emissions reversing their downward trend in most NGHGI-aligned scenarios by mid-century and result in the LULUCF sector becoming a net source of emissions by 2100 in about 25% of both the 1.5 °C and 2.0 °C scenarios. Global and regional ambition implications More ambitious mitigation action is required to meet the global emission benchmarks derived from scenarios when assessed using the NGHGI conventions compared with model-based conventions (Extended Data Table 2 and Extended Data Fig. 1).The NGHGI-aligned pathways result in earlier net-zero CO 2 emissions by 1-5 years for the 1.5 °C and −1 to 7 years for the 2.0 °C scenarios (Fig. 3a).Emission + + + = Enabling like-for-like comparison between the two conventions Scienti c models (red) do not currently match NGHGIs (green) resulting in different emissions estimates. To align them, indirect uxes (blue) that occur on all land considered managed in NGHGIs, simulated with vegetation models, need to be added to direct uxes (red) calculated with bookkeeping models. Alignment factor Misalignment between NGHGIs and scientific models Vegetation models Simulate uxes caused by environmental changes (for example, carbon fertilization) Scientific modelling convention Based on models of the carbon cycle We use the term 'unmanaged' to describe land not considered managed by NGHGIs to be consistent with previous literature, but recognize that this includes land that has been managed by indigenous and traditional communities for centuries to millienia 38,39 .In this study, we estimate the alignment factor to translate between both conventions (the indirect flux considered in NGHGIs but not in models, blue).This factor will change over time based on future land-use decisions and overall mitigation efforts because of, for example, changing atmospheric CO 2 levels. NGHGI convention reductions in 2030 relative to 2020 are between 3 and 6 percentage points greater for both pathway categories (Fig. 3b).The assessed cumulative net CO 2 emissions to global net-zero CO 2 also decreases by 15-18% for both the 1.5 °C and 2.0 °C scenarios (Fig. 3c) because of extra land-based carbon removal when using the NGHGI conventions. Although the NGHGI-aligned benchmarks strengthen, they are still consistent with the climate assessment of the IPCC.All land-use fluxes (direct and indirect) are included in the physical climate models used by the IPCC-that is, the temperature outcomes of each pathway are the same even if flux components are accounted for differently by models and inventories.When considering the additional land sink following the conventions of the NGHGIs, however, multiple dynamics interact that contribute to the revisions of the benchmarks, including the change in historical emission baseline, the enhanced anthropogenic land sink compared with what was reported by IAMs and declining sequestration from that additional sink in the future. Parties to the UNFCCC use the net land CO 2 flux reported in the NGHGIs as a basis to assess compliance with their NDCs and track the progress of their long-term emission reduction strategies under the Paris Agreement 2,29,30 as with previous climate pacts 31 .Historically, NDCs have been compared with scenario-based estimates of needed emission reductions by either aligning the IPCC-assessed pathways to NGHGIs with constant offsetting methods 1 or excluding LULUCF emissions entirely 9,29 .Comparing our results with one of the most recent aggregate NDC estimates 1 (Methods and Extended Data Fig. 2), we find that the gap between unconditional NDCs and a median 2.0 °C outcome is approximately 18% larger, whereas our assessment of the gap between unconditional NDCs and a median 1.5 °C outcome is around 4% smaller (Extended Data Fig. 3).It is therefore important to incorporate a dynamic estimation of indirect fluxes when assessing national climate targets because their changing role in achieving these targets depends on domestic land-management decisions as well as the overall strength of global mitigation (Fig. 4). Aligning pathways to inventory-based LULUCF accounting conventions can additionally affect how equitable mitigation action is understood, as around 60% of the historical NGHGI adjustment falls in Non-Annex I countries 26 .Assessed regionally, 1.5 °C-consistent emission reductions are higher for developed countries, whereas they are slightly lower in most developing regions when assessing scenario outcomes using the NGHGI-based conventions (Extended Data Fig. 4).In the 2.0 °C pathways, the NGHGI alignment results in more stringent 2020-2030 emission reductions globally compared with the unadjusted pathways, as the strength of the indirect flux continues to grow with increasing atmospheric carbon concentrations.This strengthening most directly affects regions with large forested areas such as Latin America and Russia, whereas others such as the Organisation for Economic Co-operation and Development (OECD) countries and Asia, on average, see a decrease in emission reductions.Our results span both positive and negative values across many regions, showcasing the diversity of future responses to land-sink changes and complexities when setting both equitable and ambitious climate targets based on national inventories.They also highlight the risk of over-dependence on land sinks to measure mitigation progress using national inventory conventions against ambitious climate targets. Considering carbon removal In most 1.5 °C and 2.0 °C pathways, hundreds of gigatonnes of CO 2 are removed from the atmosphere over the course of this century, with ultimate levels dependent on the strength of near-term mitigation action 17,32,33 .Because our assessment relies on a bookkeeping model that explicitly tracks land carbon reservoirs, we are able to isolate Article those LULUCF fluxes that effectively constitute carbon removals from carbon emissions (for example, deforestation), thereby quantifying total land-based carbon dioxide removal (CDR) consistently across scenarios and filling a gap in the IPCC Sixth Assessment Report (see footnote 53 in ref. 17) as well as underlying scenario database that constitutes a widely used resource in the climate change community 34 .Scenarios see a marked increase by 2030 in CDR from the LULUCF sector, resulting in around 60% higher removals of CO 2 by 2030 compared with the 2020 levels in the 1.5 °C pathways and 10% higher removals in the 2.0 °C pathways (Fig. 5a).Taken together with engineered (non-LULUCF) CDR options, models deploy 2.6 [1.4-3.2]Gt CO 2 yr −1 (interquartile range) and 0.7 [0.3-2.5]Gt CO 2 yr −1 additional CDR between 2020 and 2030 in the 1.5 °C and 2.0 °C pathways, respectively.Land-based sinks account for nearly 100% of current CDR.By 2030, in the 1.5 °C pathways, 95% [88-98%] of total CDR is delivered by land-based sinks (Fig. 5b).By 2100, CDR from LULUCF accounts for about 30% [21-42%] of the annual total. Although deep mitigation scenarios assessed by the IPCC show a notable and continued dependence on land-based removals over the whole century, LULUCF removals of the same pathways aligned to NGHGIs would peak by mid-century and decline thereafter.Over time, the reduced effectiveness of indirect removals counterbalances the gains from direct removals 35 (Extended Data Fig. 5), maintaining the overall direct and indirect removals at around 6-7 Gt CO 2 yr −1 by mid-century.The 1.5 °C pathways cumulatively sequester around 20% more carbon from direct removals but 20% less carbon from indirect removals compared with the 2.0 °C pathways over that period (Extended Data Fig. 6).Considering the changing dynamics of indirect carbon removals included in NGHGIs can dramatically change the estimated carbon removals on land over time.Although the 1.5 °C scenarios show growth in total assessed net land removal by 2030 (Fig. 5c), the scenarios aligned with current policies approximately double removals compared with the 1.5 °C and 2.0 °C scenarios by mid-century, because of the increasing strength of indirect removals (notably through strong CO 2 fertilization) (Fig. 5d). Thus, although the addition of a larger 'managed land' sink in NGHGIs may reduce the reported levels of present-day national emissions in some cases, maintaining the strength of this carbon sink on these land areas may pose a fundamental challenge in the long term.Not only do estimates of needed progress in anthropogenic emission reductions risk being masked by natural sink enhancement in the near term, but even the maintenance of existing natural sinks requires additional efforts to remove carbon, for example, through the expansion of forest areas, from the NGHGI accounting perspective.In other words, the future effort needed to achieve or maintain net-zero economy-wide emissions would be underestimated using NGHGI accounting conventions as the indirect contribution to land sinks loses efficacy and may eventually become a net source of emissions in low-warming scenarios. Balancing practicality and policy advice We provide here an estimation of the LULUCF emissions consistent with NGHGI accounting conventions for all IPCC-assessed scenarios that provide sufficient land-use cover information using probabilistic and constrained estimates from a single established model, OSCAR.Repeating this work with additional models would increase robustness by averaging model biases and structural uncertainties, although this would require land-use scenario information at a much finer resolution than the five regions. Because the pathways are aligned with the NGHGI conventions by re-allocating indirect carbon fluxes caused by environmental changes to anthropogenic fluxes, our results do not change any climate outcome or mitigation benchmark produced by the IPCC, but provide a translational lens to view those outcomes from the perspective of national emissions reporting frameworks as deployed by the UNFCCC parties.For example, the fact that we find net-zero timings for the 1.5 °C pathways advance by up to 5 years compared with the IPCC-assessed benchmarks does not imply that 5 years have been lost in the race to net-zero, but that following the reporting conventions for natural sinks used by parties to the UNFCCC results in net-zero needing to be reached 5 years earlier to match the modelled benchmarks.Our results reinforce the importance of a rapid decline in fossil fuel and industry emissions in this decade while limiting reliance on nature-based solutions that can weaken over time to keep global temperature rise within the limit prescribed in the Paris Agreement.Importantly, this 'new' net-zero year is conceptually consistent with the meaning of balancing of sources and sinks of greenhouse gases (GHGs) as stipulated in Article 4 of the Agreement (although in the context of CO 2 ).Yet it occurs before the climatological milestone that results in halting further warming, as is the case of the net-zero year under the scientific modelling convention.Understanding and addressing how these different frameworks can be mutually interpreted is a fundamental challenge for evaluating progress towards the Paris Agreement, given the reality that carbon removals from anthropogenic and natural land-based processes cannot be estimated separately by the NGHGIs, which are typically based on direct observations.The outcomes presented here highlight that the conventions by which land-based carbon removals are considered have important implications for NDC assessment and transparency, operationalization of removals under carbon markets as laid out in Article 6.4 of the Paris Agreement and monitoring, reporting and verification of these removals. The policy and scientific communities can take steps to meet this challenge by reconciling terms, definitions and estimates of land-based CO 2 fluxes in four concrete ways.First, climate targets can be formulated explicitly for areas of critical mitigation action, including gross CO 2 emission reductions without LULUCF, net land-based removals, engineered carbon removals and non-CO 2 GHG emission reductions, allowing for parties to define their expected contributions and to measure progress in each domain separately.Second, parties can clarify the nature of their deforestation pledges, because direct and indirect carbon fluxes vary greatly in different forest types 36 .Third, scientific and practitioner communities can convene discussions on how to enhance monitoring, reporting and verification of LULUCF fluxes to better align estimates from both groups.Fourth, IAM teams can provide their individual assumptions and estimates for direct LULUCF emissions and removals, including the indirect flux component consistent with the NGHGIs 37 and their assumptions about the land-use contribution of NDCs and long-term strategies.Future IPCC assessments could either use such scenario data if available or use an approach such as that presented here to provide a holistic scenario assessment aligned with the NGHGIs and better inform necessary collective action to meet global climate goals. Although science and policy processes continue to co-evolve, informing one another, a full reconciliation of the conceptual discrepancies outlined here will take time.However, the first iteration of the GST will be completed by the end of 2023 and new NDCs will be formulated soon thereafter, necessitating earlier compatibility between national targets and benchmarks estimated by global models.Our results provide estimates and a line of evidence that can be directly used by parties and the UNFCCC to meaningfully compare aggregated national targets and mitigation benchmarks.No matter what the reporting conventions are, the near-term action that is needed to meet the Paris Agreement is clear: emissions must peak as soon as possible and reduce markedly this decade.This message must not be lost in the translation between different concepts of anthropogenic land carbon fluxes. 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Methods Selection of AR6 scenarios As part of its Sixth Assessment Report, IPCC Working Group III authors analysed more than 2,200 scenarios for potential inclusion in its mitigation pathway assessment 40 .Of those, 1,202 were eventually vetted: deemed to have provided enough detail to allow a climate analysis using the climate assessment architecture of the IPCC 41 .Those scenarios were then divided into different scenario categories based on their peak and end-of-century temperature probabilities 34 . In this study, we focus on three scenarios: C1, C2 and C3 as defined in AR6 of the IPCC (ref.40).C1 scenarios are as likely as not to limit warming to 1.5 °C and have been interpreted as consistent with the 1.5 °C long-term temperature goal of the Paris Agreement as outlined in Article 2 (ref.42), although arguments have been made that further delineation should be made into scenarios that do and do not achieve net-zero CO 2 emissions to better reflect its Article 4 (ref.43).We assess outcomes from the 2.0 °C C3 scenarios given their historic policy relevance, their capability to show progress towards 1.5 °C and their use in examining climate impacts beyond what is envisioned by the Paris Agreement.We also highlight mitigation outcomes of C2 scenarios, also called high overshoot scenarios, which are as likely as not to limit warming to 1.5 °C in 2100 but are likely to exceed 1.5 °C in the interim period.Such pathways are nominally similar in mitigation and impact assessment with C3 scenarios until at least mid-century 43 . For this analysis, we require that scenarios have been vetted by the IPCC climate analysis framework and provide a minimum set of land-cover variables such as Land Cover|Cropland, Land Cover|Forestry and Land Cover|Pasture.We analyse the presence of each of these variables and their combination in Extended Data Table 3 at the global, IPCC 5-region (R5) and IPCC 10-region (R10) levels.Balancing concerns of greater regional detail and greater scenario coverage, we perform our analysis based on the R5 regions (Extended Data Table 4) given that nearly all models with full global variable coverage also provide detail at the R5 regional level for the C1-C3 scenarios. To understand how well our scenario subset containing R5 land-cover variables corresponds statistically to the full database sample of the C1-C3 scenarios, we perform a Kolmogorov-Smirnov test over key mitigation variables of interest including GHG and CO 2 2030 emission reductions, median peak warming, median warming in 2100, year of median warming, cumulative net CO 2 emissions throughout the century, cumulative net CO 2 until net-zero and cumulative net negative CO 2 after net-zero (Extended Data Fig. 7).For all variables, the Kolmogorov-Smirnov test is not able to determine whether the R5 subset comes from a different distribution than the full database sample, whereas it is able to determine the non-R5 subset is different for peak warming and cumulative net CO 2 emissions, both of which are shown in Extended Data Fig. 8.These results indicate that the subset of about 75-80% of all the C1-C3 scenarios we chose to perform our analysis will result in sufficiently similar macro-mitigation outcomes to represent such outcomes from the original distribution of scenarios. Reanalysis with OSCAR We use OSCAR v.3.2: a version structurally similar to the one used for the 2021 Global Carbon Budget (GCB) 44 , albeit used here with a regional aggregation that matches the R5 IPCC regions.We first run a historical simulation (starting in 1750 and ending in 2020) using the same experimental setup as for the 2021 GCB 5, 44 , with the updated input data used in ref. 36.This historical simulation is used not only to initialize the model in 2014 for the scenario simulations but also to constrain the Monte Carlo ensemble (n = 1,200) using two values (instead of one in the GCB): the cumulative land carbon sink in the absence of land-cover change over 1960-2020 and the NGHGI-compatible emissions averaged over 2000-2020.The former is a constraint of 135 ± 25 Gt CO 2 yr −1 (ref.44).The latter is a constraint of −0.45 ± 0.77 Gt CO 2 yr −1 , using ref. 2 as a central estimate and combining uncertainties in ELUC and SLAND from the GCB.All physical uncertainties in this section are 1 standard deviation (1σ).All values reported in the main text and figures are obtained using the weighted average and standard deviation of the Monte Carlo ensemble, using these two constraints for the weighting 5 . To run the final scenario simulations over 2014-2100, OSCAR needs two types of input data: (1) CO 2 and local climate projections and (2) land use and land-cover change projections.The former mostly affects the land carbon sink (that is, the indirect effect), whereas the latter mostly affects the bookkeeping emissions (that is, the direct effect).OSCAR follows a theoretical framework 45 that enables a clear separation of both direct and indirect effects.Only the direct effect is reported annually in the GCB.Note that we do not re-evaluate the land-cover change albedo effect because this was already included in the original AR6 database climate projections. Atmospheric CO 2 time series is taken directly from the database, as the median outcome estimated by the Model for the Assessment of Greenhouse Gas Induced Climate Change (MAGICC).However, local climate temperature and precipitation changes are not directly available.These are, therefore, computed using the internal equations of OSCAR 46 , and the time series of global temperature change and species-based effective radiative forcing (ERF) from the database (same source).The missing components of the global ERF were treated as follows.Black carbon on snow and stratospheric H 2 O start at a historical level in 2014 (ref.47) and follow the same relative annual change as the ERF of the scenario from black carbon and CH 4 , respectively.Contrails are assumed constant after 2014.Solar forcing is assumed to follow the same pathway common to all Shared Socioeconomic Pathways (SSPs).Volcanic aerosols are assumed to be constant and equal to the average of the historical period (that is, to have a zero ERF).Finally, we apply a linear transition over 2014-2020 between the observed and projected CO 2 and climate, so that these variables are 100% observed in 2014 and 100% projected in 2020.We note that the observed and projected CO 2 are virtually indistinguishable over that period but the observed and projected regional climate changes do differ by up to a few tenths of a degree.We further note that, because only median atmospheric CO 2 , ERF and global temperature are used as input, we do not sample and report the full physical uncertainty of the Earth system, but only the biogeochemical uncertainty from the terrestrial carbon cycle in response to these median outcomes. Land use and land-cover change input data for OSCAR have three variables: the land cover change per se, wood harvest data (expressed in carbon amount taken from woody areas without changing the land cover) and shifting cultivation (a traditional activity consisting of cycles of cutting forest for agriculture, abandoning to recover soil fertility and then returning).Wood harvest and shifting cultivation information are not provided in the database; so we use proxy variables to extrapolate the historical 2014 values.Wood harvest is scaled using the Forestry Production|Roundwood variable, and shifting cultivation is scaled using Primary Energy|Biomass|Traditional as a proxy of the development level of a region.When scenarios did not report these proxy variables, we assumed a constant wood harvest or shifting cultivation in the future, because these are second-order effects on the global bookkeeping emissions. Land-cover change is split between gains and losses that are deduced directly as the year-to-year difference (gain if positive, loss if negative) using the following land-cover variables of the database: Land Cover|Forest, Land Cover|Cropland, Land Cover|Pasture and Land Cover|Built-up Area (built-up area is assumed to be constant if not available).Land-cover change in the remaining biome of OSCAR (non-forested natural land) is deduced afterwards to maintain a constant land area.To build the transitions matrix required as input by OSCAR, it is then assumed that the area increase of a given biome occurs at the expense of all the biomes that see an area decrease (within the same region and at the same time step), in proportion to the share of total area decrease of the biomes.By construction, this approach provides only net land-cover transitions because it is impossible to have gain and loss in the same year, in a given region.Therefore, and because our historical data account for gross transitions but scenarios do not, we add to this net transitions matrix a constant amount of reciprocal transitions equal to their average historical value over 2008-2020 to obtain a gross transitions matrix.Finally, the three land use and land-cover change input variables follow the same linear transition over 2014-2020 as the CO 2 and climate forcings. We extract two key variables (and their subcomponents) from these scenario simulations: the bookkeeping emissions (ELUC in the GCB) and the land carbon sink (SLAND in the GCB).Following the approach in ref. 4, the adjustment flux (that is, the indirect flux included in the NGHGIs but not included by the IAMs, also called the factor in the main text) required to move from bookkeeping emissions to NGHGI-compatible emissions is calculated as the part of the land carbon sink that occurs in forests that are managed.Therefore, we obtain the adjustment flux by multiplying the value of SLAND simulated for forests by the fraction of (officially) managed forests.We set this fraction to the one estimated by ref. 4 for 2015, which also allows us to deduce the area of managed and unmanaged (that is, intact) forest in our base year.We then estimate how the area of intact forest evolves in each scenario, assuming that forest gains are always managed forest (that is, they do not change intact forest area) and that half of the forest losses are losses of intact forest with the other half being losses of the managed forest.This fraction is deduced from ref.48 that estimated that around 92 Mha of intact forest disappeared between 2000 and 2013, whereas the FAO Global Forest Resources Assessment 2020 reports about 170 Mha of gross deforestation over the same period.We acknowledge, however, that applying a global and constant value for this fraction is a coarse approximation that should be refined in future work, possibly using information from the scenario database itself.This assumption also implies that, as long as there is a background gross deforestation (as is the case here, given the added reciprocal transitions), countries will report more and more managed forest area.This is not necessarily inconsistent with the Glasgow Declaration on Forest made at COP26, as its implications in terms of pristine forest conservation are unclear 36 .The subcomponents of the bookkeeping emissions are extracted following the land categories defined in ref. 2, and we consider that the net flux happening in the forest land category, excluding shifting cultivation, is the direct contribution to land CDR.The indirect contribution to land CDR would be exactly the adjustment flux described above. The re-analysed bookkeeping net emissions (that is, direct effect) show an average deviation of −87 Gt CO 2 for C1 scenarios and −63 Gt CO 2 for C3 scenarios from the reported emissions in the database, accumulated over the course of the century.Using the best-guess transient-climate response to cumulative emissions estimated by the IPCC (ref.49), this implies that the global temperature outcomes of these scenarios would differ by about −0.04 °C and −0.03 °C, respectively, from what was reported in the IPCC report, if our estimates of bookkeeping emissions were used instead of those reported by the IAM teams. Furthermore, after re-allocating the indirect effect in managed forest (to align with the NGHGIs), we observe a 4.4 ± 1.0 Gt CO 2 yr −1 difference between the aligned and unaligned historical LULUCF emissions over 2000-2020.This number is at the lower end of the latest 6.4 ± 1.2 Gt CO 2 yr −1 provided in the 2022 GCB 3 .Compared with the 6.7 ± 2.5 Gt CO 2 yr −1 difference reported in ref. 2, and correcting for the absence of organic soils emissions in our simulations with OSCAR (about 0.8 Gt CO 2 yr −1 ), OSCAR can explain about 75% of the observed difference.Although OSCAR typically produces fairly central estimates of the direct effect 3 , its estimates of the indirect effect show a biased high CO 2 fertilization 50 . Comparing adjusted pathways with current policy and NDC estimates We use the latest available estimate of aggregate NDCs from ref. 1 to compare with the NGHGI-adjusted global pathways.The 1.5 °C and 2.0 °C pathways we use are the same as previously discussed: the IPCC C1 and C3 pathways with sufficient land cover detail at the R5 region.We additionally re-analyse the current-policy pathways from the IPCC AR6 database.These correspond to pathways consistent with the current policies as assessed by the IPCC, or the P1b pathways as per the AR6 database metadata indicator Policy_category_name. We incorporate an endogenous estimation of the indirect effect with OSCAR, which varies over time based on land-cover pattern changes and changes to carbon-cycle dynamics and carbon fertilization.As such, we compare our central estimate of global GHG emissions in 2015, approximately 49.4 Gt CO 2 -equiv to that in ref. 1, 51.2 Gt CO 2 -equiv, resulting in a difference of 1.8 Gt CO 2 -equiv.We then apply this offset value (1.8 Gt) to all estimations of 2030 emission levels in ref. 1 to provide comparable levels with our pathways.This ensures that the NDC targets calculated based on national inventories become comparable with the NGHGI-adjusted modelled pathways. Extended Data Table 1 | Indirect LULUCF flux estimates aligned with NGHGIs Median values and interquartile ranges of the indirect flux in Gt CO 2 yr −1 estimated by OSCAR per R5 IPCC region (see Extended Data Table 4 for region definitions).This value is computed for every scenario with sufficient land-use data (see Methods) in each model region for every point in time.This value constitutes the 'NGHGI Adjustment Factor' and is computed and added to each scenarios' estimated direct LULUCF flux values to quantify emissions pathways from global models aligned with NGHGI LULUCF reporting conventions. Fig. 1 | 1 Fig. 1 | Difference in present estimates of LULUCF carbon fluxes under NGHGI and model-based accounting conventions.Schematic showing the difference in accounting conventions between NGHGIs (green) and scientific models (bookkeeping models in red and vegetation models in blue).Models such as IAMs are based on bookkeeping approaches and consider direct fluxes due to land use (for example, wood harvest) and land-cover changes.Additional indirect fluxes due to evolving environmental conditions can be estimated by processed-based vegetation models.NGHGIs consider a wider managed land area and are generally based on physical observations, and thus include both direct and indirect fluxes.We use the term 'unmanaged' to describe land not considered managed by NGHGIs to be consistent with previous literature, but recognize that this includes land that has been managed by indigenous and traditional communities for centuries to millienia38,39 .In this study, we estimate the alignment factor to translate between both conventions (the indirect flux considered in NGHGIs but not in models, blue).This factor will change over time based on future land-use decisions and overall mitigation efforts because of, for example, changing atmospheric CO 2 levels. Fig. 2 | 2 Fig. 2 | Land-use emissions in re-analysed IPCC pathways with modelbased and NGHGI-based accounting conventions.a,b, Land-use emissions pathways before and after alignment to match NGHGIs for 1.5 °C (a) and 2,0 °C (b) pathways.Historical estimates 2,3 are shown with carbon-cycle uncertainty (1σ), and the median of scenario pathways are shown with the scenario interquartile range in shaded plumes.Pathways consistent with modelbased convention are shown in red, whereas the NGHGI convention is shown in green.c, Comparing the two conventions results in a difference between re-analysed and NGHGI-adjusted pathways-that is, an alignment factor, which evolves as a function of the strength of land-based climate mitigation. Fig. 3 | 3 Fig. 3 | Changes in global mitigation benchmarks across assessed scenarios.a-c, Scenario-wise distributions of the estimated change in the net-zero CO 2 year (a), 2020-2030 CO 2 emission reductions (b) and cumulative emissions until net-zero CO 2 (c) between the re-analysed model-based and the NGHGI LULUCF accounting conventions are shown for 1.5 °C (blue, IPCC category C1), 1.5 °C-OS (green, IPCC category C2) and 2.0 °C (purple, IPCC category C3) scenarios.A positive value indicates that the benchmark comes later (for net-zero years) or is higher (for cumulative emissions) in the model-based framework compared with the NGHGI-based framework, whereas a negative value indicates that the benchmark is higher in the NGHGI-based framework (for emission reductions).Across all benchmarks, NGHGI-based accounting tends to result in more stringent outcomes (earlier net-zero years, higher emission reductions and lower cumulative emissions to net-zero CO 2 emission).A comparison with the original AR6 benchmarks is shown in Extended Data Fig.1.a.u., arbitrary units. Fig. 4 | 4 Fig. 4 | The future role of indirect fluxes in national climate targets.In a future with strong mitigation action in line with the goals of the Paris Agreement (bottom row), stabilizing or even decreasing atmospheric CO 2 will result in a weakening of the indirect sink (blue arrows), whereas a future with weak mitigation action will see the indirect sink increase (as long as CO 2 fertilization dominates over climate feedbacks, top row).The direct component of LULUCF fluxes (red arrows) is due entirely to land-use management decisions (columns).Future estimates of net LULUCF emissions (green arrows) will differ between conventions depending on how much overall mitigation occurs and how much land-based mitigation occurs, which can have unexpected consequences on national climate target achievement. Fig. 5 | 5 Fig. 5 | CDR characteristics in mitigation and current-policy pathways.a, Net land-use carbon removal levels from direct fluxes (green bars) are compared with non-land CDR (brown bars) and total levels (summing land-use and CDR, grey bars) with whiskers denoting the interquartile range of each estimate across 1.5 °C and 2.0 °C scenarios.Here, non-land CDR comprises technologies included in the IAM pathways assessed in AR6 other than those due solely to land-use change, such as bio-energy with carbon capture and storage, direct air capture of CO 2 with storage and enhanced mineral weathering.b, The share of land-based CDR reduces over time across both 1.5 °C and 2.0 °C pathways with the median (solid line) and interquartile range (shaded area) shown for the population of scenarios assessed.The direct Extended Data Fig. 1 | 1 Scenario-wise mitigation benchmark shift.The change between estimates of mitigation benchmarks for 1.5 C (blue, IPCC category C1), 1.5C-OS (green, IPCC category C2), and 2 C (purple, IPCC category C3) scenarios.Original values from the AR6 database (which follows IAM reporting conventions) are shown as circles whereas values derived from reanalyzed scenarios in this study (in line with NGHGI reporting conventions) are shown as triangles.The estimates of the year of global net-zero CO 2 (panel a), emissions reductions between 2020 and 2030 (panel b), and cumulative CO 2 emissions (panel c) are shown.Each pair of dots and triangles represents results from a single scenario, with scenarios ordered along the y-axis based on the values in the original AR6 dataset.Extended Data Fig. 2 | NGHGI-adjusted global GHG pathways compared with NDCs and current policies.The interquartile range shown and median highlighted is plotted together with current estimates of 2030 aggregated national climate target levels and current policy estimates from den Elzen et al. (2022).Extended Data Fig. 3 | The 2030 emissions gap between current policies and pledges.1.5 C and 2 C as assessed in this study and by den Elzen (2022) is compared against levels of current policies, conditional NDCs, and unconditional NDCs as reported in den Elzen (2022).Median estimates of all values are used to compute the respective emission gaps.Extended Data Fig. 4 | The change in the estimated 2030 emission gap between due to alignment to NGHGI conventions.The total magnitude, left, relative value, right.Each bar represents the median value with the interquartile range of the estimate across scenarios.These changes occur differently across different regions between pathways following model-based conventions and adjusted pathways following NGHGI-based conventions.A positive value means that the gap is larger when considering both (i.e. when aligned to NGHGIs), and a negative value means the gap is smaller.Regions labels conform to IPCC 5-region labels for Asia, Latin America, Middle East and Africa, the OECD and EU, and the Reformed Economies, respectively (Extended Data Table 4).Extended Data Fig. 5 | Gross carbon removal levels.Gross carbon removal levels from LULUCF (reanalyzed with OSCAR) by direct effects (green) and indirect effects (purple) across 1.5 C and 2 C pathways.Interquartile ranges of each estimate are shown by error bars.Extended Data Fig. 6 | Cumulative carbon sequestered on land starting from 2020.Gross cumulative carbon removal levels starting from 2020 from LULUCF (reanalyzed with OSCAR) by direct effects (green) and indirect effects (purple) across 1.5 C and 2 C pathways.Removals in both categories increase by midcentury, but at different levels.Both pathway categories see similar total cumulative removal levels by the end of the century with varying strength of indirect removals.Extended Data Fig. 7 | K-S test of scenario distributions.Kolmogorov-Smirnov (K-S) test results for key mitigation indicators for the full set of C1-C3 scenarios, those scenarios having all land-cover variables defined at the R5 region level, and those not having all land-cover variables defined at the R5 region level.The null hypothesis of the K-S test is that two dataset values are from the same distribution.For all indicators derived from scenarios including land-cover variables data at the R5 region level, we can not reject the null hypothesis (p > 0.05).Some indicators of the scenario set without land-cover data (not used in this analysis) do reject the null hypothesis.Extended Data Fig. 8 | Mitigation metrics from scenario subsets.Key mitigation metrics where scenarios without R5 region coverage (in red) cannot replicate the full database outcome.The blue bar presents the outcome for the full database, scenarios with global values of land-cover variables and R5 values are shown in yellow, and scenarios with land-cover variables at the R5 region are shown in green.The red bar shows how the distribution changes when considering the population of scenarios without full variable coverage ('No R5 all'). Differences stem from de nitions of managed land and the carbon uxes that are included Land with extensiveOther land serving production,Land with limited orhuman activityecological and social functionsno human activityManagedManagedManagedUnmanagedBookkeeping modelsHumanClimateTrack carbon uxes caused directly byDirect uxesIndirect uxeshuman activities Impact of indirect fluxes on ability to achieve national climate targetsFuture national land carbon balance under NGHGI convention shift with global CO 2 levels CO 2CO 2Stylized uxesUnchanged land-based mitigationIncreased land-based mitigationNo additional mitigation effort inStrong mitigation effort pursued inthe land sector of a countrythe land sector of a countryCarbon sinksCarbon sourceNetCarbon sinksCarbon sourceNetCO 2 removalsCO 2 emissionsemissionsCO 2 removalsCO 2 emissionsemissionsCO 2Direct+Indirect+Direct=NGHGIDirect+Indirect+Direct=N GHGILow global mitigationCO 2 levels in line with current climate policiesParis Agreement alignmentNational land sector Global emissionsParis Agreement alignmentNational land sector Global emissionsCO 2Direct+Indirect+Direct=NGHGIDirect+Indirect+Direct=NGHGIHigh global mitigation CO 2 levels in line with the Paris AgreementParis Agreement alignmentNational land sector Global emissionsParis Agreement alignmentNational land sector Global emissionsCO 2 Care is needed when national climate targets rely on indirect fluxes Direct uxes are consistent with the degree of mitigation in the land sector.Indirect uxes depend on how environmental conditions (for example, CO 2 , climate) change over time, which is in turn dependent on global mitigation efforts.Under the NGHGI convention, a Paris-consistent world could lead to weaker indirect removals, masking increased direct ones. © The Author(s) 2023 Acknowledgements We thank M. Sanz and C.-F. Schleussner for the comments on an initial draft of the paper, M. Beer and F. Spagopoulou from the Designers for Climate for expert support on visualizations, and the reviewers whose comments improved the quality of the paper.We acknowledge the funds received from the Horizon Europe research of the European Union and the innovation programme RESCUE, grant agreement no.101056939 (M.J.G. and T.G.); the Horizon 2020 research of the European Union and the innovation programme ESM2025-Earth System Models for the Future, grant no.101003536 (T.G. and Z.N.); and the ERC-2020-SyG Article GENIE grant of the European Union, grant no.951542 (M.J.G., W.F.L., J.M. and K.R.).The views expressed are those of the writers and may not under any circumstances be regarded as stating an official position of the European Commission.Data availabilityAll data generated and analysed here are available at GENIE Scenario Explorer (https://data.ece.iiasa.ac.at/genie).Code availabilityOSCAR is an open-source model available at GitHub (https://github.com/tgasser/OSCAR).Source code for all analysis files is available at GitHub (https://github.com/iiasa/gidden_ar6_reanalysis).Extended Data Table 2 | Updated mitigation benchmarksNet mitigation outcomes from scenarios: (a) as assessed by the IPCC in AR6, (b) with direct effects of LULUCF reanalyzed by OSCAR, and (c) including both direct and indirect effects of LULUCF (i.e.aligned to NGHGIs).All values provided as medians with 5 th −95 th percentile ranges in parentheses.Extended Data Table 3 | Variable coverage of scenariosFraction of AR6 database scenarios with land-use variables of interest, per scenario category.Author contributions M.J.G., T.G. and K.R. contributed to the conceptualization; M.J.G., T.G., G.G., I.J. and Z.N. devised the methodology; M.J.G. and T.G. helped in the investigation; T.G. provided the software support; M.J.G. helped with visualization; M.J.G. wrote the original draft; and M.J.G., T.G., G.G., N.F., I.J., W.F.L., J.M., Z.N., J.S. and K.R. reviewed and edited the paper.Competing interestsThe authors declare no competing interests.Additional information Supplementary informationThe online version contains supplementary material available at https://doi.org/10.1038/s41586-023-06724-y.Correspondence and requests for materials should be addressed to Matthew J. 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SNORA56-mediated pseudouridylation of 28 S rRNA inhibits ferroptosis and promotes colorectal cancer proliferation by enhancing GCLC translation Xiaoli Zhu xiaolizhu@tongji.edu.cn Department of Laboratory Medicine Shanghai Tenth People's Hospital of Tongji University 200072ShanghaiChina Chang Xu Chang Xu Zhixuan Bian Department of Laboratory Medicine Shanghai Tenth People's Hospital of Tongji University 200072ShanghaiChina Zhixuan Bian Chang Xu Zhixuan Bian Department of Laboratory Medicine Shanghai Children's Medical Center School of Medicine Shanghai Jiao Tong University 200127ShanghaiChina College of Health Science and Technology School of Medicine Shanghai Jiao Tong University 200025ShanghaiChina Shanghai Key Laboratory of Clinical Molecular Diagnostics for Paediatrics 200127ShanghaiChina Sanya Women and Children's Hospital Managed by Shanghai Children's Medical Center 572000SanyaChina Xinyue Wang Chang Xu Zhixuan Bian Department of Laboratory Medicine Shanghai Tenth People's Hospital of Tongji University 200072ShanghaiChina Na Niu Department of Laboratory Medicine Shanghai Tenth People's Hospital of Tongji University 200072ShanghaiChina Li Liu Department of Laboratory Medicine Shanghai Children's Medical Center School of Medicine Shanghai Jiao Tong University 200127ShanghaiChina College of Health Science and Technology School of Medicine Shanghai Jiao Tong University 200025ShanghaiChina Shanghai Key Laboratory of Clinical Molecular Diagnostics for Paediatrics 200127ShanghaiChina Yixuan Xiao Department of Laboratory Medicine Shanghai Children's Medical Center School of Medicine Shanghai Jiao Tong University 200127ShanghaiChina College of Health Science and Technology School of Medicine Shanghai Jiao Tong University 200025ShanghaiChina Shanghai Key Laboratory of Clinical Molecular Diagnostics for Paediatrics 200127ShanghaiChina Jiabei Zhu Department of Laboratory Medicine Shanghai Children's Medical Center School of Medicine Shanghai Jiao Tong University 200127ShanghaiChina College of Health Science and Technology School of Medicine Shanghai Jiao Tong University 200025ShanghaiChina Shanghai Key Laboratory of Clinical Molecular Diagnostics for Paediatrics 200127ShanghaiChina Nan Huang Department of Laboratory Medicine Shanghai Tenth People's Hospital of Tongji University 200072ShanghaiChina Yue Zhang Department of Central Laboratory Shanghai Tenth People's Hospital of Tongji University 200072ShanghaiChina Yan Chen Department of Laboratory Medicine Shanghai Tenth People's Hospital of Tongji University 200072ShanghaiChina Qi Wu Department of Laboratory Medicine Shanghai Tenth People's Hospital of Tongji University 200072ShanghaiChina Fenyong Sun Department of Laboratory Medicine Shanghai Tenth People's Hospital of Tongji University 200072ShanghaiChina Qiuhui Pan panqiuhui@scmc.com.cn Department of Laboratory Medicine Shanghai Children's Medical Center School of Medicine Shanghai Jiao Tong University 200127ShanghaiChina College of Health Science and Technology School of Medicine Shanghai Jiao Tong University 200025ShanghaiChina Shanghai Key Laboratory of Clinical Molecular Diagnostics for Paediatrics 200127ShanghaiChina Sanya Women and Children's Hospital Managed by Shanghai Children's Medical Center 572000SanyaChina SNORA56-mediated pseudouridylation of 28 S rRNA inhibits ferroptosis and promotes colorectal cancer proliferation by enhancing GCLC translation E35ED358CBD7E68493288CDB262AD9A410.1186/s13046-023-02906-8Received: 9 September 2023 / Accepted: 16 November 2023Colorectal cancerSNORA56ProliferationPseudouridylationFerroptosisGCLCBiomarkerTherapy Background Colorectal cancer (CRC) is one of the most common malignancies and is characterized by reprogrammed metabolism.Ferroptosis, a programmed cell death dependent on iron, has emerged as a promising strategy for CRC treatment.Although small nucleolar RNAs are extensively involved in carcinogenesis, it is unclear if they regulate ferroptosis during CRC pathogenesis.MethodsThe dysregulated snoRNAs were identified using published sequencing data of CRC tissues.The expression of the candidate snoRNAs, host gene and target gene were assessed by real-time quantitative PCR (RT-qPCR), fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) and western blots.The biological function of critical molecules was investigated using in vitro and in vivo strategies including Cell Counting Kit-8 (CCK8), colony formation assay, flow cytometry, Fe 2+ /Fe 3+ , GSH/GSSG and the xenograft mice models.The ribosomal activities were determined by polysome profiling and O-propargyl-puromycin (OP-Puro) assay.The proteomics was conducted to clarify the downstream targets and the underlying mechanisms were validated by IHC, Pearson correlation analysis, protein stability and rescue assays.The clinical significance of the snoRNA was explored using the Cox proportional hazard model, receiver operating characteristic (ROC) and survival analysis.ResultsHere, we investigated the SNORA56, which was elevated in CRC tissues and plasma, and correlated with CRC prognosis.SNORA56 deficiency in CRC impaired proliferation and triggered ferroptosis, resulting in reduced tumorigenesis.Mechanistically, SNORA56 mediated the pseudouridylation of 28 S rRNA at the U1664 site and promoted the translation of the catalytic subunit of glutamate cysteine ligase (GCLC), an indispensable rate-limiting enzyme in the biosynthesis of glutathione, which can inhibit ferroptosis by suppressing lipid peroxidation. Background Colorectal cancer (CRC) is the second leading cause of cancer deaths worldwide, and its incidence and mortality are increasing [1].CRC diagnosis relies on imaging and the detection of plasma biomarkers, such as carcinoembryonic antigen (CEA) and carbohydrate antigen199 (CA199), but these methods have limited sensitivity and specificity [2].Histological analysis, which is used for pathological staging and to guide subsequent management, is not suitable for extensive CRC screening because of its invasiveness.Consequently, CRC patients with poor prognosis result from the lack of effective therapeutic targets and accurate biomarkers for early detection.Currently, CRC is mainly treated through surgery combined with chemotherapy, radiotherapy, and immunotherapy.However, CRC has a high risk of metastasis and recurrence because of drug resistance [3].Thus, a better understanding of the mechanisms that underlie CRC progression is needed for the development of biomarkers for early diagnosis, as well as effective therapies. Small nucleolar RNAs (snoRNAs), a family of conserved non-coding RNAs with a length of 60-300 nucleotides, are mainly derived from the introns of host genes.Based on their structural elements, snoRNAs are classified as box C/D snoRNAs or box H/ACA snoR-NAs, which interact with evolutionarily conserved ribonucleoproteins to modulate the processing of ribosomal RNAs (rRNAs) through 2'-O-methylation and pseudouridylation, respectively, thereby regulating ribosome subunit maturation and translation [4].Recent studies have shown that snoRNAs are frequently dysregulated in various cancers and that they contribute to tumorigenesis and cancer progression through diverse mechnisms [5][6][7].This implies that snoRNAs might have therapeutic value.For example, SNORA38B plays an oncogenic role in non-small cell lung cancer by binding to E2F1 and regulating the GAB2/AKT/mTOR pathway to affect immunotherapy sensitivity [8].Moreover, SNORD12C/78 functions in CRC pathogenesis by guiding 2'-O-Methylation at Gm3878 and Gm4593 sites, thereby increasing oncogene translation [9].Notably, because snoRNAs are stable in body fluids [10], they have the potential for use as non-invasive biomarkers for early CRC detection and prognosis prediction. The finding that CRC initiation and progression is accompanied by flexible metabolic reprogramming may lead to the identification of metabolic therapeutic targets and have an impact on treatment responses [11].Moreover, the metabolic adaptation involving ferroptosis has been proposed as a potential therapeutic target in CRC [12].Ferroptosis, as a programmed cell death, is mainly regulated by system Xc − , which imports cystine for the synthesis of reduced glutathione (GSH).Through the catalytic action of glutathione peroxidase 4 (GPX4), GSH suppresses the formation of lipid peroxides and inhibits ferroptosis.GCLC, the rate-limiting enzyme in GSH synthesis, suppresses ferroptosis by catalyzing the ligation of cysteine to glutamate, therefore promoting CRC metastasis [13].GCLC is reported to maintain glutamate homeostasis during cystine starvation and to protect from ferroptosis in a glutathione-independent manner, implying that changes in GCLC function are crucial for ferroptosis resistance [14].Past studies have revealed that high GCLC expression contributes to oxaliplatin detoxification in CRC peritoneal metastases derived organoids [15], highlighting the suppression of GCLC as a potential strategy for CRC treatment.Although GCLC expression is driven by the transcription factor, nuclear factor erythroid 2-related factor 2 during several pathogeneses [16,17], the translational regulation of GCLC during CRC is not fully understood.Notably, recent studies indicate that non-coding RNAs regulate ferroptosis [18,19].Indeed, studies have identified ferroptosis-associated long noncoding RNA signatures for predicting the prognosis of various cancers [20][21][22].However, the relationship between snoRNAs and ferroptosis is unclear. This study investigated SNORA56, which is overexpressed in CRC tissues and cells.SNORA56, located in an intronic region of dyskerin 1 (DKC1), is 129 nucleotides long and correlated with CRC prognosis.We first used in vitro and in vivo strategies to confirm that SNORA56 promotes CRC proliferation while suppressing ferroptosis.We identify GCLC, the rate-limiting enzyme in GSH biosynthesis, as a major downstream target of SNORA56, which promotes GCLC translation by mediating the pseudouridylation of 28 S rRNA, thereby inhibiting ferroptosis and promoting CRC survival.These findings provide novel insights into the roles of SNORA56 in CRC. of Sciences (Shanghai, China).HEK-293T, SW480, and Caco2 cells were cultured in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin (PS, NCM Biotech, China).LoVo cells were cultured in F-12 K medium (Gibco, USA) supplemented with 10% FBS and 1% PS.HCT8, HCT116, HT29, and HIEC cells were cultured in RPMI-1640 (Gibco, USA) containing 10% FBS and 1% PS.All cell lines were cultured in a humified incubator, at 37 °C and 5% CO 2 . CRC and paired para-cancerous tissues, as well as plasma samples, were collected at Shanghai Tenth People's Hospital between 2020 and 2023.Ethical approval for the study was granted by the ethics committee of Shanghai Tenth People's Hospital.Two pathologists confirmed CRC diagnosis and provided detailed clinicopathological information. Plasmids, antisense oligonucleotides (ASOs), and cell transfection Human SNORA56 (NR_002984.1),GCLC (NM_001197115.2), and the full-length cDNAs for the SNORA56 mutants, MUT1 (mutated from AGUUAUCC to UCAAUAGG) and MUT2 (mutated from GGGAG to CCCUC) were synthesized by Ke Lei Biological Technology (Shanghai, China) and cloned into the pCDNA3.1 vector.The SNORA56 cDNA was also cloned into the pLVX-AcGFP vector for simultaneous expression and the product named LV-SNORA56.SNORA56-targeting sgRNAs (sgSNORA56) were designed using CRISPOR [23] and cloned into the lentiCRISPRv2 plasmid (sgNC).ASOs were purchased from RIBOBIO corporation (Guangzhou, China).The plasmids and ASOs were transiently transfected using Lipofectamine 2000 (Invitrogen, USA) according to manufacturer instructions.For stable overexpression or knockdown, packaged plasmids (psPAX2 and pMD2.G) and the target plasmid were cotransfected into HEK-293T cells followed by lentivirus harvesting at 24, 48, and 72 h.The indicated cell lines were then incubated overnight with the lentivirus and polybrene (Santa Cruz, USA).Stably transfected cells were selected through continuous treatment (two weeks) with puromycin (Invitrogen, USA) at 2 µg/ml. Quantitative reverse transcription PCR (RT-qPCR) Total RNA was isolated from cells or plasma using Trizol (Invitrogen, USA).RNA was extracted from CRC tissues using a FastPure Cell/Tissue Total RNA Isolation kit V2 (Vazyme, Nanjing) following the manufacturer's protocol.RNA was retrotranscribed using a PrimeScript ™ RT reagent Kit with or without gDNA Eraser (RR037A, RR047A, TaKaRa, Japan).RT-qPCR analysis was done using TB Green® Premix Ex Taq ™ II (RR820A, TaKaRa, Japan) on a QuantStudio Dx Real-Time PCR system (Thermo, USA) using U6 to normalize snoRNAs expression and 18 S as the reference gene for all other genes.Relative RNA levels were determined using the 2 -ΔΔCt method. Cell proliferation assays Cell Counting Kit-8 (CCK8, Beyotime, China) was used to assess cell proliferation.Briefly, cells were seeded into 96-well plates at a density of 1,000 cells/well.At indicated time points, the medium was replaced with fresh medium containing 10% CCK8 reagent, and absorbance read at 450 nm on a SpectraMax iD5 multimode plate reader (Molecular Devices, USA) after a three-hour incubation.For colony formation analysis, cells were plated on 12-well plates at a density of 1,000 cells/per well and cultured for one to two weeks.They were then fixed with 4% paraformaldehyde (Biosharp, China) and stained with 1% crystal violet for visualization. Animal experiments Four-week-old nude mice (male) were purchased from Charles River Corp (Beijing, China) and subcutaneously injected with 1 × 10 7 HT29 sgNC or sgSNORA56 cells on either flank.Tumor growth was monitored every other day and tumor volume was calculated using the formula: ab 2 /2, where a = length and b = width).For the IKE sensitivity assay, 20 male mice aged four weeks were subcutaneously injected with HT29 sgNC or sg SNORA56 cells on either flank, and tumor volumes were monitored every two days.When the mean volume reached 90 mm [3], the mice were randomly divided into two subgroups and intraperitoneally injected with IKE (Selleck, USA) at 50 mg/kg, daily for 2 weeks.For injection, the IKE was dissolved in a solution of 65% D5W (5% dextrose in water, Biosharp, China), 5% Tween-80 (Selleck, USA) and 30% PEG-300 (Selleck, USA).Control mice were treated with the solvent only.The mice were euthanized at the end of the study, followed by tumor harvesting and tumor weight measurement. Western blot analyses Proteins were extracted from cells by incubating them on ice for 10 min in RIPA buffer (Beyotime, China) supplemented with phosphatase and protease inhibitors (Sangon Biotech, Shanghai, China).The lysate was then cleared by centrifugation at 12,000 revolutions per minute, for 15 min, at 4 °C.Protein concentration was determined using a BCA protein assay kit (NCM Biotech, China).Proteins were then boiled in 6× loading buffer (Beyotime, China) for 10 min, and equal amounts resolved using SDS-PAGE.They were then transferred onto nitrocellulose membranes, blocked with 5% BSA in TBST, and then incubated with indicated primary antibodies (anti-GCLC and anti-GAPDH) at 4 °C, overnight. They were then washed thrice with TBST and incubated with secondary antibodies at room temperature for one hour.Finally, the protein signal was visualized on an Odyssey imaging system (LI-COR, USA). Immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) Paraffin-embedded tissues or CRC tissue microarrays were used for IHC analysis.Briefly, following deparaffinization, hydration, antigen retrieval, and blocking, slides were incubated with antibodies against Ki67 or GCLC at 4 °C overnight.Next, the slides were incubated with biotinylated secondary antibody and peroxidaselabeled streptavidin, and the signal was developed using the diaminobenzidine chromogenic substrate.For FISH analysis, a SNORA56-specific digoxin-labeled probe was synthesized by Biosune Biotech Corp (Shanghai, China).The FISH experiments on CRC tissue microarray and panoramic scanning were done by Runnerbio Corp (Shanghai, China).The staining intensity were independently determined by two blinded pathologists. 4D label-free qualitative proteomics Proteins were extracted from the HCT8 sgNC and sgS-NORA56 cells and label-free qualitative proteomics carried out by PTM Bio Corp (Shanghai, China).Briefly, the tryptic peptides dissolved in solvent A (0.1% formic acid and 2% acetonitrile in water) were directly loaded onto a homemade reversed-phase analytical column.The peptides were then separated in solvent B (0.1% formic acid in acetonitrile) at a constant flow rate, using a nanoElute UHPLC system (Bruker Daltonics).Next, the peptides were subjected to capillary electrophoresis, followed by mass spectrometry on a timsTOF Pro system (Bruker Daltonics), which was done in the parallel accumulation serial fragmentation mode.Data were processed using the MaxQuant search engine (v.1.6.15.0) and referenced against the human SwissProt database.FDR was adjusted to < 1%. Glutamate cysteine ligase (GCL) activity assay Fresh cells (more than 10 7 ) were ultrasonically lysed on ice.A GCL enzyme activity assay kit (BC1210, Solarbio, China) was then used to assess GCL activity according to manufacturer instructions via absorbance readings.The GCL activity was normalized through relative cell count. Measurements of iron concentration and the GSH/GSSG ratio An iron assay kit (ab83366, Abcam, UK) was used to measure total iron, Fe 2+ , and Fe 3+ .Iron concentration was calculated using the following formula: iron concentration (µM) = iron content (nmol)/sample volume (µL) × dilution factor.GSH and GSSG assay kits (BC1175 and BC1180, respectively, Solarbio, China) were used to determine the GSH/GSSG ratios according to manufacturer guidelines. Flow cytometry Lipid peroxidation and cell death were assessed as previously described [24].Briefly, cells were collected and stained with BODIPY-C11 (Invitrogen, USA) at 37 °C avoiding light, for 30 min, followed by lipid peroxidation measurement using flow cytometry.For live cell analysis, cells were incubated with propidium iodide (Invitrogen, USA) for five min at room temperature avoiding light followed by flow cytometry. Polysome profiling A total of 2 × 10 7 cells in a lysis buffer (50 mM Tris-HCl pH7.4, 100 mM NaCl, 5 mM MgCl 2 , 100 µg cycloheximide, and 1% Triton X-100) supplemented with a protease inhibitor cocktail and an RNase inhibitor, were incubated on ice for 15 min.The supernatants were then collected by centrifuging at 12,000 revolutions per minute for 10 min, at 4 °C.The lysates were then loaded into 10-50% sucrose density gradients prepared using a BioComp Gradient Master Model 108 (BioComp, Canada) and then separated by ultracentrifugation using an SW41Ti rotor (Beckman Coulter, USA) at 23,000 g for three hours at 4 °C.The centrifuged solution was then divided into 13 equal fractions and their absorbances measured simultaneously at 260 nm, from top to bottom on an automatic separation and analysis system (Bio-Comp, Canada). O-propargyl-puromycin (OP-Puro) assay The incorporation of OP-Puro into nascent polypeptides indicates protein synthesis activity.Cells growing on sixwell plates were treated with puromycin at 20 µg/mL for two hours and then harvested for protein extraction and western blot analysis using anti-puro antibody (Millipore, USA). Protein stability assay GCLC protein stability was examined as described previously [6].Briefly, SNORA56-silenced or SNORA56-overexpressing cells were treated with cycloheximide (CHX, Sigma, USA) at 100 µg/ml to block translation and then harvested at 0, 4, 8, 12, 24 h after treatment.Proteins were then extracted, followed by western blot analysis of GCLC level. Statistical analyses All experiments were done in triplicate.Data analyses were done using SPSS version 25.0 (IBM, Germany) and GraphPad Prism version 9.0 (San Diego, USA).Variables with normal distribution were compared using the t-test for two groups, or one-way analysis of variance (ANOVA) for multiple groups.The Wilcoxon rank test was used to analyze paired data.Tumor growth curves were analyzed using two-way ANOVA (Bonferroni's test).Correlation analysis was done using the Spearman rank test.Chi-square or Fisher's exact test were used to compare clinical characters in SNORA56 high and low expression groups.Kaplan-Meier analysis was used for survival analysis and the survival differences were compared using the Log-rank test.The Cox proportional hazards model was used to analyze the impact of variables on survival.Receiver operating characteristic (ROC) curve analysis and area under curve (AUC) were used to assess diagnostic potential.Results are presented as the mean ± standard deviation.P < 0.05 indicates statistically significant differences.*, **, and *** indicate P < 0.05, < 0.01, and < 0.001, respectively. Results SNORA56 is highly expressed and correlated with poor prognosis in CRC To determine the differential expression of snoRNA in CRC, we performed snoRNA sequencing on five CRC and its adjacent non-tumor tissues in our previous work, then compared our data with two published snoRNA datasets from CRC tissues [25,26].This analysis identified the snoRNAs, SNORA1, SNORA56, SNORA27, and SNORD18B, as being differentially upregulated in all three datasets (Fig. 1A).Next, we used RT-qPCR to validate their expression in 47 paired CRC and corresponding adjacent non-tumor tissues.SNORA56, as the most upregulated, was selected for further investigation (Fig. 1D, S1A-C).SNORA56, which is derived from the tenth intron of the DKC1 gene, is 129 nucleotides long and contains the conserved H/ACA box (Fig. 1B-C).Pan-cancer analysis of The Cancer Genome Atlas (TCGA) dataset revealed that SNORA56 was most significantly enriched in CRC (Figure S2). Next, we used RT-qPCR and FISH assay to validate SNORA56 upregulation in various CRC cell lines and CRC tissue microarrays, respectively.Our analyses revealed that SNORA56 levels in CRC cell lines (HT29, HCT8, HCT116, SW480 and Caco2, except for LoVo) were markedly higher than in the normal human intestinal epithelial cell line (HIEC) (Fig. 1E).Moreover, FISH revealed significantly higher SNORA56 levels in CRC tissues compared to the adjacent non-tumor tissues (Fig. 1I-K, S1D), suggesting that SNORA56 might influence CRC progression.Furthermore, analysis of the expression of DKC1, from which SNORA56 is derived, revealed that as with SNORA56, DKC1 mRNA levels were frequently upregulated in CRC tissues and cells (Fig. 1F-G).Moreover, in CRC tissues, there was a significant positive correlation between SNORA56 and DKC1 at the transcriptional level using Spearman rank correlation analysis (r = 0.5856, P < 0.001, Fig. 1H), implying that SNORA56 is co-transcribed with DKC1.Because DKC1 is proposed as a CRC prognostic biomarker [27], we evaluated the potential role of SNORA56 in CRC prognosis using TCGA_COAD data from SNORic [28].This analysis found that CRC patients with higher SNORA56 levels had a poorer 5-year survival rate (Fig. 1L), indicating that SNORA56 is involved in CRC pathogenesis and highlighting SNORA56 as a potential biomarker for CRC prognosis. SNORA56 promotes CRC cell proliferation in vitro and in vivo To assess the biological function of SNORA56 in CRC, we transiently downregulated its expression using two independent antisense oligonucleotides (ASOs) in HCT116 and HCT8 cells.Moreover, we generated HIEC and CRC cell lines (HCT8 and HT29) in which SNORA56 was stably up-or down-regulated using lentiviral transduction of overexpressed plasmids (LV-NC and LV-SNORA56) or CRISPR/Cas9 system (sgNC and sgSNORA56), respectively.Transfection efficiency was assessed using RT-qPCR (Fig. 2A-C, S3A).Notably, stable SNORA56 overexpression or silencing did not affect DKC1 levels, suggesting that SNORA56 functions independently of DKC1 (Fig. 2D, S3B).Next, CCK-8 and colony formation analyses of the proliferative potential of CRC cells in vitro revealed that SNORA56 depletion disrupts CRC cell viability (Fig. 2E-J), whereas its upregulation markedly promotes proliferation (Figure S3C-D).Moreover, a xenograft nude mouse model of CRC using HT29 sgNC and sgSNORA56 cells was constructed through subcutaneous injection (Fig. 2K).Results revealed that the weights and volumes of tumors from the SNORA56deficient cells (sgSNORA56) were markedly lower than in those from the control (sgNC) (Fig. 2L-M).Consistent with this observation, Ki67 levels, a canonical proliferative marker, in SNORA56 depleted xenografts were apparently decreased with the analysis of IHC, which confirmed that SNORA56 significantly promoted CRC cell proliferation in vivo (Fig. 2N). SNORA56 drives tumorigenesis by promoting 28 S rRNA maturation and translation Because snoRNAs canonically function in ribosome modification, we first examined the effect of SNORA56 on rRNA maturation.According to the snoRNA Orthological Gene Database (snOPY), SNORA56 guides 28 S rRNA U1664 pseudouridylation via a base-pairing interaction between its flanking regions and rRNA (Fig. 3A).Structure analysis revealed that although the U1664 site is located in the large ribosomal subunit, it is far from the ribosome's functional domain (Fig. 3B), implying that SNORA56 might alter ribosomal conformation.To investigate SNORA56-mediated pseudouridylation, we mutated two interacted flanking regions of SNORA56 to their complementary matched sequences respectively to disrupt the association between SNORA56 and 28 S rRNA as previously described (Fig. 3A, MUT1 and MUT2) [6,30].We then examined the expression levels of SNORA56 after transfection with its mutants or non-mutated control (WT) (Fig. 3E).Also, qPCR analysis using specific primers against unprocessed or total rRNAs revealed that SNORA56 silencing markedly increased the levels of immature 28 S rRNA (Fig. 3C-D, S4B), whereas SNORA56 overexpression reduced their level (Figure S4A).However, SNORA56 levels did not have observable effects on 18 S rRNA maturation (Figure S4C-E), which is consistent with the interaction between SNORA56 and 28 S rRNA not 18 S rRNA.Furthermore, polysome profiling and OP-Puro analysis of ribosomal activity showed that SNORA56 markedly enhances the translation capacity of the ribosome in CRC cells (Fig. 3F-G, S4F-G). Next, to determine whether the tumorigenic role of SNORA56 is mediated by its interaction with 28 S rRNA, we overexpressed SNORA56-WT, MUT1 or MUT2, in SNORA56-depleted HCT8 and HT29 cells respectively (Fig. 3E, S4H-J).This analysis revealed that overexpressing SNORA56-WT, and not the mutants, restored the levels of 28 S rRNA (Fig. 3D, S4B), implying that interaction between SNORA56 and 28 S rRNA is essential for 28 S rRNA maturation.Notably, SNORA56 mutants failed to rescue SNORA56 silencing-induced proliferation both in vitro and in vivo (Fig. 3H-O), suggesting (E-F, H-I SNORA56 upregulates GCLC protein expression by activating its translation To identify potential downstream targets of SNORA56 in CRC, we carried out a proteomic analysis on sgNC and sgSNORA56 cells (Fig. 2C-D).This analysis uncovered 258 differentially expressed proteins, of which 120 were upregulated and 138 were downregulated (Fig. 4A-B).Clusters of Orthologous Groups/Eukaryotic Orthologous Groups (COG/KOG) functional classification showed that 10 of the downregulated proteins are involved in translation, ribosomal structure, and biogenesis (Figure S5A).Moreover, KEGG functional enrichment analysis revealed that the downregulated proteins were enriched for the ribosome (Fig. 4C), further indicating that SNORA56 is involved in ribosome activation.Notably, the downregulated proteins were enriched for metabolic processes associated with ferroptosis, such as glutathione and cysteine and methionine metabolism (Fig. 4C), suggesting that SNORA56 might influence ferroptosis by regulating metabolism. To test this possibility, we focused on the protein GCLC, an indispensable rate-limiting enzyme in the biosynthesis of glutathione.GCLC is downregulated upon SNORA56 silencing and it may inhibit ferroptosis by decreasing cellular peroxide levels.Consistently, transient or stable SNORA56 silencing in CRC cells significantly reduced GCLC protein levels (Fig. 4D, S5B).Furthermore, we found that SNORA56 deficiency significantly decreased the activity of glutamate cysteine ligase (Fig. 4E-F, S5C), whereas SNORA56 overexpression markedly upregulated GCLC protein and enzymatic levels (Fig. 4D, F).Moreover, IHC analysis of CRC tissue microarray revealed GCLC upregulation in paired CRC and adjacent non-tumor tissues (Fig. 4G).Further analysis indicated a significant positive correlation between GCLC protein level and SNORA56 staining intensity (Fig. 4H).Next, validation analysis of the relationship using other paired CRC tissues revealed that as with SNORA56, GCLC protein levels were markedly upregulated in tumors (Fig. 4K).However, GCLC mRNA levels were unaffected by SNORA56 (Fig. 4I-J, S5D-E), implying that SNORA56 regulates GCLC posttranscriptionally or translationally.To test this, GCLC protein stability was assessed upon treatment with cycloheximide (CHX) in HT29 and HCT8 cells to block protein synthesis and evaluate protein degradation.This analysis revealed no clear differences in GCLC protein half-life in conditions of SNORA56 deficiency or overexpression (Fig. 4L-M, S5F), suggesting that SNORA56 regulates GCLC translation and not degradation.To test this possibility, we transfected stable SNORA56-silenced HCT8 and HT29 cells with SNORA56-WT or with 28 S rRNA binding-impaired SNORA56 mutants.Importantly, the SNORA56 mutants failed to rescue GCLC protein expression (Fig. 4N), indicating that the SNORA56induced maturation of 28 S rRNA was required for the high GCLC protein levels in CRC. SNORA56 inhibits ferroptosis and promotes proliferation by upregulating GCLC protein in CRC Because GCLC is thought to function in peroxide clearance, we investigated if SNORA56 regulates ferroptosis in CRC.Lipid reactive oxygen species (ROS) and cell death were assessed using flow cytometric analysis of HCT116, HCT8 and HT29 cells after transient or stable SNORA56 silencing.Notably, our data showed that SNORA56 deficiency markedly caused lipid ROS accumulation accompanied by increased cell death (Fig. 5A-D, S6A-D), suggesting that SNORA56 is involved in ferroptosis inhibition in CRC cells.Because labile iron contributes to ferroptosis by directly oxidizing lipids via Fenton reaction and acting as a cofactor for lipid oxidizing enzymes [31], we assessed the Fe 2+ /Fe 3+ ratio to validate the role of SNORA56 in ferroptosis regulation.This analysis revealed a marked decrease in the Fe 2+ /Fe 3+ ratio in the SNORA56 knockdown group (Fig. 5E, S6E), which is consistent with peroxidation in the cellular microenvironment.Similarly, the GSH/GSSG ratio was reduced by SNORA56 silencing (Fig. 5F, S6F), indicating that SNORA56 depletion triggers ferroptosis in CRC cells.However, SNORA56 overexpression in HIEC cells increased both ratios and resistance to erastin, a canonical ferroptosis inducer that blocks the system Xc -antiporter (Fig. 5E-F, L, S6G-H).Notably, only the ferroptosis inhibitor, ferrostatin-1 (Fer-1) reversed lipid ROS elevation and cell death following SNORA56 deficiency, and both the necroptosis inhibitor, necrosulfonamide, and the apoptosis inhibitor, Z-VAD-FMK, could not prevent CRC cell death (Fig. 5G-J).Moreover, analysis of the impact of SNORA56 on ferroptosis sensitivity in CRC revealed that SNORA56 downregulation markedly sensitized CRC cells to ferroptosis in a dose-dependent manner, whereas SNORA56 overexpression caused ferroptosis resistance (Fig. 5K-L, S6L).These observations highlight targeting both SNROA56 and ferroptosis inducers as a promising strategy for CRC treatment.Additionally, cell viability assays revealed that SNORA56 knockdown suppressed cell growth, which could be almost entirely restrained after treatment with Fer-1 (Figure S6I-K), implying that SNORA56 facilitates cell proliferation, at least partly, through ferroptosis resistance.These data illustrate that SNORA56 mediates ferroptosis resistance and highlight SNORA56 as a critical target for regulating ferroptosis sensitivity in CRC cells. Since GCLC is a key rate-limiting enzyme in GSH synthesis (Fig. 5M), we next evaluated whether SNORA56mediated ferroptosis inhibition depends on GCLC protein expression.To determine the hypothesis, we generated a GCLC overexpression system after silencing SNORA56 and verified transfection efficiency using RT-qPCR and western blotting (Figure S6M-P).As expected, GCLC overexpression rescued GCL enzyme activity in HT29 cells (Fig. 5N, S6Q), and remarkably, CRC cell proliferation was reversed upon GCLC overexpression (Fig. 5O-P, S6R-S).Moreover, lipid ROS, cell death, and ferroptosis sensitivity were partially reversed under GCLC retrieved (Fig. 5Q-R, S6T-U, X-Y), indicating that SNORA56 contributes to ferroptosis resistance in CRC mediated by GCLC.Considering SNORA56 facilitates GCLC translation via the 28 S rRNA pseudouridylation, we transfected with SNORA56-WT, MUT1 or MUT2 respectively in HCT8 and HT29 sgSNORA56 cells and examined the ferroptosis.As expected, SNORA56 mutants with depleted 28 S rRNA binding capability failed to recover the ferroptosis resistance (Fig. 5S-T, S6V-W).Next, we examined the pharmacological effect of DL-Buthionine-Sulfoximine (BSO), which inhibits glutathione synthesis and induces ferroptosis, in SNORA56overexpressing HIEC cells.This analysis revealed that BSO markedly suppressed SNORA56-induced elevation of GCL enzyme activity (Fig. 5U), as well as SNORA56driven proliferation and ferroptosis inhibition in HIEC cells (Fig. 5V-Y), indicating that SNORA56 promotes CRC tumorigenesis by enhancing GSH protein synthesis. SNORA56 is a promising CRC diagnostic biomarker and therapeutic target To further investigate the correlation between SNORA56 expression and the clinical characteristics of CRC patients, we obtained SNORA56 expression data from the TCGA_COAD dataset (n = 394) from SNORic, along with clinical data from the UCSC Xena website and analyzed it using chi-square tests or Fisher's exact tests to illustrate the potential relationship between SNORA56 level and clinical index.These analyses revealed a significant correlation between SNORA56 expression and histological type (P = 0.015), venous invasion (P = 0.040), and distant metastasis (P = 0.002) (Table S1-2).Importantly, Cox proportional hazards regression analysis identified high SNORA56 expression as an independent risk factor for CRC patients' overall survival (HR = 2.223, P = 0.021, Fig. 6A).Because snoRNAs are stable in the plasma, we assessed SNORA56 levels in the plasma of CRC patients (age: 66.61 ± 11.55, n = 48) vs. healthy subjects without underlying gastrointestinal disease (age: 57.72 ± 8.67, n = 48).This analysis revealed higher SNORA56 levels in CRC plasma (Fig. 6B).We then collected 154 paired CRC tissues to evaluate the diagnosis efficacy of SNORA56.Consistent with this, SNORA56 levels were upregulated in CRC compared to the corresponding adjacent non-tumor tissues (Fig. 6C).To evaluate the diagnostic efficacy of SNORA56, analyses of the ROC curve were performed in CRC tissues and plasma, and the area under the curve (AUC) were 0.6759 and 0.7572, respectively (Fig. 6D-E), indicating that plasma SNORA56 has significant potential for CRC diagnosis.Notably, combining CEA or CA199, the canonical plasma-based CRC biomarkers, with SNORA56 markedly increased their AUC values (Fig. 6E-F), implying improved diagnostic value. Finally, we examined the therapeutic potential of targeting SNORA56 in CRC.Because SNORA56 is involved in ferroptosis resistance, we first generated a xenograft model of CRC by subcutaneously injecting HT29 sgNC and sgSNORA56 cells into nude mice.Next, we intraperitoneally injected the mice with imidazole ketone erastin (IKE), an erastin substitute that is stable in vivo.Notably, knocking down SNORA56 markedly enhanced the sensitivity of the HT29 cells to IKE, characterized by significant suppression in tumor volumes and weights (Fig. 6H-J), highlighting the anti-CRC therapeutic potential of targeting SNORA56 while inducing ferroptosis.Moreover, GCLC and Ki67 staining was significantly weaker in the HT29 sgSNOR56 xenograft tumors than in the control, and this effect was enhanced by IKE (Fig. 6K-L), implying that SNORA56 exerts its effects on ferroptosis resistance and proliferation inhibition via GCLC.Together, these data highlight the diagnostic and therapeutic potential of SNORA56 in CRC. Discussion Previous studies have reported that snoRNAs regulate rRNA modification, RNA splicing, and translation to affect cell fate, mainly through RNA 2'-O-methylation and pseudouridylation.The function of snoRNAs is dysregulated in various cancers and is a promising biomarker for cancer diagnosis and prognosis.Here, we for the first time, show that SNORA56 plays critical roles in CRC pathogenesis.Our data show marked SNORA56 upregulation in CRC was correlated with poor prognosis, which is consistent with its host gene DKC1 27 .Notably, SNORA56 stimulated ferroptosis resistance and promoted CRC proliferation in vivo and in vitro independently of DKC1. Pseudouridylation, the most common posttranscriptional modification in non-coding RNAs, is mainly executed by box H/ACA snoRNAs that have two hairpins containing an internal pseudouridylation loop.SNORA56 is reported to mediate 28 S rRNA pseudouridylation at the U1664 site via base-pair interactions [32].Interestingly, a recent report proposed that H/ACA pseudouridylation loops can consecutively synthesize two pseudouridines [33], which offers versatility in function of SNORAs apart from stabilizing RNA structure canonically.Our results revealed that SNORA56 mutants with complementary pairing interaction regions that lack rRNA binding activity, are associated with increased levels of unprocessed 28 S rRNA and reduced carcinogenesis, indicating that the oncogenic roles of SNORA56 in CRC are partly attributable to its effect on ribosome maturation.As expected, SNROA56 silencing, which impaired pseudouridylation at site 28 S-U1664, globally suppressed translation.However, our structural analyses showed that site 28 S-U1664 is located far from the ribosome's decoding region, indicating that SNORA56 might not directly affect translation and that it was more likely to regulate translation by altering ribosome conformation and promoting ribosome structural stability. Because the formation of pseudouridine affects ribosomal biogenesis and translation [34], we performed proteomics analysis using CRC cells and found that several ribosomal proteins were markedly decreased upon SNORA56 silencing.Notably, we found that ribosomal proteins, especially 60 S-associated factors like RPL4, RPL28, RPL35A, and RPL17, were markedly downregulated during SNORA56 deficiency, further highlighting the importance of SNORA56 in ribosome biogenesis in CRC.Analysis of ribosome function using polysome profiling and the OP-Puro assay showed that upon SNORA56 depletion, the 60 S subunit was inactivated, which was accompanied by reduced nascent peptide levels.Together, these findings indicated that SNORA56mediated 28 S-U1664 pseudouridylation is required for ribosome assembly and global translation in CRC cells. Next, we assessed the effect of SNORA56 on the translation of GCLC, one of its main downstream candidates.This analysis confirmed that SNORA56 elevated GCLC protein levels but did not affect its transcription.Moreover, SNORA56 did not significantly influence GCLC protein stability, indicating that its effect on GCLC is not dependent on protein degradation.Specifically, GCLC protein levels fully relied on the binding of the SNORA56 pseudouridylation loop to 28 S rRNA, implying that SNORA56 may affect GCLC translation by regulating U1664 pseudouridylation.Hundreds of pseudouridylation mRNA sites have been identified and shown to respond to environmental alterations [35].Therefore, it is possible that in some circumstances, SNORA56 promotes ferroptosis resistance and proliferation in CRC by directly inducing the pseudouridylation of specific mRNAs.Moreover, we predicted the interaction between the SNORA56 pseudouridylation loop and the GCLC mRNA sequence using the online tool, IntaRNA (http://rna.informatik.uni-freiburg.de/IntaRNA/Input.jsp).In the future, we will explore whether SNORA56 regulates GCLC translation by controlling its mRNA pseudouridylation. GCL, an indispensable rate-limiting enzyme in GSH biosynthesis composed of catalyzed subunit GCLC and modifier subunit GCLM, catalyzes the ligation of glutamate and cysteine in the first step [36].In this study, we found that SNORA56 mediates CRC ferroptosis resistance and proliferation at least in part, by regulating GCLC protein expression.Intriguingly, GCLM protein also decreased after SNORA56 silencing according to our proteomics analysis, indicating that SNORA56 may play a dual promoting role both in translation and in the affinity to glutamate of GCLC.Ferroptosis, a form of programmed cell death characterized by lipid peroxidation, is triggered when the antioxidant status is compromised [31].Mounting evidence indicates that GCLC suppresses ferroptosis through the reductive effects of GSH [14,16,37].Notably, ferroptosis involves multiple metabolic processes and also influences response to cancer chemotherapy, radiotherapy, and immunotherapy [18,38,39].Thus, targeting GSH metabolism to induce ferroptosis is a promising potential strategy for CRC therapy.Although metabolic reprogramming is a hallmark of cancer, interventions that target cancer metabolism have not been highly successful in clinical trials because of its flexibility and heterogeneity.A recent study demonstrated that deubiquitination can maintain protein homeostasis, which allows cancer cells to survive when GSH is depletion [40].Therefore, to identify potential therapeutic strategies, it is necessary to determine the mechanisms that underlie cancer metabolic adaptation.Numerous studies have indicated that the transcription of GCLC is controlled by nuclear factor erythroid 2-related factor 2 signaling [37,41,42], and that it correlates with prognosis in various cancers [43,44].Here, our findings propose a novel regulatory mechanism through which SNORA56 enhances GCLC translation, thereby inhibiting ferroptosis and promoting CRC proliferation.Thus, combining SNORA56 targeting and ferroptosis induction may be a potential CRC treatment strategy.However, the precise reasons for the regulation of SNORA56 expression, and the efficacy of such combined therapy, need further investigation. Conclusions Taken together, our data show that SNORA56 is a key factor in CRC pathogenesis (Fig. 7).We found that SNORA56 was markedly elevated in CRC tissue and plasma and that it is a promising biomarker for CRC diagnosis and prognosis.Our study validated that SNORA56 stimulates CRC ferroptosis resistance and promotes CRC proliferation in vitro and in vivo.Importantly, we found that SNORA56 pseudouridylated 28 S rRNA at the U1664 site, which activated GCLC translation.GCLC, a rate-limiting enzyme in GSH synthesis, inhibited ferroptosis, thereby further enhancing CRC proliferation.These findings provide novel insights into SNORA56 regulation and highlight it as a potential target for combined CRC therapy.Fig. 7 The proposed model of how SNORA56 regulates GCLC translation, thereby inhibiting ferroptosis and promoting proliferation.SNORA56, which was significantly upregulated in CRC, pseudouridylates 28 S rRNA at site U1664, thereby promoting ribosome maturation.Consequently, SNORA56 triggers GCLC translation, which drives ferroptosis resistance and proliferation in CRC. List of Abbreviations Fig. 1 1 Fig. 1 SNORA56 upregulation in CRC correlates with poor prognosis.(A) A Venn diagram of three published cohorts reporting upregulation of snoRNAs in CRC tissues.Cohort 1 was obtained from the published article [25].Cohort 2 is from the Gene Expression Omnibus dataset GSE20916 26 .Cohort 3 is from our previously published work [29].(B) Visualization of the genomic location of SNORA56 in its host gene, DKC1, on the UCSC Genome Browser.(C) A schematic representation of the structure of SNORA56.(D, F) The relative expression of SNORA56 and DKC1 in 47 paired CRC and adjacent non-tumor tissues was revealed using RT-qPCR.(E, G) The relative expression of SNORA56 and DKC1 in HIEC, HT29, HCT8, HCT116, SW480, Caco2, and LoVo cells was determined using RT-qPCR.(H) Analysis of the correlation between the expression of SNORA56 and DKC1.(I) SNORA56 FISH analysis and hematoxylin & eosin (H&E) staining in CRC tissue microarray.The mean density of SNORA56 signals was measured using Image Pro Plus.(J-K) SNORA56 staining intensity and the IHC score of CRC tissue microarray.(L) Kaplan Meier curve of the 5-year survival analysis in SNORA56 high and low groups using TCGA_COAD datasets from the SNORic database Fig. 2 2 Fig. 2 SNORA56 promotes CRC proliferation in vitro and in vivo.(A-C) The efficiency of SNORA56 silencing in CRC cell lines upon the indicated transfections was determined using RT-qPCR.(D) RT-qPCR analysis of DKC1 mRNA levels in HT29 cells stably transfected with sgNC and sgSNORA56.(E-F,H-I) CCK8 analysis of the proliferation of CRC cells upon the indicated transfections.(G, J) Colony formation analysis in CRC cells upon transient or stable SNORA56 knockdown vs. the negative control.(K) Tumors from nude mouse subcutaneous xenografts bearing HT29 sgNC cells (right) and sgSNORA56 cells (left).(L-M) Tumor weights and growth curves based on tumor volume measurements every two days.(N) IHC analyses of H&E and Ki67 in the xenograft tumors.Ki67 scores were calculated in HT29 sgNC and sgSNORA56 xenografts.Scale bar: 100 μm )Fig. 3 3 Fig. 2 SNORA56 promotes CRC proliferation in vitro and in vivo.(A-C) The efficiency of SNORA56 silencing in CRC cell lines upon the indicated transfections was determined using RT-qPCR.(D) RT-qPCR analysis of DKC1 mRNA levels in HT29 cells stably transfected with sgNC and sgSNORA56.(E-F,H-I) CCK8 analysis of the proliferation of CRC cells upon the indicated transfections.(G, J) Colony formation analysis in CRC cells upon transient or stable SNORA56 knockdown vs. the negative control.(K) Tumors from nude mouse subcutaneous xenografts bearing HT29 sgNC cells (right) and sgSNORA56 cells (left).(L-M) Tumor weights and growth curves based on tumor volume measurements every two days.(N) IHC analyses of H&E and Ki67 in the xenograft tumors.Ki67 scores were calculated in HT29 sgNC and sgSNORA56 xenografts.Scale bar: 100 μm Fig. 4 4 Fig. 4 SNORA56 upregulates GCLC protein by activating its translation.(A) Differential protein expression in the sgSNORA56 vs. the sgNC group.(B) Volcano plot of differentially expressed proteins.(C) KEGG pathway enrichment analysis of significantly downregulated proteins.(D) GCLC protein levels in HCT116, HCT8, HT29 and HIEC cells were determined using western blotting after indicated transfections.(E-F) GCL enzyme activity in HT29 and HIEC cells with indicated transfection.(G) IHC analysis of GCLC levels in CRC tissue microarray.Mean GCLC density was determined using Image Pro Plus.Scale bar: 100 μm.(H) Analysis of the correlation between the SNORA56 and GCLC protein levels using CRC tissue microarray.(I-J) RT-qPCR analysis of relative GCLC mRNA levels in HT29 and HIEC cells upon indicated transfections.(K) Western blot analysis of GCLC protein levels in paired CRC tissues.(L-M) Western blot analysis of GCLC protein stability in SNORA56-silenced HCT8 and HT29 cells vs. control cells after CHX treatment for indicated durations.Relative band intensities were measured on ImageJ.(N) Western blot analysis of GCLC protein levels in HT29 and HCT8 cells after indicated transfections.GCLC protein levels were normalized to GAPDH. Fig. 5 5 Fig. 5 SNORA56 causes GCLC-mediated CRC ferroptosis resistance and proliferation.(A, C, S, X, Q) HCT116, HCT8, HT29, and HIEC cells were transfected as indicated, stained with the BODIPY C11 probe, and subjected to flow cytometry for lipid ROS detection.(B, D, T, Y, R) HCT116, HCT8, HT29, and HIEC cells were subjected to indicated transfections, followed by propidium iodide staining and cell death analysis using flow cytometry.(E) The relative Fe 2+ /Fe 3+ ratios in HT29 and HIEC cells after indicated transfections.(F) The relative GSH/GSSG ratios in HT29 and HIEC cells after indicated transfections.(G-J) Lipid ROS levels and cell death in HCT116 and HCT8 cells transfected with SNORA56 ASOs and control ASO, followed by treatment with DMSO, Fer-1, necrosulfonamide, and Z-VAD-FMK, were measured using flow cytometry.(K-L) The cell viability of HT29 and HIEC cells after indicated transfections and treatment with various erastin concentrations for 24 h, was determined using the CCK8 assay.(M) A schematic representation of GSH synthesis.(N, U) GCL enzyme activity in HT29 and HIEC cells after indicated treatment.(O-P, V-W) CCK8 and colony formation assays were used to assess the proliferation of treated HT29 and HIEC cells Fig. 6 6 Fig. 6 SNORA56 is a promising CRC diagnostic biomarker and therapeutic target.(A) Forest plot of the overall survival hazard ratios using TCGA_COAD data.(B) Relative levels of plasma SNORA56.(C) Relative SNORA56 expression in 154 paired CRC tissues was revealed by RT-qPCR.(D-E) ROC curve analysis of SNORA56 in CRC plasma and tissues.(F-G) ROC curve analysis of the CRC diagnostic potential of CEA or CA199 when combined with SNORA56.(H) Tumors from a nude mouse xenograft model that was subcutaneously injected with HT29 sgNC (right) and sgSNORA56 (left) cells and then treated with IKE or solvent respectively.(I) Plots of tumor growth measurements taken on indicated days.(J) Tumor weights.(K) IHC analysis of GCLC and Ki67 in xenograft tumors after treatment with IKE or the solvent.Scale bar: 100 μm.(L) GCLC and Ki67 IHC scores in indicated xenografts AcknowledgementsNot applicable.Data AvailabilityThe datasets used or analyzed during the current study are available from the corresponding author on reasonable request.FundingThis study was sponsored by the National Natural Science Foundation of China (81930066, 82172357, 82293662), the Innovation Group Project of Shanghai Municipal Health Commission (2019CXJQ03) and the Key project of Shanghai "Science and Technology Innovation Action Plan" (22JC1402300).Declarations Ethics approval and consent to participateEthical approval for the study was granted by the ethics committee of Shanghai Tenth People's Hospital.And all animal studies were approved by the Animal Research Ethics Committee of Shanghai Tenth People's Hospital.Consent for publicationNot applicable.Competing interestsThe authors declare that they have no competing interests.Publisher's NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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Study on the association between adverse drug reactions to opioids and gene polymorphisms: a case-case-control study Jing Yang School of Medicine and Pharmacy Ocean University of China QingdaoChina Department of Pharmacy Shandong Medical College JinanChina Department of Pharmacy Shandong Provincial Third Hospital Cheeloo Col-lege of Medicine Shandong University JinanChina Ying-Zi Sun Department of Pharmacy Shandong Provincial Third Hospital Cheeloo Col-lege of Medicine Shandong University JinanChina Qun-Fang Li Department of Pharmacy Shandong Medical College JinanChina Zheng Fu Department of Pharmacy Shandong Medical College JinanChina Yu-Yao Guan Department of Pharmacy Shandong Provincial Third Hospital Cheeloo Col-lege of Medicine Shandong University JinanChina Chao Song Department of Pharmacy Shandong Provincial Third Hospital Cheeloo Col-lege of Medicine Shandong University JinanChina Lei Zheng zhenglei8501@163.com Department of Pharmacy Shandong Provincial Third Hospital Cheeloo Col-lege of Medicine Shandong University JinanChina Study on the association between adverse drug reactions to opioids and gene polymorphisms: a case-case-control study 69A6A6B425885717DCA7DAF201F8278610.1186/s40360-023-00708-4Received: 23 January 2023 Accepted: 15 November 2023OpioidsGene polymorphismAdverse drug reactions (ADRs)Individualized medicationCancer pain Objective Adverse drug reactions (ADRs) caused by opioid drugs show individual differences.Our objective was to explore the association between gene polymorphism and ADRs induced by opioid drugs.Methods Evidence-based medical data analysis was conducted for genes related to ADRs induced by opioid drugs to select target genes.Sixty patients with cancer pain who had ADRs after taking opioid drugs (morphine, codeine, oxycodone) and 60 patients without ADRs after taking opioid drugs were used as the experimental group and control group, respectively.Then, we used polymerase chain reaction (PCR) or in situ hybridization to detect target genes.By combining with clinical data such as age, sex, dosage and duration of medication, the effect of gene polymorphism on the ADR of patients after taking opioid drugs was statistically analysed.ResultsBased on a database search and evidence-based medical data, we identified CYP2D6*10, CYP3A5*3, ABCB1, and OPRM1 as target genes for detection.The results of statistical analysis showed no significant difference in genotype distribution between the experimental group and the control group (p > 0.05).However, if 32 patients with ADRs after taking oxycodone and 32 controls were selected for comparison, the SPSS22.0 and SNPStats genetic models showed that the ABCB1 (062rs1045642) CT and TT genotypes correlated with the occurrence of ADRs (p < 0.05): the total number of CT + TT genotypes in the experimental group was 29 (90.62%), with 11 (34.37%)CT + TT genotypes types in the control group.ConclusionPolymorphism of ABCB1 (062rs1045642) is related to ADRs caused by oxycodone, and the incidence of ADRs is higher with the allele T. Polymorphism of ABCB1 is expected to become a clinical predictor of ADRs to oxycodone, and attention should be given to the occurrence of serious ADRs in patients with ABCB1 (062rs1045642) CT and TT genotypes. Opioid analgesics are currently the main treatment for moderate to severe pain.However, individual differences in reactions to these drugs are large, often including nausea, vomiting, lethargy, constipation and addiction, and even respiratory inhibition (RD) and other ADRs.In addition to some cases of drug abuse, the occurrence of the above ADRs is more related to the individual heterogeneity of patients, such as gene polymorphism [1].At present, it is believed that the genes related to opioid pharmacokinetics and pharmacodynamics include μ opioid receptor (OPRM1) (A118G), catechol-O-methyltransferase (COMT), CYP2D6, CYP3A4*1G, CYP3A5*3, ATP-binding cassette transporter (ABCB1); other gene polymorphisms may also be related to the effective analgesic dose of opioids, drug abuse and ADRs.However, the current main problem is that although there are preliminary studies on the correlation between gene polymorphisms and ADRs, there is a lack of high-quality and large-standard clinical research; research to date is mainly on postoperative analgesia patients, and there are some contradictory conclusions [2].Therefore, the main objective of our study was to determine the correlation between gene polymorphisms (SNPs) and the ADRs caused by opioid drugs used by cancer pain patients and to explain differences in ADRs to opioid drugs among individuals from a genetic perspective, which is helpful to guide individualized clinical drug use [3]. Methods Design and setting The study was conducted at a 1400-bed tertiary university teaching hospital.This study was approved by the institutional review board of our hospital and complied with the Helsinki Declaration.Each patient provided written consent for study participation before enrolment.The patients were hospitalized from January to December 2022.Sixty patients with cancer pain who had ADRs after taking opioid drugs (morphine, codeine, oxycodone) and 60 patients without ADRs after taking opioid drugs were used as the experimental group and control group, respectively. Screening candidate test genes We searched the release guidelines of the Dutch Pharmacology Working Group (DPWG) and Clinical Pharmacology Implementation Consortium (CPIC), pharmacogenetics (PharmaGKB, FDA, NCCN and OncoKB), PubMed, and Chinese literature (CNKI, Wanfang, China Biomedical Literature Database (CBM)) databases to collect gene loci that may be related to reported adverse reactions of opioid drugs.Then, we preliminarily summarized and analysed the relevant gene loci, determined the recommended level (ABCD level) with reference to CPIC, and carried out gene detection for the target population. Screening study population The Adverse Drug Reaction Monitoring System (CHPS) and the electronic medical record database were used to retrieve the target genes of patients who had adverse drug reactions after use of opioid analgesics in a Class III hospital.The inclusion criteria were as follows: (1) cancer pain patients who experienced adverse drug reactions after using opioid drugs for pain relief; (2) patients with clear awareness and no mental or intellectual disabilities; and (3) patients who voluntarily signed an informed consent form.The exclusion criteria were as follows: (1) liver or kidney dysfunction; (2) long-term use of nonsteroidal antipyretic and analgesic drugs or corticosteroids; (3) history of drug abuse; (4) history of opioid allergy; and (5) history of mental illness or neurological disorder.This study was approved by the Ethics Committee of Shandong Provincial Third Hospital, and all patients signed the informed consent form. ADR evaluation criteria The evaluation criteria for the causal relationship of ADRs adopted the WHO-UMC evaluation method, as follows: ① whether there is a reasonable time relationship between the time sequence of medication and the occurrence of ADRs (ADR occurs after medication); ② whether it is known and whether the suspected ADR conforms to the ADR type known for the drug; ③ excluding others, whether the suspected ADR can be explained by the patient's pathological state, combined medication, and the effect of therapy; ④ whether the suspected ADR decreases or disappears after the drug is stopped or the dose is reduced; and ⑤ whether the same reaction occurs again after contact with the suspected drug again (recurrence of ADRs can confirm the causal relationship).According to the above criteria, the evaluation results were divided into "affirmative (①-⑤), very likely (①-④), possible, possibly unrelated, to be evaluated, and unable to be evaluated".Those who were evaluated as "affirmative and very likely" were included in the study. Genotype detection After gargling, the patient used a disposable sampling swab to scrape the mucosa of the sidewall of the oral cavity and collected the exfoliated cells as the monitoring sample (noninvasive, more acceptable to the patient than blood sampling).Samples that were not immediately tested were stored at 2-8 °C for no more than 7 days.The specific detection method was as follows.First, 400 μl of L-sample extraction solution (CQ-ENH type, mainly composed of 0.74% ammonium chloride solution) was added to an oral pharyngeal swab tube, shaken and mixed well, allowed to stand for 1 minute, and then transferred to a 1.5 mL centrifuge tube.After L treatment, 1.0 μl of the sample solution was added to the reagent tube wall; the tube was centrifuged at 12000 r/min for 5 minutes and used for testing.Single-nucleotide polymorphism analysis was performed using PCR or fluorescence in situ hybridization.The PCR method was used to detect CYP2D6, and all other genotypes were detected using the in situ hybridization method.The in situ hybridization method was performed using a multichannel fluorescence quantitative analyser Fascan 48S (Shaanxi XZZ No. 20182400043) and a universal genetic testing reagent (Jinan Guangyin Medical Technology Co., Ltd.).Two independent Z-type probes were hybridized with the target sequence in series.The signal amplification precursor sequence binds to 28 bases in the upstream region of the double Z-type probe, causing a change in the threedimensional configuration of the sequence, thereby amplifying the signal and ultimately outputting genotype detection results. Statistical methods SPSS 22.0 software was used for statistical analysis of data, and the Shapiro Wilk method was used for normality testing of measurement data.Measurement data that conformed to a normal distribution are represented by x ± s, and two independent sample t tests were used for intergroup comparisons.Count data are expressed in terms of examples and percentages or rates, and comparisons between groups were performed using χ2 inspection.The Hardy-Weinberg balance test and correlation analysis between gene polymorphisms and opioidinduced constipation were conducted using the SNPStats program (https:// www.snpst ats.net/snpstats/start.htm).The correlation analysis results were corrected using the Kolmogorov-Smirnov method.In correlation analysis, statistically significant gene loci and general data such as age, sex, medication dosage, and medication duration were used as variables.Multivariate logistic regression analysis was used to predict the occurrence of opioidinduced constipation.Receiver operating characteristic (ROC) curves were drawn to analyse the effectiveness of each factor in predicting opioid-induced constipation.Inspection level α = 0.05. Results Identified genes to be tested Based on a comprehensive database search and evidencebased medical data, 4 target gene loci with strong correlations were identified, including CYP2D6 (008rs1065852), CYP3A5*3 (058rs776746), ABCB1 (062rs1045642), and OPRM1 (047rs1799971).Briefly, we describe the relevant conclusions of the study on the relationship between the above gene loci and adverse drug reactions to opioids and preliminarily determine the degree and grade of correlation with reference to the evidence of PharmGKB.See Table 1 for details. General information of patients The study population was patients admitted to a tertiary hospital from January to December 2022.They were all cancer patients with pain who used opioid drugs (including oxycodone, morphine and codeine) for analgesia.Patients with adverse ADRs (specifically including 38 cases of constipation, 18 cases of nausea and vomiting, and 8 cases of chest tightness and suffocation) after use of drugs were used as the experimental group, and patients without ADRs were randomly selected as the control group, with 60 cases in each group for gene detection.There was no significant difference in age, height, body mass, daily dosage of drugs or duration of medication between the two groups (P > 0.05).See Table 2 for details. CYP2D6 (008rs1065852) The toxicity of codeine is easily increased in the ultrafast metabolic type.Polymorphism at this site may be related to the gastrointestinal system damage, nervous system damage, respiratory system damage and other toxic reactions caused by opioid drugs. 1A CYP3A5*3 (058rs776746) The toxicity of opiates is easily increased in the ultrafast metabolic type.It may be related to the gastrointestinal system damage, nervous system damage, respiratory system damage and other toxic reactions caused by opioid drugs. 2A ABCB1 (062rs1045642) The ABCB1 gene encodes the P protein, and opioid drugs are the substrate of P protein; polymorphism is related to the respiratory depression caused by opioid drugs and may also be related to increases in other system damage. 3 OPRM1 (047rs1799971) Some studies report that polymorphism at this site is related to the gastrointestinal system damage, respiratory system damage and other toxic reactions caused by opioid drugs. Distribution of genotype frequency and allele frequency The allele detection of four genes, CYP2D6 (008rs1065852), CYP3A5* 3 (058rs776746), ABCB1 (062rs1045642), and OPRM1 (047rs1799971), is shown in Table 3 and was consistent with Hardy-Weinberg equilibrium (P > 0.05).However, statistical analysis and comparison of genotype distribution between the experimental group and the control group showed no significant difference (P > 0.05). Correlation between genotype and ADRs caused by oxycodone To further evaluate correlations for different drugs, a total of 32 patients with oxycodone (sustained-release tablets or sustained-release capsules) were statistically analysed separately from all 60 patients.First, SPSS 22.0 was used for differential analysis.The results showed that for ABCB1 (062rs1045642, C > T), significantly more patients with adverse drug reactions carried the CT and TT genotypes than CC genotype (P < 0.05, Table 4). In addition, SNPStats (https:// www.snpst ats.net/ snpst ats/ start.htm) was used for genetic model analysis.The correlation analysis results were corrected by the Kolmogorov-Smirnov method.The P value of each SNP was multiplied by the number of genetic markers analysed, and the corrected P < 0.05 indicated that the correlation between the site and adverse reactions was significant.The results also showed that the CT genotype and TT genotype with the T allele of ABCB1 (062rs1045642, C > T) correlated with the occurrence of adverse reactions after use of oxycodone.See Table 5 for the specific results. Discussion Opioids are the most common analgesics used for moderate to severe pain and are closely related to the quality of life of cancer patients.However, there are large individual differences in clinical application of these drugs, and the reactions to analgesics of different patients vary greatly.In addition to different analgesic effects, adverse reactions of analgesics (such as nausea, vomiting, lethargy, constipation, and respiratory depression) and even susceptibility to addiction have great individual differences.The reasons for this sensitivity difference include both genetic and nongenetic factors.Genetic factors mainly cause individual differences by affecting the pharmacokinetics (metabolic enzymes and transporters, etc.) and pharmacodynamics (receptors and signal transduction pathways) of opiates [4,5].Preliminary studies have been conducted on the correlation between the efficacy and ADRs of opioid analgesics and gene polymorphisms, mainly including OPRM1 (A118G), COMT, CYP3A4*1G, CYP3A5*3, CYP2D6, and ABCB1, among others [6][7][8].Relevant studies suggest that the ADRs caused by use of opioids may be closely related to gene polymorphisms of patients.Therefore, to further evaluate relevance in the Asian population, we selected CYP2D6, CYP3A5*3, ABCB1 and OPRM1 as the target genes for population testing.Unfortunately, no differences were found in the above gene loci when the gene polymorphisms of patients who used morphine, codeine and oxycodone were evaluated; however, if the patients with ADRs after use of oxycodone were counted separately, the analysis result showed that carrying the CT genotype and TT genotype with the T allele of ABCB1 (062rs1045642, C > T) correlated significantly with the occurrence of adverse drug reactions after use of this drug.OPRM1 is the main receptor of most opioid drugs currently used and is also the key target for the effects of analgesia as well as tolerance and dependence.Therefore, the OPRM1 polymorphism is the main factor affecting the efficacy of opioid drugs.There are multiple mutations in the OPRM1 gene.For example, A118G is a common SNP.Clinical research shows that this mutation significantly affects the clinical efficacy of opioid drugs [9].That study found that the analgesic effect of opioid drugs in G118 allele carriers was significantly decreased, and adverse reactions such as respiratory inhibition, nausea and vomiting were less common than in A118 allele carriers.Mague et al. found that G118 carriers were more sensitive to pain; it has also been reported that pain is related to adverse events (constipation, delirium, dizziness, nausea, pain, postoperative nausea and vomiting, itching, respiratory insufficiency, lethargy, urinary retention and vomiting) after use of morphine or oxycodone [10,11].However, it should be noted that different results have been reported.For example, some studies report that for patients with severe cancer pain who have not previously used opioid drugs, subjects with the 118G allele need a high dose to control pain, but with no difference in the adverse reactions observed (including nausea, vomiting, constipation, dizziness) [12].No differences were found in our study.CYP2D6 is one of the most important oxidative metabolic enzymes in the CYP450 family.Common opioid analgesics such as codeine and tramadol exert pharmacological effects through demethylation by CYP2D6 into active metabolites as precursors.Current research suggests that CYP2D6 gene mutation is essential for codeine and tramadol, especially for the CYP2D6 ultrafast metabolic type.It is recommended that codeine or tramadol should not be used to avoid the risk of severe poisoning.However, no ultrafast metabolic type was found in this study, and no difference was detected.Hydroxycodone is mainly metabolized into oxymorphone and noroxycodone in the liver and intestinal wall through CYP2D6 and CYP3A4/5, respectively.In general, it is believed that CYP2D6 genotype does not affect cancer pain patients treated with oxycodone [13].Another associated gene, CYP3A5, is the strongest oxidase in the CYP3A pathway that affects metabolism of oxycodone, and CYP3A5*3 is a common mutation site.Polymorphism of this gene can affect the plasma distribution of noroxycodone (a metabolite with a weak analgesic effect of oxycodone), which will lead to an increase or decrease in its dose and an increase in the probability of adverse drug reactions.At present, there are few studies on CYP3A5 compared with CYP2D6.Some studies have shown that individuals carrying the CYP3A5*3/*3 genotype are more likely to develop tolerance to oxycodone, but with no mention of adverse drug reactions.It is also generally believed that AA metabolism is fast and easily increases toxicity according to metabolic type [14,15].However, no significant differences were found in our study. The ABCB1 (ATP binding cassette subfamily B member -1) gene encodes P-glycoprotein.ABCB1 gene polymorphisms may have a close relationship with the pain perception of cancer patients, thus affecting use of opioid drugs by patients.Some scholars have shown that ABCB1 3435C > T (rs1045642) patients with different β genotypes have differences in levels of endorphins, resulting in differences in the degree of pain.It has also been suggested that respiratory depression caused by postoperative anaesthesia is related to adverse drug reactions.The incidence of respiratory depression after using fentanyl in patients with the 1236TT, 2677TT and 3435TT genotypes is high, with a deep degree of inhibition [14].Genotype is also related to a decrease in P-gp protein transport function.That study found that expression of P-gp in the duodenum of CC carriers is more than 2 times higher than that of TT carriers and that the ABCB1 (rs1045642) T/T genotype can lead to a decrease in the drug clearance rate and the possibility of adverse reactions caused by an increase in the blood concentration of the drug [16,17].Therefore, the CT + TT genotype was present at a significantly higher rate among patients in the oxycodone adverse drug reaction group than the CC genotype, which may be related to the slow metabolism of oxycodone and increase in blood drug concentration. Conclusion Our preliminary study shows that the CT and TT genotypes with the T allele of ABCB1 (062rs1045642, C > T) are related to the occurrence of adverse reactions after oxycodone use and are expected to become a clinical predictor of adverse reactions to oxycodone drugs.Overall, the occurrence of severe constipation should be given more attention in patients who have been taking drugs for a long time.However, the limitation of this study is that the number of patients included was small, which cannot greatly reflect the overall impact of population polymorphism, and more cases will be included in future work.In general, cancer patients with pain are greatly affected by tumour type and stage, leading to sensitivity and heterogeneity issues.Further research will include the same tumour type and stage to eliminate this impact. Table 1 1 Screening of candidate detection genotypes/SNPs Genotype/SNP Relationship with ADRs of opioid drugs PharmGKB evidence level Table 2 2 Comparison of general patient data GroupnMale/Female Age (x¯ ± s), year Height (x¯ ± s), cm Weight (x¯ ± s), cm Daily doseDuration of(Calculated bymedication,morphine), mgdayExperience group 60 32/2864.21 ± 8.12166.45 ± 7.8067.33 ± 11.23102.05 ± 10.2599.25 ± 5.20Control group60 30/3062.18 ± 9.36168.23 ± 8.0165.45 ± 10.02100.10 ± 10.5085.30 ± 8.65p > 0.05 Table 3 3 Genotype distribution of the study population GroupCYP2D6CYP3A5*3ABCB1OPRM1(008rs1065852,C > T)(058rs776746,A > G)(062rs1045642,C > T)(047rs1799971,A > G)CCCTTTAAAGGGCCCTTTAAAGGGExperience group n2017233620424261028257%33.33% 28.33% 38.33% 60.00% 33.33% 6.67%40.00% 43.33% 16.67% 46.67% 41.67% 11.67%Control groupn152619351873125430228%25.00% 43.33% 31.67% 58.33% 30.00% 11.67% 51.67% 41.67% 6.67%50.00% 36.67% 13.33%χ 20.1680.1210.9130.092p> 0.05> 0.05> 0.05> 0.05 Table 4 4 Genotype distribution of patients using oxycodone ABCB1(062rs1045642,C > T) Table 5 5 SNPStats genetic model analysis Genotype/SNPExperience groupControl grouppOR(95%CI)AICBICABCB1(062rs1045642,C > T)CC3(9.38%)21(65.63%)1135.4129.8CT + TT29(90.62%) *11(34.37%)0.0180.21(0.09-1.15) AcknowledgementsWe would like to thank the Department of Pharmacy, Shandong Provincial Third Hospital, Shandong University, Jinan, where the study was carried out.Availability of data and materialsThe datasets generated and/or analysed during the current study are available in the Annotare 2.0 repository, [https:// www.ebi.ac.uk/ fg/ annot are/ edit/ 17619/].FundingThis work was supported by Jinan Municipal Bureau of Science and Technology Clinical Medical Science and Technology Innovation Plan (No.202134016); the special fund project for clinical research for therapeutic drug monitoring of the Shandong Medical Association (YXH2020ZX047); Project of Shandong ADR monitoring centre (2021SDADRKY03); Shandong Provincial Natural Science Foundation (ZR2022MH250); Shandong Provincial Natural Science Foundation (ZR2023MG064); and Shandong Medical and Health Development Plan (202213010928).Authors' contributionsJing Yang: PhD.Topic selection and Manuscript writing.E-mail:15853 199531@ 163.com.Yingzi Sun: PhD.Research idea, study design, statistical analysis.E-mail:11074 6228@ 163.com.Qunfang Li: MD.Data analysis, E-mail:11074 6228@ qq.com.Zheng Fu:MD.Data analysis and interpretation.E-mail:fuzhe ng202 212@ 163.com.Yu-Yao Guan: MD.Evidence collection.E-mail:guyuy ao198 412@ 163.com.Chao Song (*Corresponding author): MD.Manuscript writing and clinical pharmacist E-mail:sdsjt yyyjk lcyx@ 163.com.Lei Zheng (*Corresponding author): MD.Manuscript writing and financial support.E-mail:zheng lei85 01@ 163.com.Declarations Ethics approval and consent to participateAll patients signed the informed consent form.The study was performed in accordance with relevant guidelines and was approved by the Ethics Committee of Shandong Provincial Third Hospital (KYLL-20211115).Consent for publicationNot applicable.Competing interestsThe authors declare no competing interests.Publisher's NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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Abdominal compartment syndrome as a complication of endoscopic carbon dioxide insufflation in a patient with malignant bowel obstruction: a case report Taro Tanabe tatanabe1209@outlook.jp 0000-0001-8128-5519 Genki Tsukuda 0000-0001-8128-5519 Takahiro Hobo Noboru Yokoyama Haruhiro Inoue 1 Digestive Diseases Center, Showa University Koto Toyosu Hospital, 5-1-38 Toyosu, Koto-ku, Tokyo 135-8577, Japan Abdominal compartment syndrome as a complication of endoscopic carbon dioxide insufflation in a patient with malignant bowel obstruction: a case report 0F51CE9958CEBDE57E11A51A7F5645EF10.1186/s40792-023-01783-9Received: 10 July 2023 Accepted: 15 November 2023Abdominal compartment syndromeSelf-expandable metal stentColonic obstructionPneumoperitoneumCase report Background A self-expandable metal stent is often placed as a bridge to elective surgical treatment of left-sided malignant obstruction of the colon because it allows for primary anastomosis without the need for a temporary stoma, which has a positive impact on the patient's quality of life.However, although a relatively safe procedure, colonic stenting can have complications that require emergency surgery.This case report describes a rare case of abdominal compartment syndrome that occurred as a complication of endoscopic insufflation during colonic stenting.Case presentationThe patient was a 72-year-old woman who presented complaining of several days of constipation and loss of appetite.Computed tomography of the abdomen revealed obstruction of the sigmoid colon by a tumor.There were no symptoms or computed tomography findings to suggest perforation.Therefore, an attempt was made to insert a self-expandable metal stent.Acute respiratory disturbance and a change in consciousness occurred during the stenting procedure, with marked abdominal distention.Abdominal compartment syndrome was diagnosed and treated by decompressive laparotomy.ConclusionsTo the best of our knowledge, this is the first reported case of abdominal compartment syndrome as a complication of endoscopic insufflation during colonic stenting.The possibility of abdominal compartment syndrome should be considered if acute respiratory disturbance or altered consciousness occurs during endoscopic procedure in a patient with malignant bowel obstruction. Background Self-expandable metal stent (SEMS) implantation is being used increasingly in the management of malignant colorectal obstruction, not only for palliative purposes, but also for preoperative treatment in patients who are candidates for surgery [1].The clinical guidelines for obstructing colonic and extracolonic cancer published by the European Society of Gastrointestinal Endoscopy in 2014 did not recommend insertion of a SEMS as a bridge to surgery in a patient with potentially curable left-sided obstructive colon cancer [2]; however, the 2020 update to the guidelines recommends stent insertion as an alternative to emergency resection [3] because of its increasing success rate and a decrease in the rate of early complications [4].Although stenting of the colon is considered a relatively safe procedure with a mortality rate of approximately 1% [5], it can have important complications that require emergency surgery [4][5][6]. Abdominal compartment syndrome (ACS) is defined as sustained intra-abdominal pressure above 20 mmHg with new onset of organ dysfunction [7].The major risk factors for ACS are large-volume fluid resuscitation for sepsis, shock, and other inflammatory conditions, such as pancreatitis [8]. We have encountered a case of ACS without perforation caused by tension pneumoperitoneum that occurred because of carbon dioxide insufflation during stenting for malignant colonic obstruction. Case presentation A 72-year-old woman (height, 148cm; weight, 50kg; BMI, 22.8) presented to our hospital complaining of constipation and loss of appetite in the previous few days.She did not appear to be unwell with a blood pressure of 128/75 mmHg, a heart rate 93 beats/min, a body temperature of 36.6 °C, and a respiratory rate of 22 breaths/min with 99% oxygen saturation in room air.Her Glasgow Coma Scale (GCS) was E4V5M6.Physical examination revealed a distended but non-tender abdomen.Laboratory investigations did not reveal any obvious abnormalities.A computed tomography (CT) scan confirmed marked colonic dilatation caused by cancer in the sigmoid colon (Fig. 1).There were no metastatic lesions.The diagnosis was obstructive sigmoid colon cancer without impending sepsis.The decision was made to insert a SEMS. Endoscopy showed a full circumferential tumor that was almost completely occluding the sigmoid colon and making it difficult to pass a guidewire (Fig. 2).Forty minutes after the start of endoscopy, the patient became cyanotic, unresponsive, and developed a severely distended abdomen (Fig. 3) with a blood pressure of 69/42 mmHg, a heart rate 50 beats/min, and a respiratory rate of 33 breaths/min with 59% oxygen saturation.Her level of consciousness and oxygenation did not improve after withdrawal of sedation and immediate termination of the insertion procedure.The patient was emergently intubated, but ventilation was difficult, her tidal volume was under 200 mL with pressure support of 15 cm H2O.Urgent CT, compared to pre-stent insertion (Fig. 4A and B), demonstrated marked bowel dilatation and pneumoperitoneum without spillage of contrast (Fig. 4C and D). Based on the marked abdominal dilatation, difficulty ventilation, and CT findings, a diagnosis of ACS was made clinically, and emergency decompressive laparotomy was performed. Almost immediately after the laparotomy, the markedly dilated intestinal tract was ejected from the body cavity, and respiratory status improved (Fig. 5).There was no obvious intestinal perforation or contamination of the abdominal cavity.Based on the intraoperative findings, the dilated intestinal tract due to insufflation during stenting procedure was the cause of the ACS, and the The image shows a full circumferential tumor that was almost completely occluding the sigmoid colon, making it difficult to pass a guidewire colon was decompressed by resecting the appendix and aspirating the intestinal contents at the same site, followed by a Hartmann's procedure. The patient's condition Improved promptly after surgery.She was extubated on the day after surgery, discharged from the intensive care unit on the following day and discharged from hospital about two months after surgery.There was no functional impairment discovered at the outpatient clinic after 6 months of discharge. Discussion This report has two important clinical teaching points: (1) ACS can occur as a complication of endoscopic carbon dioxide insufflation and (2) ACS should be suspected if a sudden change in level of consciousness or respiratory disturbance occurs during endoscopic procedure in a patient with malignant bowel obstruction.Colonic stenting as a bridge to surgery is recommended as one of the treatment options for malignant colonic obstruction in the European guidelines [3].However, while colonic stenting is considered a relatively safe procedure, the stent-related mortality rate is approximately 1% [9], and complications that require emergency surgery, such as colonic perforation, occur in 3.7% to 4.8% of cases [6,[9][10][11].Therefore, candidates for colonic stenting should be selected carefully, given that they are often already in poor condition.This is the first report of ACS as a complication of endoscopic insufflation during colonic stenting.In this case, the sigmoid colon cancer acted as a one-way valve, trapping the air delivered by the colonoscope and causing remarkable intestinal dilation and pneumoperitoneum without perforation, resulting in ACS.Pneumoperitoneum has been reported to occur at rates of 0.3%-1% in diagnostic colonoscopy and 3% in therapeutic colonoscopy [12].Most cases of pneumoperitoneum without perforation do not require surgical intervention; however, there has been a report of a case of ACS caused by pneumoperitoneum without perforation [13].In patients with malignant colonic obstruction, intra-abdominal pressure can be high even before colonoscopy is started; therefore, careful attention to air delivery is needed during colonic stenting. Perforation at the time of stent insertion is a major complication requiring surgery and often manifests as abdominal pain or subcutaneous emphysema one hour to one day later [6,14].However, in a case of ACS, acute respiratory failure and a change in consciousness occurs during the procedure.Therefore, ACS should be suspected when these changes occur during stent insertion.Marked abdominal dilatation on physical examination is important for diagnosis, and pneumoperitoneum without contrast leak on CT may be a useful finding. ACS is usually caused by rapid addition of volume [15], in which case primary abdominal closure after decompressive laparotomy is often difficult [7,16,17].However, when ACS is caused by endoscopic manipulation, primary abdominal closure can be achieved because gas is the main cause of the increase in intra-abdominal pressure. Conclusions We have reported the first case of ACS as a complication of endoscopic carbon dioxide insufflation during colonic stenting.Acute respiratory failure or change in consciousness during this procedure should raise suspicion for ACS. Fig. 1 1 Fig. 1 Computed tomography scan obtained before colonic stenting.The scan shows wall thickening in the sigmoid colon (yellow arrows) and dilatation of the colon and small intestine on the oral side of it Fig. 2 2 Fig. 2 Colonoscopic image obtained at the time of colonic stenting.The image shows a full circumferential tumor that was almost completely occluding the sigmoid colon, making it difficult to pass a guidewire Fig. 3 Fig. 4 34 Fig. 3 Physical examination on completion of colonic stenting revealed a significantly distended abdomen Fig. 5 5 Fig. 5 Clinical photograph obtained at the time of emergency laparotomy.The image shows overflow from a markedly dilated intestinal tract and the wound immediately after the abdomen is opened.There is no obvious intestinal perforation or intra-abdominal contamination AcknowledgementsWe thank Edanz (https:// jp.edanz.com/ ac) for editing a draft of this manuscript.Availability of data and materialsAll data on which the conclusions of this case report are based are included in the present publication.FundingThis report was prepared without financial support.AbbreviationsACSAbdominal compartment syndrome CT Computed tomography SEMS Self-expandable metal stent Author contributions TT and GT managed the patient and conceived the idea for the report.TT wrote the initial version of the manuscript.HI, NY, and TH coordinated and helped draft the manuscript.All authors read and approved the final version of the manuscript.Declarations Ethics approval and consent to participateEthical approval was waived by the ethical review committee at Showa University Koto Toyosu Hospital.The authors certify that the study was performed in accordance with the ethical standards as laid down in 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.Written informed consent was obtained from the patient.Consent for publicationWritten informed consent was obtained from the patient for publication of this case report and any accompanying images.A copy of the written consent is available for review by the Editor-in-Chief of this journal.Competing interestsThe authors declare they have no competing interests.Publisher's NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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Effects of oxidative stress regulation in inflammation-associated gastric cancer progression treated using traditional Chinese medicines A review MM aBo Chen 0009-0005-8109-5551 MM bXinqian Dong 0009-0005-8109-5551 MM a ,Jinl Zhang MD bWei Wang MM a ,Yujiao Song MM a ,Xitong Sun MM a ,Kangning Zhao MDZhen Sun 0009-0005-8109-5551 a Shandong University of Traditional Chinese Medicine, Jinan, People's Republic of China, b Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, People's Republic of China. Jinan 250014, People's Republic of China Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Effects of oxidative stress regulation in inflammation-associated gastric cancer progression treated using traditional Chinese medicines A review F49DF28AA6154E5E01FB432FFDB5F52510.1097/MD.0000000000036157Received: 15 August 2023 / Received in final form: 25 October 2023 / Accepted: 26 October 2023gastric cancergastritisnatural prescriptionsNF-κBNrf2oxidative stress Gastric cancer (GC) is a global public health concern that poses a serious threat to human health owing to its high morbidity and mortality rates.Due to the lack of specificity of symptoms, patients with GC tend to be diagnosed at an advanced stage with poor prognosis.Therefore, the development of new treatment methods is particularly urgent.Chronic atrophic gastritis (CAG), a precancerous GC lesion, plays a key role in its occurrence and development.Oxidative stress has been identified as an important factor driving the development and progression of the pathological processes of CAG and GC.Therefore, regulating oxidative stress pathways can not only intervene in CAG development but also prevent the occurrence and metastasis of GC and improve the prognosis of GC patients.In this study, PubMed, CNKI, and Web of Science were used to search for a large number of relevant studies.The review results suggested that the active ingredients of traditional Chinese medicine (TCM) and TCM prescriptions could target and improve inflammation, pathological status, metastasis, and invasion of tumor cells, providing a potential new supplement for the treatment of CAG and GC. Introduction Gastric cancer (GC) is a common malignant disease of the digestive system that seriously threatens human health and poses an economic burden on families and society.Although the incidence of GC has significantly decreased, it remains the third most common cause of cancer-related deaths worldwide. [1]With the rapid improvement in medical treatment, the treatment of GC is increasing; however, patient survival rates are still unsatisfactory, and the economic cost remains high.Therefore, reducing the incidence of GC is an issue that requires urgent resolution.Oxidative stress, as one of the important pathogeneses of chronic atrophic gastritis (CAG) and GC, promotes the development of the disease in many respects and actively participates in the inflammatory process, causing continuous damage to the gastric mucosa and ultimately leading to GC progression.Oxidative stress promotes CAG and GC progression through 2 processes: overexpression of oxidases, manifesting as an increase in reactive oxygen species (ROS), activation of the nuclear factor-kappa b (NF-κB) signaling pathway, accumulation of lipid peroxidation end products nitric oxide (NO) and malondialdehyde (MDA), and up-regulation of pro-inflammatory factors such as TNF-α, IL-I, and IL-8.These lead to aggravated gastric mucosal injury and increased pathological malignancy.Second, antioxidant enzyme deficiency meanss the physiological oxidative stress caused by oxidases cannot be counteracted, leading to inhibition of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, downregulation of superoxide dismutase (SOD), glutathione (GSH) and other antioxidant enzyme contents, and finally destruction of the cell DNA structure.[4] An increasing number of studies have shown that oxidative stress can enhance the inflammatory expression of CAG, aggravate inflammatory infiltration of the gastric mucosa, and increase the risk of CAG carcinogenesis.In addition, the inhibition of oxidative stress damage to the gastric mucosa by reducing Helicobacter pylori-associated inflammation prevents subsequent GC development. [5]Experiments have also verified that the upregulation of antioxidants exerts anticancer activity to prevent GC. [6,7] The above studies showed that oxidative stress plays an important role in CAG and GC.Inhibition of oxidative stress can not only inhibit the occurrence of inflammation and hinder further carcinogenesis of CAG, but also intervene in GC by regulating this pathway (Fig. 1). Observations The role of oxidative stress in the course of gastric "inflammation carcinoma" 2.1.1.MDA, SOD, and GSH as important biomarkers in gastric "inflammation carcinoma."10] It has been reported that oxidative stress damage is more severe with higher MDA content. [11]It has been found that H pylori (Hp) acts as a pro-inflammatory mediator, inducing DNA methylation, activating oxidative stress pathways, upregulating MDA content, and damaging the gastric mucosa leading to transformation of gastric "inflammation-carcinoma." [12] Notably, in patients with CAG and patients with intestinal metaplasia MDA content is significantly increased compared with that in healthy patients, and pathological changes are positively correlated with gastric mucosal injury. [13,14]Liu et al found that oxidative stress could be significantly alleviated and gastric mucosal injury could be reduced by inhibiting MDA expression. [15]It has been reported that there are higher MDA levels in GC cells and higher MDA levels diminishes the antioxidant capacity of the body.Normal gastric tissue cannot then resist the infiltration of cancer cells, resulting in extensive spread of cancer cells in the gastric wall and accelerated tumor progression. [16]The above studies showed that an increase or decrease in MDA levels promotes or inhibits disease progression and that MDA plays an important role in measuring the level of oxidative stress.MDA levels can indirectly reflect the degree of gastric mucosal injury, be used as an evaluation index for disease development, and at the same time assess GC prognosis.Glutathione (GSH) is a small peptide consisting of glutamate, cysteine, and glycine, which exists in both reduced (GSH) and oxidized (GSSG) forms. [17,18]As an important antioxidant in cells, GSH mediates the regulation of target signaling molecule expression to maintain redox homeostasis and protect host cells from damage including damage caused by lipid peroxidation. [19]SH is oxidized to GSSG by glutathione peroxidase, while oxidative stress metabolites are reduced to nontoxic hydroxyl moieties, which in addition rapidly metabolize and decompose the resulting harmful substances and avoid the destruction of intact cell membrane structure and function by oxidative stress, and this effective defense is attributed to the actions of GSH via synthesis and redox pathways.[20,21] Matsuoka detected gastric epithelial cells in patients with CAG and GC infected with Hp, and the results showed that GSH levels decreased to different extents in different patients, but decreased most significantly in patients with GC, indicating that GSH expression in the stomach was positively correlated with the degree of carcinogenesis of gastric epithelial pathology and that gastric mucosal injury was more severe in patients with GC. [22] It has been reported that the virulence protein factor CagA secreted by Hp enables the nuclear translocation of transcriptional regulatory proteins to antagonize the regulation of GSH antioxidant enzymes.[23] Velmurugan et al found that improving antioxidant capacity, such as that of GSH, inhibited oxidative stress damage to the gastric mucosa of atrophic gastritis model rats.[24] Chen et al found that GSH can promote metabolic remodeling of cells to inhibit GC cell metastasis.[25] The above studies showed that CAG and GC progression was related to the decrease of GSH antioxidant enzymes.Increasing GSH content could effectively inhibit the persistent damage caused by ROS and other oxides to the gastric mucosa, reduce the risk of carcinogenesis, and effectively inhibit GC metastasis and recurrence. Superoxide dismutase is an antioxidant metalloprotease naturally found in organisms. [26]It performs its important antioxidant role by effectively removing ROS from the body and protecting cells from ROS-induced oxidative damage. [27]SOD is a key enzyme involved in free radical detoxification in the body.Under normal physiological conditions, ROS decompose to generate oxygen (O 2 ) and hydrogen peroxide (H 2 O 2 ).Meanwhile, H 2 O 2 generates O 2 and H 2 O through the enzymatic action of catalase (CAT). [28]If the SOD content decreases or is insufficient, the scavenging ability of free radicals decreases, which can lead to the accumulation of a large number of peroxidation products leading to damage to normal mucosal tissues.Therefore, the upregulation of SOD levels can strengthen the ability of the body for defense and repair of free radical damage. [29]It has been reported that SOD regulation is an important factor affecting tumor progression and survival in conditions including various gastrointestinal diseases. [30]Xiao demonstrated that increasing SOD content could improve the protection and repair ability of the gastric mucosa, alleviate the level of oxidative stress, and reduce the risk of pathological intestinal metaplasia and malignant transformation of the gastric mucosa. [31]Another study showed that SOD2 polymorphisms were associated with an increased GC risk. [32]The results suggest that SOD has potential value in monitoring the degree of inflammatory injury to the gastric mucosa and gastric tumor formation. In summary, MDA, GSH, and SOD have been used as biomarkers to evaluate oxidative stress in a large number of in vitro and in vivo experiments and can indirectly reflect the degree of gastric mucosal injury and GC progression and prognosis.Therefore, we can effectively inhibit oxidative stress-induced damage to the gastric mucosa by inhibiting the overexpression of MDA and ROS and increasing the SOD and GSH levels, thus further inhibiting the progression of gastric "inflammation-carcinoma."This would improve the prognosis of patients with GC, reduce the resistance to chemotherapeutic drugs, improve the overall response rate of treatment, and improve patient survival rates. NO as an important inflammatory mediator in the course of gastric "inflammation cancer." NO is a free radical produced by nitric oxide synthase (NOS) via the oxidative pathway. [33]It is an important regulatory substance involved biological processes such as apoptosis, immunity, and oxidative stress. [34]Normally, NO levels maintain normal activity to protect the mucosal barrier from damage by toxic metabolites. Under pathological conditions, elevated NO levels destroys the main components of cells, inhibits protein synthesis, damages DNA molecules, destroys mucosal integrity, and ultimately promotes carcinogenesis. [35]In addition, when stimulated by inflammatory molecules, inducible nitric oxide synthase (iNOS) produces high concentrations of NO which promotes tumor angiogenesis and abnormal cell proliferation and inhibits the physiological apoptosis of cells. [36]Notably, NO inhibits leukocyte proliferation, leading to tumor growth and spread. [37]ifferences in NOS concentrations therefore have the ability to promote or inhibit disease.Studies have shown that NO plays an important role in maintaining the integrity of the gastric mucosa and microvascular barrier function.Normal levels of NO can regulate gastric mucosal blood flow, enhance the recovery and defence abilities of the mucosa, and can improve the ability of the gastric mucosa to clear toxic substances and resist oxygen free radicals.Excessive NO levels activate oxidative stress pathways, up-regulate inflammatory levels, accelerate DNA mutations, change the normal cell architecture and function, reduce the self-clearance ability of gastric mucosa, and increase the risk of carcinogenesis. [38,39]Liao et al observed higher levels of iNOS and NO-induced damage in patients with GC.In addition, some pickled meats, fumigated products, ham, and other food products can increase endogenous NO production, cause lipid peroxidation, and promote GC formation. [40,41]tudies also report that the downregulation of NO metabolite concentration in gastric juice could effectively reduce the degree of inflammatory infiltration and atrophy of the gastric mucosa, improve the histopathological status, and downregulate oxidative stress levels. [42]1.3.Nrf2 and NF-κB as important signaling pathways in the course of gastric "inflammation cancer."Nrf2 is a potent transcriptional activator that is commonly expressed in various organs and tissues and is involved in the upregulation of antioxidant proteins, improvement of antioxidant capacity, and exertion of biological effects against cytotoxicity and oxidative damage.[43,44] Physiologically, Nrf2 and cytoskeleton-associated protein (Keap1) are present in the cytoplasm as inactive dimers through the binding of the Neh2 region at the N-terminus, thus maintaining enzymes and antioxidants that protect and stabilize cells at basal expression levels.[45] In pathological conditions, Nrf2 dissociates from Keap1 and translocates into the nucleus, where the Neh1 region forms a heterodimeric complex with proteins such as Maf which then binds to antioxidant response element promoter sites to initiate the expression of Nrf2 downstream target genes to resist deleterious stimuli.[46,47] Numerous studies have shown that Nrf2 plays an important role in protecting the gastrointestinal tract against oxidative stress and chemical damage to the gastrointestinal mucosa.[48] Yanaka demonstrated that activating Nrf2 expression could upregulate antioxidant enzyme activity, improve the antioxidant capacity of the body, and protect and repair the gastric mucosa from oxidative stress damage during Hp infection.[49] Farkhondeh et al showed that Nrf2 reduces GC recurrence rates and drug resistance through the antioxidant pathway, which has significant implications for GC treatment.[50] In addition, Zheng et al confirmed that Nrf2 is associated with GC infiltration and can be used as an indicator of prognosis.[51] The above studies suggest that intervening in the expression of the key regulator Nrf2 can effectively inhibit the expression of upstream and downstream target molecules of the oxidative stress pathway and reduce the inflammatory response.Nrf2 expression may also be used to monitor the malignant transformation of disease, which has significant implications for the development of drugs using the Nrf2 antioxidant pathway to treat various malignant diseases. Nuclear factor-κB (NF-κB) is another important transcriptional regulator whose family consists of 5 transcriptional regulators, each of which has an N-terminal Rel homology domain responsible for its binding to DNA as well as dimerization, which differentially impacts cellular activity. [52,53]his signaling pathway can bidirectionally regulate the biological processes of cells, meaning it can both regulate the oxidative stress pathway to remove abnormal cells and inhibit neoplastic disease progression, but also promote abnormal proliferation and differentiation of cells, leading to malignant transformation of neoplastic diseases. [54]The NF-κB signaling pathway acts on cytokines in both classical and non-classical pathways to maintain orderly cell signaling.Inactive NF-κB is present in the cytoplasm in a complex that binds to the inhibitory protein IκB into a trimer (p50-p65-IκB), which binds to NF-κB dimers mainly in the cytoplasm and plays an important role in signaling responses. [55,56]Classical signaling pathways are activated by IKK complexes (IKKβ, IKKα, and NEMO) that phosphorylate IκB when stimulated by cytokines including inflammatory factors (e.g., IL-Iβ) or tumor necrosis factor (e.g., TNF-α).Phosphorylated IκB dissociates from the trimeric complex, is degraded by the proteasome after ubiquitination modification, and releases the NF-κB/Rel complex.Activated NF-κB/Rel complexes translocate into the nucleus where they interact with other transcription factors to induce target gene expression. [57,58]In the non-canonical signaling pathway, the NF-κB2p100/Rel B complex stays in the cytoplasm in an inactive state and activates the kinase NIK through receptors (e.g., LTβR).Activated NIK in turn activates the IKKα complex, which phosphorylates the carboxy-terminal amino acids of p100.Phosphorylated p100 is degraded to P52 after ubiquitination, eventually forming an active p52/Rel heterodimer complex that promotes the conduction of cell signals after transferring the nucleus. [59,60]It has been shown that the activation of NF-κB pathway can promote the occurrence and development of CAG and GC. [61]In addition, ROS induces NF-κB to promote IL-8 transcription in gastric epithelial cells, promoting the inflammatory response of the gastric mucosa, up-regulating oxidative stress and change the pathology of the gastric mucosa.[64] The above studies have shown that oxidative stress plays a dual role in the course of gastric "inflammation-carcinoma." Activation of oxidative stress pathways promotes the progression of inflammation associated gastric carcinoma, where inhibition of key oxidative stress pathways effectively halts this progression by ameliorating gastric mucosal injury.Currently, the therapeutic effect of Western medicine alone on CAG and GC is unsatisfactory.Recently, a large number of in vivo and in vitro experiments have demonstrated the therapeutic effects of traditional Chinese medicines (TCM), which often have multiple active components and drug targets and few adverse effects, which is advantageous in the treatment of diseases. Active ingredients of Chinese herbs regulate oxidative stress and gastric "inflammation-cancer" As mentioned earlier, NF-κB and Nrf2 signaling pathways are important transcriptional regulators and play a key role in the course of CAG.In addition, key marker proteins of the oxidative stress signaling pathway, such as MDA, SOD, and GSH, can also reflect the degree of gastric mucosal damage.With the development of molecular biology, network pharmacology, and other technologies, it is possible to reveal the pharmacological mechanism of a drug in the treatment of a disease at the molecular level.This provides a theoretical basis for the qualification and quantification of the effects of TCM in the treatment of diseases.An increasing number of active ingredients have been identified in TCM preparations, shedding light on new approaches for treating various cancers.They have prominent advantages such as a single composition and a clear mechanism of action, and play a significant role in the treatment of diseases.The following compounds are of positive significance in intervening in gastric "inflammation-carcinoma" progression by targeting and regulating NF-κB, Nrf2, and other signaling pathways and key proteins to inhibit oxidative stress.The data in this study were obtained from published articles; therefore, no ethical issues related to patients were involved.Therefore, approval from an ethics committee was not required (Fig. 2 and Table 1). 2.2.1. Pathological damage to gastric mucosa decreased through regulation of ROS, MDA, SOD, and GSH.The extract of Ginkgo biloba (EGb) is a natural plant component and nutraceutical derived from the leaves of Ginkgo biloba with antioxidant and blood circulation-boosting effects. [85]Previous studies have shown that EGb regulates neurotransmitters, delays neuronal degeneration, improves vascular microcirculation and body tolerance. [86,87]Jiang et al treated N-methyl-N'-nitro-Nnitrosoguanidine (MNNG)-treated rats with EGb by gavage to observe EGb effects in an animal model of gastric cancer. [65]he results showed that the extract enhanced SOD activity, inhibited MDA expression in a dose-dependent manner, and downregulated the expression of c-myc, bcl-2, and fasL gastric mucosal oncogenes.EGb may exert its actions via a reduction in the degree mucosal inflammation, intestinal metaplasia, and gastric mucosal dysplasia by regulating cell proliferation and apoptosis and upregulating antioxidant activity, which inhibits the progression of precancerous gastric lesions.EGb is therefore a potential therapeutic agent for CAG treatment.Patchouli is the dried aerial part of Pogostemon cablin (Blanco) Benth.Owing to its unique aroma and ability to treat various diseases, it is widely used in cosmetics and medicine. [88]Patchouli alcohol (PA), a sesquiterpenoid, is an important component of the essential oils extracted from patchouli plants. [89]Modern pharmacological studies have shown that PA has good antioxidant, analgesic, anti-inflammatory, and antidepressant effects, as well as multiple biological activities. [90]Previous studies have shown that PA can inhibit Hp activity and exert antioxidant effects during CAG treatment. [91]Xie et al found that PA inhibited ROS production, decreased the expression levels of inflammatory factors such as TNF-α, IL-2, and IL-4, up-regulated CAT and SOD expression level, and down-regulated the MDA and NO contents in gastric epithelial cells (GES-1) cultured with Helicobacter pylori urease. [66]This suggests that PA protects against H pylori ureaseinduced GES-1 cell injury.Its protective mechanism may be related to the attenuation of apoptosis, reduction of intracellular ROS, and improvement of antioxidant and anti-inflammatory capacities, which have potential implications for PA-mediated oxidative stress reduction for the treatment of CAG.Allicin, an active compound derived from garlic, has anti-inflammatory, antioxidant, and anticancer effects. [92]Chen et al found that allicin improved the pathological state of intestinal metaplasia and restored blood flow in rat gastric mucosa.In addition, it also downregulated the expression of TNF-α, IL-6, IL-1β, and MDA and increased SOD and CAT activities.This may be related to the inhibition of the JAK2/STAT3 signaling pathway activation by allicin. [67]Baicalin (BI) is an active flavonoid isolated from TCM Scutellarin baicalinase, which has outstanding antiinflammatory, antioxidant, and immunomodulatory effects. [93]iu demonstrated that BI had a protective effects on the gastric mucosa of CAG model rats and could promote the serum expression of NO and SOD, promote CAG cell apoptosis, and relieve oxidative stress.This mechanism was related to the activation of the OPG/RANKL axis. [68]Curcumin is an active substance extracted from the rhizome of Curcuma longa and has demonstrated antitumor, antioxidant, and antiproliferative effects. [94]Liang et al demonstrated the effects of curcumin on BGC-823 human gastric cancer cells, showing that curcumin induced the ROS-mediated ASK1-MKK4-JNK cascade and led to apoptosis in BGC-823 cells, which is of potential significance in the treatment of GC. [69] Carvacrol is a monoterpene phenol produced by abundant aromatic plants and is used as a food and cosmetic additive.Modern pharmacological studies have shown that carvacrol exerts anti-inflammatory, antioxidant, and anticancer effects. [95]Gunes et al investigated the gastric mucosal protective effect of carvacrol in an MNNG-induced GC rat model, and found that carvacrol effectively reduced the levels of pro-inflammatory factors such as IL-6, IL-1β, TNFα, and TGF-β, reduced ROS activity, downregulated oxidative stress, and reduced the extent of inflammatory infiltration in the gastric mucosa of model rats, thus inhibiting GC progression. [70]2.2.Regulation of NF-κB, Nrf2 signaling pathways prevents gastric "inflammation-carcinoma" transformation.Galloyl paeoniflorin (GPF) is an important chemical component isolated from part of the peony root.Modern pharmacological studies have confirmed that GPF has medicinal value due to its anti-inflammatory, antioxidant, and antitumor effects.It is widely used in the treatment of various diseases such as osteoporosis, cerebral ischemia, and allergic asthma.[96][97][98][99][100] A recent study found that GPF could treat CAG and reduce Hpinduced gastric mucosal injury and pathological scores.[71] The mechanism is related to activating the expression of Nrf2 and its downstream target genes to counteract oxidative stress and reduce the expression of pro-inflammatory factors such as TNFα, IL6, and IL8, while upregulating SOD, HMOX1, and NQO1 levels and reducing MDA levels.Resveratrol is one of the most intensively studied and widely used polyphenols, and is present in a variety of plants.[101] Evidence suggests that resveratrol has pharmacological effects including anti-inflammatory, anticancer, antioxidant, antiaging, and cardioprotective properties.[102] Among these numerous biological effects, its antioxidant properties are the most prominent.It exerts these effects by scavenging or inhibiting the synthesis of oxygen free radicals, inhibiting lipid peroxidation, regulating the activity of related antioxidant enzymes, and stabilizing the oxidation-antioxidant balance, leading to beneficial effects in CAG.[103,104] Resveratrol relieves gastric mucosal injury by down-regulating IL-8, iNOS, and MPO levels, thus inhibiting IκBα phosphorylation, blocking NF-κB activation, increasing the expression levels of Nrf2 and HO-1, and reducing the degree of gastric mucosal injury in Hp-induced gastritis mice.This suggests that the antioxidant mechanism of resveratrol may be related to the Nrf2 and NF-κB pathways.[72] Red ginseng is widely consumed as a traditional health-promoting food, while also playing an important role in the pharmaceutical field.[105] Ginsenosides are the active components in red ginseng and have numerous biological effects, such as improving microcirculation and immunity and possess anti-inflammatory, anti-viral, and anti-oxidant properties.[106] Cho et al investigated the effect of red ginseng extract (RGE) on Hp-induced human gastric epithelial cells (AGS) and showed that RGE inhibited oxidative stress in Hp-infected AGS cells and reduced gastric mucosal injury.Its mechanism of action may be that RGE downregulates NADPH oxidase, ROS production, and IL-8 expression by inhibiting the NF-κB pathway and blocking JAK 1/STAT 2 activation. RGE signficantly improved the pathological state of AGS cells by regulating the expression of related cytokines, thereby preventing gastric epithelial cell carcinogenesis in a time-and dose-dependent manner.[73] Rhubarb is recorded in Shennong Ben Cao Jing and is the dried root and rhizome of Rheum officinale, R. palmatu, and R. tanguticum, medicinal plants of the Liao family.[107] Because it has purging effects such as, clearing heat, purging fire, cooling and detoxifying blood, reducing blood stasis, unblocking meridians, and removing dampness and yellowing, it has a therapeutic effect in digestive, endocrine, cardiovascular, and malignant diseases.110] Liu et al found that in CAG model mice, the levels of proinflammatory factors TNF-α, COX-2, IL-6, and IL-1β, MDA content was increased, and SOD expression was downregulated, indicating that the decrease in antioxidant enzymes in the gastric mucosa.This may have been caused by the inhibition of the Nrf2 and MAPK signaling pathways.[74] After intervention with emodin, the Nrf2 and MAPK signaling pathways were activated, the expression of inflammatory protein factors was inhibited, and antioxidant enzyme activity was increased to protect the structural integrity of the gastric mucosa.This revealed the molecular mechanism by which emodin prevents oxidative stress-induced damage to the gastric mucosa, providing a further evidence for the clinical application of this drug in diseases affecting the gastric mucosa.Propolis is a natural resin extracted from various plants with anti-inflammatory, antioxidant, and anticancer activities.Song et al found that gastric mucosal injury was significantly improved in CAG model mice treated with propolis, accompanied by downregulated inflammatory factors (IL-8, TNF-α, and IL-1β) and decreased NO expression.Western blotting analysis results showed that propolis also significantly inhibited IκBα and p65 phosphorylation, thereby inhibiting p65 translocation from the cytoplasm to the nucleus to reduce expression of pro-inflammatory target genes in model mice.Its therapeutic mechanism is related to inhibition of NF-κB signaling pathway and alleviation of oxidative stress.[111] Astaxanthin, a naturally fat-soluble carotenoid, can be extracted from various seafoods and is used in the treatment of various diseases owing to its antioxidant, anticancer, DNA repair, and anti-apoptotic properties.[112] Kim et al found in AGS cells infected with Hp that treatment with astaxanthin significantly inhibited ROS activity, decreased IL-8 expression, and blocked NF-κB pathway activation.[75] This suggests that astaxanthin can alleviate oxidative stress and inflammatory damage to the gastric mucosa and effectively alleviate inflammatory infiltration.Kirenol has been found to be a natural diterpenoid with anti-inflammatory and anticancer properties.[113] Kirenol downregulated lipid peroxidation, increased enzymatic and nonenzymatic antioxidants, suppressed TNF-α, IL-2, and PGE6 expression, and reduced tumor incidence in MMNG rats via an action mechanism related to kirenol inhibition the of NF-κB signaling pathway.[76] Crocin is a carotenoid extracted from the medicinal plant, saffron.Previous studies have shown that crocin can be used as a protective agent against gastric mucosal injury.In addition, crocin has lower toxicity and a better safety profile than the anticancer agent 5-fluorouracil (5-FU).[114] Luo et al treated GC cell lines AGS and SGC-7901 using crocin.The results showed that crocin exerted anticancer effects by activating the classical Nrf2 signaling pathway and improving antioxidant capacity. Ginge is a common TCM that is widely used because of its anti-inflammatory, anticancer, and antioxidant effects and pharmacological activity in immune disorders.[77,115] Mansingh et al found that aqueous ginger extract (AGE) was able to upregulate SOD, glutathione peroxidase and GR contents, decrease TNF-α and IL-6 expression, and alleviate oxidative stress and inflammatory factor expression associated with GC in N-methyl-N-nitrosourea (MNU)-treated rats, and its mechanism of action was associated with NF-κB.[78] Rosmarinic acid (RA) is a phenolic compound extracted from plants such as rosemary and mint.It has anti-inflammatory, anti-cancer, antioxidant, and other pharmacological properties. [116]ng et al used gastric cancer cells (SGC-7901) to demonstrate that rosmarinic acid exerts anticancer activity by regulating the Nrf2/HO-1 related signaling pathways, increasing ROS and MDA activities, and activating oxidative stress to promote gastric cancer cell apoptosis.[79] 2.2.3.Regulating other signaling pathways to intervene in GC progression.Dehydrocostus lactone (DHCL) is derived from the dried roots of Aucklandia lappa Decne.It is a member of the family Asteraceae and has anti-inflammatory and antitumor effects.[117] Shao et al showed that DHCL inhibits SGC-7901 proliferation, promotes intracellular ROS production, activates the Bax/Bcl-2 mediated mitochondrial apoptosis pathway, and induces GC cell apoptosis in a dosedependent manner to inhibit GC progression.[80] Cucurbitacin D (CuD) is a tetracyclic triterpenoid extracted from members of the family Cucurbitaceae, and various studies have shown that it has anti-inflammatory, hepatoprotective, and antitumor activities.[118] Zhang et al showed that CuD could upregulate ROS expression and increase NO content in human gastric cancer cell lines (AGS, SNU1, and Hs2T), while also triggering apoptosis pathways, leading to higher ROS and NO production, thereby accelerating gastric cancer cell apoptosis.[81] This inhibitory effect of CuD on GC progression was achieved by inducing iNOS/NO signaling and inhibiting the Akt pathway.Syringic acid (SA) is a natural phenolic compound present in herbs and edible plants and has anti-inflammatory, antioxidative, and anti-angiogenic effects. [119] Peiet al found that SA could effectively inhibit GC progression through targeted inhibition of mTOR/AKT signaling pathway, significantly reduce p53 and Bcl -2 expression, enhance antioxidant capacity through mitochondrial membrane potential, up-regulate caspase-3 and caspase-9 activities, and down-regulate IL-6 and TNF-α levels.[82] Licochalcone A (LA), a flavonoid derived from the root of Glycyrrhiza uralensis, has anti-inflammatory, anticancer, and lipid-and blood glucose-regulatory effects.[120] Hao et al found that LA upregulates MDA expression, decreases the GSH/GSSG ratio, increases ROS activity, and inhibits GC cell proliferation.[83] Furthermore, LA induces ROS-mediated MAPK activation, inhibits the PI3K/AKT signaling pathway, and prevents GC progression.Salvianolic acid B (Sal-B) is the active substance of the medicinal plant Salvia miltiorrhiza Bunge.Related studies have shown that Sal-B has anti-inflammatory, anticancer, and anti-fibrotic effects and promotes stem cell proliferation and differentiation.[121] In addition, Wang et al treated AGS and AGS/DDP cells with Sal-B and found that Sal-B not only initiated ROS activity and promoted apoptosis but also reduced migration, invasion, and epithelial-mesenchymal transition (EMT) in AGS and AGS/DDP cells and reduced DDP resistance in GC cells, which was achieved via regulation of the AKT/mTOR pathway.[84] The above studies have shown that various active ingredients can not only inhibit the inflammatory response by regulating the Nrf2, NF-κB, JAK1/STAT2 and MAPK signaling pathways, effectively reduce the degree of pathological damage caused by oxidative stress, but also protect the integrity of gastric mucosal structure, promote gastric mucosal self-repair, attenuate the progression of CAG, and finally reduce the rate of gastric mucosal carcinogenesis.These results have significance for guiding the study of active ingredients for targeted and precise treatment of CAG.Therefore, they are potential drugs for patients with gastric mucosal injuries.In addition, we found that oxidative stress is inextricably related to the progression, prognosis, and drug resistance of GC and inhibition or promotion of this process can have a clinical on GC development and progression.Therefore, in-depth studies should be conducted to increase the selectivity of natural prescriptions for GC treatment. Chinese herbal prescription regulates oxidative stress and gastric "inflammation-cancer" transformation 2.3.1. Increasing antioxidant factor expression and hindering GC development. Compared with conventional drug active ingredients, Chinese herbal preparations have the overall characteristics and unique advantages of multiple active components and multiple targets with actions that may be exerted via multiple pathways.Many studies have reported that Chinese herbal preparations are effective for the CAG treatment.Liu et al treated CAG patients using Huatan Xiaoyu Fang (tangerine peel, Pinellia ternata, Ji Nei Jin, Danshen, Coix seed, Cat claw grass, Puhuang, Scutellaria barbata, and Agrimonia pilosa) and found that this formula could resist oxidative stress damage and improve pathological tissue lesions by downregulating MDA and TNF-α and increasing SOD and serum epidermal growth factor levels in the body. [122]Dendrobium Yangwei Decoction (fried malt, fried licorice, Zhuru, fried Radix Paeoniae Alba, Beisha, Trichosanthes kirilowii, Ophiopogon japonicus, and Dendrobium officinale) activates blood circulation and relieves pain, nourishes yin, a nourishes the stomach meaning it is widely used in the treatment of CAG. [123]Wu et al found in an in-depth study that in the treatment of patients with CAG this preparation can reduce the levels of C-reaction protein, IL-6, TNF-α, NO, and MDA, up-regulate the contents of GSH and SOD in the gastric mucosa, significantly improve inflammatory injury, effectively relieve oxidative stress, and inhibit the progression of the course of gastric "inflammation-carcinoma." [124] Zhu found that Huagan decoction (Radix Bupleuri, Pericarpium Citri Reticulatae, Cortex Moutan, Fructus Gardeniae, Fritillaria thunbergii, Radix Paeoniae Alba, Rhizoma Coptidis, Fructus Evodiae, Rhizoma Alismatis, and Radix Glycyrrhizae) combined with folic acid improved serum PG I and SOD levels and the function and antioxidant capacity of gastric mucosal glandular cells. [125]Previous studies have confirmed that weipiling (Radix Pseudostellariae, Rhizoma Atractylodis Macrocephalae, Poria cocos, Radix Notoginseng, Rhizoma Curcumae, Herba Hedyotis diffusa, Rhizoma Monkey Mushroom, Shougong) can reverse precancerous lesions, such as CAG, and intestinal metaplasia and dysplasia. [126]Pan et al used intragastric administration of Weipiling 3.75 g/kg and 15 g/kg to gastric precancerous lesion (GPL) model mice for 8 weeks and found that this decoction could increase the protein expression of SOD, up-regulate Nrf2, NQO1, and HO-1 expression, exert anti-oxidative stress injury in a dose-dependent manner, improve gastric mucosal lesions, reduce histological scores, and increase mouse body weight. [127]hese results suggest that this decoction exerts effects through the Nrf2/NQO1/HO-1 signaling pathway to control or even reverse gastric "inflammation-carcinoma" transformation and restore the damaged gastric mucosa.Zeng et al used Jianpi Huayu Jiedu decoction (which includes Hericium erinaceus, gecko, Curcuma zedoaria, Astragalus membranaceus, Atractylodes macrocephala, Poria cocos, Pseudostellaria heterophylla, Panax notoginseng, and Hedyotis diffusa)) in rats with GPL.They found that this decoction could down-regulate ROS, MDA, up-regulate SOD antioxidant activity, and inhibit the invasion and migration of cancer cells, achieve the purpose of repairing damaged gastric mucosal tissues, reversing intestinal metaplasia through regulating the PI3K/AMPK-mTOR-ULK1 pathway. [128]iu et al confirmed that Shenqi Xiaopi Decoction (Radix Astragali, Radix Codonopsis, Rhizoma Pinelliae, Rhizoma Atractylodis Macrocephalae, Radix Magnoliae Officinalis, Fructus Aurantii Immaturus, Radix Muxiang, Radix Bupleuri, Herba Hedyotis diffusa, cardamom, and Radix Glycyrrhizae) upregulated GSH expression by downregulating MDA, P53 protein, CEA, CA72-4, and IL-8 in the serum of CAG patients. [129]u et al found that Ophiopogon japonicus decoction (lentil, raw licorice, Polygonatum odoratum, Sha Shen, Ophiopogon japonicus, Trichosanthes) can reduce the expression of TNFα, MDA, and CDX2, increase SOD, epidermal growth factor, MLT, and PGI content, effectively reduce body inflammation, reduce oxidative stress-related factor levels, and achieve good efficacy in clinical treatment of CAG. [130]Some studies have demonstrated that gastritis formula (Pinellia ternata, Scutellaria baicalensis, Coptis chinensis, Zingiber officinale, Codonopsis pilosula, and Radix Glycyrrhizae), used to treat CAG patients, resulted in reduced IFN-β, IL-23, and MDA levels, increased SOD antioxidant capacity, decreased inflammatory responses, reduced levels of oxidative stress, and improved pathological signs of the gastric mucosa. [131][134] These formulations have achieved good therapeutic effects in vivo and in vitro reducing the production of a large number of inflammatory factors as well as oxidative metabolites through the regulation of important signals of oxidative stress and has far-reaching significance in enhancing the repair ability of damaged gastric mucosa and reducing the pathological score of gastric mucosa, laying a certain foundation for the study of the molecular mechanism of such drugs in the treatment of GPL. Inhibition of inflammatory response, reduction of drug resistance, and improvement of GC prognosis. An increasing number of studies have confirmed that herbal medicines enhance the efficacy of chemotherapy, radiotherapy, targeted therapy, and immunotherapy for neoplastic diseases.they may also mitigate the adverse effects of these therapies.Banxia Xiexin Decoction (BXD) was first recorded in the book "Miscellaneous Diseases of Typhoid Fever" during the Eastern Han Dynasty.In clinical practice, BXD is used to treat various inflammatory diseases and cancers. [135]After intragastric administration of BXD to GC model rats, MDA, SOD, and GSH contents were regulated, oxidative stress was promoted to induce apoptosis, thereby inhibiting GC cell activity and blocking GC progression.Its mechanism of action is attributed to the inhibition of the Wnt/β-catenin pathway. [136]In addition, Yu et al confirmed that Gancao Ganjiang decoction could inhibit GC progression using network pharmacology and molecular docking technology, and the mechanism may be related to its action on TNF, p53, and other signaling pathways by regulating oxidative stress; however, this needs to be verified by further experiments. [137]ou et al reported inhibited NO, IL-6, and C-reaction protein expression in the serum of patients with primary GC after a 2-month treatment with Yiqi Jianpi Jiedu decoction, which indicated that the decoction could obtain a higher shortterm efficacy in patients with primary GC, reduce the level of inflammatory factors, and did not increase the incidence of toxicity, rendering it worthy of clinical application. [138]Pan et al reported that Weifuchun capsule extract upregulated Bax and MDA activities, downregulated Bcl-2, SOD, and GSH-Px levels, promoted gastric cancer cell apoptosis, and effectively inhibited gastric cancer progression.In addition, significant changes in these proteins were reported compared to use of 5-FU alone. [139]In addition, She et al confirmed that Pingwei pills were able to upregulate SOD, Nrf2, and HO-1 protein and mRNA expression and enhance cellular antioxidant capacity for effective GC treatment. [140]After SGC-7901 cells were treated with Weiwei'an, the levels of MDA and LDH were inhibited and the activity of SOD was enhanced.It also alleviated oxidative stress and inflammatory factor expression and altered the tumor survival environment.The therapeutic effect of Weiwei'an was achieved by inhibition of the NF-κB pathway. [141]Ye et al found that Tongfu Huoxue decoction effectively promoted the recovery of postoperative gastrointestinal function and improved GC prognosis in patients after radical gastrectomy for gastric cancer to a degree that made it worthy of clinical application.These affects were achieved through downregulating MDA and upregulating SOD activity, thereby protecting intestinal mucosal barrier function and reducing oxidative stress. [142]The above results suggest that these Chinese herbal prescriptions have significant impact in promoting or inhibiting oxidative stress to intervene GC progression and prevent the recurrence and metastasis of tumor cells.In the future, more Chinese herbal prescriptions may become candidates for the treatment of GC (Fig. 2 and Table 2). Conclusions CAG is an important stage in the occurrence of GC, although the disease state of CAG has not been clearly defined in TCM.The condition can be best categorized by of "fullness and epigastric pain" according to the clinical manifestations of patients.In TCM theory, the pathogenesis of CAG can be summarized with typical clinical symptoms such as stomachache, bloating, hiccup, and belching and based in the category of "spleen deficiency."In TCM, the saying is that "vital qi exists in, evil cannot be dry, evil together, its qi will be virtual."Deficiency of qi in the spleen and stomach of the human body, inability to transport the essence of the water valley, decreased self-regulation ability of Shengqing Jiangzhuo, inability to remove toxic metabolites, and disturbance of the microenvironment in the body lead to the accumulation of damp-heat toxic pathogens in the gastrointestinal tract.Under normal physiological conditions, the human body is in the dynamic balance of yin and yang and life activities proceed in an orderly fashion.,If yin or yang cannot be balanced, it will lead to the occurrence of diseases, which is the same as the impact of oxidative stress on the course of CAG and GC elaborated above.Inflammatory factors such as "NF-κB, IL-1, and TNF-α," like "toxic pathogens" in TCM theory, destroy the normal structure of gastric mucosal tissue and cause persistent damage to the gastric mucosa, and a precancerous microenvironment is formed and accelerate CAG development into GC.Based on this theory, TCM therapy can be used to improve symptoms, regulate the above inflammatory factors and metabolites, improve the antioxidant capacity of the body or inhibit oxidative stress, so that balance ("yin and yang harmony") is restored, the gastric mucosal pathological score is reduced, the course of gastric "inflammation-cancer," is inhibited and the incidence of GC and the metastasis of tumor cells is effectively reduced. A large body of evidence has shown that oxidative stress plays a key role in the course of CAG and GC and can affect patient outcomes and prognosis.Therefore, referring to TCM insight into CAG and GC treatment, there are broad prospects for intervention in the regulation of oxidative stress.In this study, we investigated the mechanism underlying the inhibition of CAG progression and the prevention of GC by regulating key protein targets of oxidative stress.In addition, the active ingredients in TCM used to treat GC can intervene in the progression, drug resistance, and prognosis of GC by bidirectionally regulating oxidative stress.At present, there are some shortcomings in the treatment of CAG and GC using TCM that mediate the oxidative stress pathway.For example, (1) Although relevant controlled studies have shown that TCM can regulate oxidative stress and intervene in CAG and GC progression, there is a lack of multi-sample and multicenter cohort studies.(2) This study mainly focused on a single pathway and target signaling molecules, and the interaction of multiple oxidative stress signaling pathways should be explored in in vivo and in vitro experiments at a later stage to enrich the means of TCM treatment.(3) There have been few studies on the mechanism of action of TCM prescriptions in the treatment of CAG and GC, and such studies should be strengthened in the future to provide more options for TCM in the treatment of these diseases.(4) In the future, more new drugs and more natural products as well as nutraceuticals should be developed using network pharmacology, biological information technology, and metabolomics technology to intervene in the progression of such diseases by intervening in oxidative stress.Taking full advantage of the desirable characteristics of TCM preparations and the experience and information gained from TCM theory will contribute to better health outcomes for patients with CAG and CG. Figure 1 . 1 Figure 1.Biological processes of oxidative stress in gastric "inflammation-carcer". Figure 2 . 2 Figure 2. Mechanism diagram of active ingredients of traditional Chinese medicine and traditional Chinese medicine compound regulating oxidative stress to intervene gastric "inflammation-cancer". CAG = chronic atrophic gastritis, CAT = catalase enzyme, GPX = glutathione peroxidase, GSH = glutathione, MDA = malondialdehyde, NF-κB = nuclear factor-Κb, iNOS = inducible nitric oxide synthase, Nrf2 = nuclear factor erythroid 2-related factor 2, ROS = reactive oxygen species, SOD = superoxide dismutase, TCM = traditional Chinese medicine. barrier function and reduces oxidative stress, thereby promoting the recovery of postoperative gastrointestinal function and improving GC prognosis ----[142] MDA↓ CAG = chronic atrophic gastritis, CAT = catalase enzyme, CRP = C-Reaction Protein, EGF = epidermal growth factor, GC = gastric cancer, GEC = gastric mucosa epithelial cell, GSH = glutathione, MDA = malondialdehyde, NF-κB = nuclear factor-κB, Nrf2 = nuclear factor erythroid 2-related factor 2, PG = prostaglandin, ROS = reactive oxygen species, SOD = superoxide dismutase, TCM = traditional Chinese medicine. Table 1 1 Active ingredients in TCM in regulating oxidative stress in gastric "inflammation-carcer." NameModelsBiological effectsResultsPathwayReferencesExtract of GinkgoMNNG model ratsSOD↑Inhibits oxidative stress and Inhibits oxidative stress and------[65]BilobaMDA↓ Bcl-2↓ FasL↓reduces the risk of gastric carcinogenesisPatchouli AlcoholCEG-1 cellsSOD↑ CAT↑Inhibits oxidative stress and reduces intracellular ROS------[66]NO↓ ROS↓ MDA↓ IL-4↓TNF-α↓ IL-2↓AllicinCAG model ratsSOD↑ CAT↑Reducing gastric mucosal lesions, inhibiting oxidativeJAK2/STAT3 pathway[67]MDA↓ IL-6↓ TNF-α↓stress and reversing intestinal chemotaxis in CAG ratsIL-1β↓BaicalinCAG model ratsSOD↑ NO↑Reduce the damage of gastric mucosa and increase theOPG/RANKL pathway[68]body weight of ratsCurcuminBGC-823 cellsROS↑Promote apoptosis and inhibit the progression of gastricASK1-MKK4-JNK[69]cancerpathwayCarvacrolGC modelROS↓IL-6↓ IL-1β↓Mitigating oxidative stress and inflammatory infiltration to-----[70]ratsTNF-α↓inhibit gastric cancerTGF-β↓GalloylpaeoniforinCEG-1 cellsSOD↑ Nrf2↑inhibit oxidative stress and inflammatory factor produc-Nrf2 pathway[71]MDA↓ IL-1β↓tion, reduce gastric mucosal damageROS↓ TNF-α↓ IL-8↓ResveratrolCAG modelNrf2↑ HO-1↑Inhibition of oxidative stress and inflammation and im-NrF2/HO-1 pathway[72]mouseIL-8↓ iNOS↓ NF-κB↓provement of gastric mucosal histopathological scoresIκBα↓Red Ginseng ExtractCEG-1 cellsiNOS↓ ROS↓ NADPH↓Inhibition of oxidative stressNF-κB/Jak1/ Stat2[73]NF-κB↓ Jak1/ Stat2↓pathwayEmodinCAG modelSOD↑Significant improvement of gastric mucosal histopatholo-Nrf2/MAPK pathway[74]mouseIL-6↓ IL-1β↓gy, inhibition of inflammation and oxidative stressMDA↓ TNF-α↓AstaxanthinAGS cellsROS↓ IL-9662↓ IL-8↓Effectively alleviating inflammatory infiltrationNF-κB pathway[75]PPAR-γ↓KirenolMNG ratsmRNA↓ TNF-α↓ IL-2↓Suppresses inflammatory expression and reduces tumorNF-κB pathway[76]PGE6↓incidenceCrocinAGS cellsAKR1Cs↑Enhances antioxidant capacity and exerts anti-cancerNrf2 pathway[77]SGC-7901 cellsCHI3L1↓effectsAqueous GingerMNU ratsSOD↑ GPx↑ GR↑Inhibiting the progression of gastric cancerNF-κB pathway[78]ExtractTNF-α↓ IL-6↓Rosmarinic acidSGC-7901 cellsROS↑ MDA↑ Bax/Bcl-2↑It promotes apoptosis of gastric cancer cells and exertsNrf2/HO-1 pathway[79]anticancer effects.DehydrocostusSGC-7901 cellsROS↑ Caspase-3↑Induction of apoptosis in gastric cancer cells to interveneBax/Bcl-2 pathway[80]LactoneBeclin1↓ LC3 I/LC3 II↑in gastric cancer progressionCucurbitacin DAGS cellsROS↑ NO ↑ Bax↑Accelerating gastric cancer cell apoptosis and interven-AKT pathway[81]SNU1 cellsCaspase-9↑ing in gastric cancer progressionHs2T cellsBcl-2↓Syringic AcidAGS cellsCaspase-3↑ Caspase-9↑Enhances antioxidant capacity, down-regulates inflam-mTOR/AKT pathway[82]IL-6↓ TNF-α↓mation levels and inhibits gastric cancer progressionLicochalcone ABGC cellsMDA↑ ROS↑Inhibits the activity of gastric cancer cellsPI3K/AKT pathway[83]GSH/GSSG↓Salvianolic acid BAGS cellsROS↑Reducing drug resistance rate and inhibiting gastricAKT/mTOR pathway[84]AGS/DDP cellsEMT↓ NF-kB↓ TNF-a↓cancer progressionIL-6↓ PGE↓ Table 2 2 TCM prescriptions in regulating oxidative stress in gastric "inflammation-cancer." NameModelsBiological EffectsResultsPathwayReferencesHuatan XiaoyuCAG patientsMLT↑ SOD↑Improvement of gastrointestinal hormone levels, reduction of body----[122]PrescriptionNO↓ MDA↓ TNF-α↓inflammation and inhibition of oxidative stressShihu YangweiCAG patientsGSH↑ SOD↑Inhibition of oxidative stress and improvement of clinical symptoms----[124]DecoctionMDA↓ NO↓CRP↓and gastric mucosal pathologyTNF-α↓ IL-6↓HuaganjianCAG patientsPG I↑ SOD↑Inhibition of persistent damage to gastric mucosa by oxidative----[125]PG II↓ IL-1β↓ IL-6↓stress and inflammationIL-17↓ MDA↓WeipilingCAG patientsNrf2↑ SOD↑ NQO1↑Promotion of the activation of Nrf2, improvement of oxidativeNrf2/NQO1/HO-1[127]HO-1↑stress and reverse the gastric "inflammation-cancer" transfor-pathwaymation to a certain extentJianpi HuayuCAG model ratsSOD↑Reversion of IM and damage of GECsPI3K/AMPK-[128]JieduROS↓ MDA↓mTOR-ULK1PrescriptionpathwayShenqi XiaopiCAG patientsGSH-Px↑Correction of oxidative damage, significantly reduction of tumor----[129]DecoctionMDA↓ P53↓ CEA↓markers and p53 proteinCA72-4↓ IL-8↓MaidongCAG patientsSOD↑ EGF↑ MLT↑ PG I↑ Effectively reduce body inflammation and reduce oxidative-----[130]DococtionTNF-α↓ MDA↓ CDX2↓stress-related factor levelsWeiyanCAG patientsSOD↑Inhibit inflammatory reaction, relieve oxidative stress and improve----[131]PrescriptionIFN-β↓ IL-23↓ MDA↓pathological signs of gastric mucosaShugan HeweiCAG patientsSOD↑Inhibition of oxidative stress and inflammatory factor expression,----[132]DecoctionMDA↓ CRP↓ IL-6↓increase of SOD activity, and improvement clinical symptomsTNF-α↓ NO↓WeifuchunCAG patientsSOD↑Inhibition of oxidative stress, up-regulation of antioxidant enzyme----[133]TabletsIL-8↓ IL-6↓expression, and reduction of gastric mucosa damageTNF-α↓ NO↓Xuefu ZhuyuCAG patientsSOD↑ CAT↑Inhibition of oxidative stress and inflammatory damage of gastric----[134]CapsuleMDA↓ EGF↓mucosa, improvement of pathologyBanxia xiexinGC model ratsMDA↑promotes oxidative stress to induce apoptosis, thereby inhibitingWnt/β-cateninDecoctionGC cell activity and blocking GC progressionpathway Chen et al. • Medicine (2023) 102:46 www.md-journal.com Gastric cancer. 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November 7th, 2023 November 7th, 20234D3E8DB5BEA00AB794A46C740B6F817210.21203/rs.3.rs-3548036/v1Traumatic Brain InjuryCritical CareNutritionFatty AcidsPolyunsaturated Fatty AcidsLipidsIn ammation BackgroundEarly evidence-based medical interventions to improve patient outcomes after traumatic brain injury (TBI) are lacking.In patients admitted to the ICU after TBI, optimization of nutrition is an emerging eld of interest.Specialized enteral nutrition (EN) formulas that include immunonutrition containing omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been developed and are used for their proposed antiin ammatory and pro-immune properties; however, their use has not been rigorously studied in human TBI populations.MethodsA single-center, retrospective, descriptive observational study was conducted at LAC + USC Medical Center.Patients with severe TBI (sTBI, Glasgow Coma Scale score ≤ 8) who remained in the ICU for ≥ 2 weeks and received EN were identi ed between 2017 and 2022 using the institutional trauma registry.Those who received immunonutrition formulas containing n-3 PUFAs were compared to those who received standard, polymeric EN in regard to baseline characteristics, clinical markers of in ammation and immune function, and short-term clinical outcomes.ResultsA total of 151 patients with sTBI were analyzed.Those who received immunonutrition with n-3 PUFA supplementation were more likely to be male, younger, Hispanic/Latinx, and have polytrauma needing non-central nervous system surgery.No differences in clinical markers of in ammation or infection rate were found.In multivariate regression analysis, immunonutrition was associated with reduced hospital length of stay (LOS).ICU LOS was also reduced in the subgroup of patients with polytrauma and TBI.ConclusionThis study identi es important differences in patient characteristics and outcomes associated with the EN formula prescribed.Study results can directly inform a prospective pragmatic study of immunonutrition with n-3 PUFA supplementation aimed to con rm the biomechanistic and clinical bene ts of the intervention. INTRODUCTION Traumatic brain injury (TBI) is a signi cant cause of morbidity and mortality that affects all populations, regardless of gender, age, ethnicity, race, and socioeconomic background.Severe TBI results in > 60,000 deaths in the United States (US) annually and over 5 million persons living with TBI-associated disability 1,2 .First-year healthcare costs are estimated to be $2 billion in survivors 2 .Despite the large health and socioeconomic impact, there are still no Level 1 recommendations for medical interventions to improve outcomes in moderate and severe TBI (sTBI) 3 .The pathophysiology of sTBI is complex, with several early pro-in ammatory molecular and cellular changes occurring in the immediate and delayed phases after trauma 4 .Although these pathways are thought to be largely bene cial to support initial reparative processes, they may also contribute to adverse clinical symptomatology, metabolic stress, and disease progression if left untreated.In this context, early in-hospital interventions targeting in ammation and metabolism may prevent secondary brain injury and systemic complications that contribute to poor clinical outcomes after TBI. Optimization of nutritional status with early (within 48-72H of admission) enteral nutrition (EN) is an emerging therapeutic strategy in critically ill and trauma populations 5,6 .The goal is to both prevent malnutrition and provide essential nutrients to promote cellular and neurologic recovery.After sTBI, malnutrition occurs in up to 76% of ICU patients and is associated with increased mortality and poor functional outcomes [7][8][9] .Its high incidence is multifactorial, with important contributions from dysphagia, increased metabolic demands, altered digestive and metabolic function, and variable feeding practices while in the ICU.Together, critical illness and malnutrition in TBI result in a pro-in ammatory, immunosuppressed and catabolic state that predisposes individuals to complications such as fever and nosocomial infection, which are both independent predictors of poor outcome 10,11 .Early nutritional support with specialized dietary formulas that include immunonutrition with omega-3 polyunsaturated fatty acid (n-3 PUFA) and amino acid supplementation is a novel approach to optimize nutrition and promote recovery after TBI; however, their clinical effect has not been rigorously studied in acute neurologic disease.In pre-clinical models, administration of in ammation-resolving bioactive lipids, including the n-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), has robust evidence for neuroprotection, anti-in ammatory properties, and improved cellular energetics 12 . The purpose of this descriptive retrospective observational study was to compare the population characteristics of sTBI patients who received EN immunonutrition with n-3 PUFA supplementation versus those who received standard, polymeric EN which did not contain the added components of immunonutrition.The supplementation of n-3 PUFAs in TBI is an appealing therapeutic strategy given their proposed anti-in ammatory effect, widespread availability, safety pro le, relative low-cost and general feasibility in implementing into ICU standards of care.When incorporated into universal strategies to optimize nutritional status, such novel approaches might lead to meaningful impact on patient and hospital outcomes.The results of this study are anticipated to inform future prospective clinical trials investigating the clinical effect of immunonutrition including n-3 PUFA supplementation in moderate-to-severe TBI and other acute neurologic diseases. METHODS Study Population This is a single-center retrospective observational study of sTBI conducted in Los Angeles, California.The single study site, Los Angeles General Medical Center (formerly named LAC + USC Medical Center at the time of patient admission) is a high-volume Level 1 trauma center and the largest safety net hospital in the Western United States.This descriptive study was considered hypothesis-generating, aimed to characterize differences in patient populations based on interventions provided as clinically indicated, with no primary hypothesis testing. The target population were patients with sTBI who received EN in the ICU.Study subjects were identi ed from the institutional trauma registry that is maintained by the Division of Trauma and Acute Care Surgery.A ve-year period between July 2017 and July 2022 was studied.Sample study subjects met the following inclusion criteria: age ≥ 18 years, diagnosis of sTBI (history and radiographic ndings consistent with TBI, Glasgow Coma Scale score [GCS] ≤ 8), ≥ 14 day ICU length-of-stay (LOS), prescribed and received EN during the majority (> 7days) of the 14-day ICU period.No time cut-off for initiating EN was used.Patients who presented with GCS = 3 with dilated pupils, or those who did not tolerate planned EN initiation due to gastric dysmotility were excluded from the analysis.These inclusion and exclusion criteria were chosen to ensure that study subjects were su ciently exposed to the intervention of interest.The study population included both isolated head trauma (IHT) and polytrauma with TBI.It was preplanned to study subgroups of IHT and polytrauma to determine how these populations might differ in their baseline characteristics and response to EN. Intervention The intervention of interest was EN using an immunonutrition formula containing n-3 PUFAs from sh oil. Those who received EN with immunonutrition, either via gastric or post-pyloric feeding tube, were compared to those who received standard polymeric EN formulas not classi ed as immunonutrition. Immunontrition formulas have been developed for their proposed anti-in ammatory properties, to promote immune system function, and support biosynthesis and protein needs in high-risk critically ill and trauma patients 13 .At the study site, EN products are supplied by Abbott Nutrition®.Peptide-based products classi ed as immunonutrition that were prescribed to study subjects were Vital High Protein™ (containing EPA = 2.3g/L, DHA = 0.9 g/L, vitamin D, C and E), Vital AF 1.2 Cal™ (containing EPA = 2.7g/L, DHA = 1.1 g/L, vitamin D, C and E) and Pivot™ (containing EPA = 2.6 g/L, DHA = 1.1 g/L, arginine, glutamine, vitamin C and E).In addition to n-3 PUFAs, formulations also contain essential vitamins and amino acids as listed to support immunomodulation, antioxidant activity and cellular peptide biosynthesis.Given the retrospective, observational nature of this study, the choice of speci c EN formulation used, time to initiation, dose and duration were determined by clinical judgement.At the study site, a clinical registered dietitian is involved in the nutritional management of ICU patients who require EN, including determining the formulation prescribed, goal dose rate to meet caloric needs, and reinitiation schedule after pauses in EN delivery.Although not protocolized at our institution, immunonutrition formulas are often chosen for patients with high protein requirements due to critical illness or wound healing as described. Data Collection and Study Variables The institutional trauma registry was queried to identify patients meeting inclusion criteria.The sample population consisted of remaining subjects after inclusion and exclusion criteria were applied.Data that was not contained in the trauma registry was retrospectively collected from the electronic medical record (EMR) by study personnel.Demographic and baseline characteristics including co-morbidities were obtained, as well as the EN product used and time-to-initiation.For outcome measures, clinical markers of in ammation were recorded, including peak 14-day values of the common acute phase reactants Creactive protein (CRP, mg/L) and white blood cell count (WBC, x10 9 /L), and measures of fever occurrence. Both the number of calendar days with fever (any occurrence of temperature > 37.8 o C) and cumulative fever burden (arithmetic sum of daily peak fever > 37 over the 14-day study period) were collected.This measure of fever burden has been validated as an independent predictor of outcomes after TBI 10 .Patient outcomes, including incidence of culture-positive infection during the 14-day ICU period, ICU and hospital LOS, early GCS recovery (post-TBI day #7 GCS), and discharge status were analyzed.The outcome measures were chosen based on data that could be feasibly collected through retrospective EMR review.This study was designated IRB exempt by the University of Southern California Institutional Review Board. Statistical Analysis Data normality for continuous variables was assessed by histogram and Shapiro-Wilk test.Univariate analyses for baseline differences by study groups were conducted by Chi-square test for categorical variables and independent t-test or Wilcoxon rank sum test for continuous variables, depending on data normality.The comparison of inpatient mortality rate was conducted using multivariate Poisson regression with log link function and adjusted for confounders which were de ned as baseline variables with large differences between groups that were clinically important but not necessarily reaching a p < 0.05 threshold.There were three variables that met the de nition of confounder to the intervention effect: age, gender, and occurrence of non-central nervous system (non-CNS) acute surgery.Using the log link function for Poisson modeling, the reported associations were interpreted as relative risks (RRs).The overdispersion assumption for Poisson regression was investigated through the goodness of t test using Pearson Chi-square/DF ratio.When this ratio was larger than 1, a negative binomial distribution was used.Differences in hospital and ICU LOS were tested by multivariate generalized linear models and adjusted for potential confounders as described above.The interaction between IHT and polytrauma subgroups and intervention groups was tested in both models.A strati ed intervention effect by IHT was illustrated for trend of effect modi cation.Missing data for laboratory values was excluded from the analysis.SAS 9.4 was used for all data analyses. RESULTS A total of 151 patients with sTBI were analyzed (Figure 1), of which 61 patients had isolated TBI and 90 patients had polytrauma with associated TBI.Baseline characteristics are shown in Table 1.In the study population, the average age was 43 years, 85% were male and 60% were classi ed as Latinx/Hispanic.94 patients (62.3%) received EN with immunonutrition containing n-3 PUFA supplementation and 57 (37.7%) patients received standard, polymeric EN formulations.Compared to the standard EN group, the treatment group who received immunonutrition with n-3 PUFA supplementation were slightly younger (41.2 v 46.5, p=0.07) and were more likely to be male and Latinx/Hispanic.The immunonutrition group more commonly sustained motor vehicle accident as the mechanism of injury and were signi cantly more likely to have polytrauma and require emergent non-CNS surgery.There were no statistically signi cant differences in baseline co-morbidities between the primary study groups and initial GCS was nearly identical.Vital AF TM was the most prescribed immunonutrition formula and was used in 47.9% of patients in the treatment group.Baseline weights and time-to-initiation of EN between study groups was similar (49.6H in the immunonutrition group v 52.8H in the standard group). In unadjusted univariate analysis, peak 14-day ICU WBC and CRP values were similar between study groups (Table 2).Both 14-day cumulative fever burden (15.1 v 15.2, p=0.75) and calendar days with fever (8.4 v 8.6, p=0.74) were no different in those prescribed immunonutrition versus standard EN formulations.The observed culture-positive infection rate was lower in the immunonutrition group compared to the standard group, but this did not reach statistical signi cance (80.9% v 86.0%, p=0.42).Individually, there was no signi cant difference in rates of cerebrospinal uid, respiratory, blood, urine or wound infections.The presence of ileus on ICU day 14 was similarly rare in both groups, and the number of calendar days with NPO status was nearly identical (3.7 days with immunonutrition v 4.0 with standard EN).Those receiving immunonutrition with n-3 PUFA supplementation gained on average 1.4kg over the 2-week period versus a 0.1kg weight loss in the standard EN group, but this difference was not statistically signi cant (p=0.41).Early GCS recovery (day #7 GCS) was similar between study groups (6.0 v 5.8, p=0.58).Both ICU and hospital LOS were decreased in the immunonutrition group, but this was not statistically signi cant.In-hospital mortality was lower in the immunonutrition group, but this was also not statistically signi cant (16.0%versus 19.3%). Clinical outcomes after adjusting for age, gender and non-CNS emergency surgery are displayed in Table 3 and Figure 2. In multivariate linear regression analysis, hospital LOS was reduced in those prescribed immunonutrition with n-3 PUFA supplementation compared to standard EN (35.3 days versus 46.0 days, RR -10.7, 95% CI -19.3, -2; p=0.02).This difference was largest in the polytrauma subgroup (36.3 days v 52.9 days, RR -16.6, 95% CI -28.3, -4.9; p=0.006).ICU LOS was not signi cantly different in the whole study population but was reduced in the polytrauma subgroup who received immunonutrition (27.8 days v35.4 days, RR -7.6, 95% CI -7.6, 95% CI -15, -0.2; p=0.04).In multivariate logistic regression, the risk of inhospital mortality was similar between study groups.The RR of mortality was 0.7 in those prescribed immunonutrition compared to standard EN (10.4% v 15.7%), but this did not reach statistical signi cance (95% CI for RR -0.6, 1.9; p=0.53). DISCUSSION In this retrospective descriptive cohort study of sTBI conducted at a single Level-I trauma center, those who received immunonutrition with n-3 PUFA-containing formulas for EN were slightly younger and more likely to be male, Hispanic/Latinx, have sustained non-CNS injury in addition to TBI, and have undergone emergent non-CNS surgery due to their injuries.The greater likelihood of being prescribed n-3 PUFAcontaining immunonutrition in patients with polytrauma and in those requiring non-CNS surgery is not unexpected, given the high protein requirements believed to be necessary to support increased metabolic demand and promote wound healing after general traumatic injury and surgery 8,14 .Further, the results also suggest that in clinical practice, the decision to prescribe EN with immunonutrition is not based on the presence of trauma alone.Speci cally, older patients, female gender, and those with isolated TBI may not be considered to require similar nutritional interventions compared to younger male patients with polytrauma.Particularly for isolated TBI, this might be an incorrect assumption, as sTBI patients may need up to 200% of their baseline metabolic requirements 15 , and with similarly high rates of malnourishment (68-76%) reported in both polytrauma and TBI populations 8,16 .In addition, given in ammation and infectious risk are universal regardless of trauma type, further clinical study is needed to better de ne the nutritional needs and clinical targets for TBI patients, especially in those at high-risk for malnutrition and poor functional outcomes. Immunonutrition with n-3 PUFA supplementation compared to standard, polymeric EN formula was not associated with any statistically signi cant differences in clinical markers of in ammation and immunity.Speci cally, peak 14-day WBC and CRP, 14-day fever burden and calendar days with fever were nearly identical between study groups.Although the incidence of culture-positive infection was lower in those receiving immunonutrition, this did not reach statistical signi cance (80.9% versus 86.0%, p = 0.42).Given the proposed mechanism of immunonutrition with n-3 PUFA supplementation is to attenuate systemic in ammation and promote immune function, these results might be interpreted as unexpected; however, several circumstances should be considered.These outcome measures were chosen because they are assessable from retrospective medical record review but are likely non-speci c and crude measures of relevant anti-in ammatory activity that may be altered by several confounding factors.The study population was also severely injured (average GCS = 4.8), which may have blunted the effect of the nutritional intervention. The culture-positive infection rate found in this study is higher than many hospital-acquired infection rates reported in moderate-severe TBI 17,18 and hospitalized trauma patients 19 .Both trauma and TBI are thought to increase the risk of in-hospital infection 20,21 , while multiple surgical interventions further increase infection risk 22 .Given the features of our study population, subjects were at high infection risk.Lastly, rates of infection in TBI populations are based on early retrospective report, while true infection risk is likely underestimated.Our reported rate of lower respiratory infection (78.2%) is comparable to recent literature described in a prospective surveillance study (72.2%) 23 .Given nosocomial infection after TBI is associated with worse outcomes 17,21 , the results also highlight the clinical problem of infection risk following TBI and the need to identify novel approaches to prevent this common complication. Despite no identi ed differences in clinical markers of in ammation and immunity, there were important differences in clinical outcomes observed between the two study groups.In multivariate linear regression analysis, EN with immunonutrition and n-3 PUFA supplementation resulted in 10.7 fewer hospital days compared to standard EN (-19.3, -2.0, p = 0.02).In pre-planned subgroup analysis, this appeared to be primarily driven by differences in those with polytrauma (-28.3,-4.9, p = 0.006).Similarly, ICU LOS was signi cantly shorter in the polytrauma subgroup.Although there was no statistically signi cant difference with in-hospital mortality between study groups in multivariate logistic regression analysis, there was a 0.7 rate ratio for mortality with treatment compared to standard nutrition (-0.6, 1.9, p = 0.53).From this study, it is unclear why immunonutrition with n-3 PUFA supplementation is associated with positive outcomes, but the results are su ciently promising to warrant prospective study in clinical e cacy trials in this subgroup of the trauma population.It is possible that immunonutrition with n-3 PUFAs positively impacts energetics and optimizes nutritional status in ways that we were not able to capture in a retrospective study.Particularly for n-3 PUFA supplementation, the pre-clinical data is robust.In rodent models of TBI, eicosanoid n-3 PUFA therapy has been demonstrated to be neuroprotective against in ammation-mediated oxidative stress and pro-apoptotic signaling after TBI [24][25][26] , and can reduce proin ammatory signaling, maintain mitochondrial integrity, and reduce infarct size in stroke models [27][28][29] .Additional amino acid components of immunonutrition, speci cally arginine and glutamine, have been shown to support the synthesis of several cellular proteins vital to oxidative metabolism, immune function, and cellular repair [30][31][32] . Although this was not a prospective study of early EN (< 48-72H from admission), prescription of enteral immunonutrition initiated with a mean of 50.8H after admission in our study population appeared safe. Although a comprehensive evaluation of ICU complications was out of the scope of this study, the use of early EN immunonutrition therapy was not associated with weight loss, increased NPO days, day 14 ileus, or increased infection risk compared to standard therapy.This was an important nding as results from the general ICU population have been con icting regarding the safety of early EN, partly due to a concern for aspiration-related respiratory infections 33 .The capability to resolve in ammation without compromising immune function is one of the positive aspects of n-3 PUFA supplementation and immunonutrition, and because these components are naturally occurring with no known serious adverse effects, their supplementation is considered safe with a large therapeutic window 12 .Early EN may be a clinically important intervention in TBI.It is estimated that every 10 kcal/kg/day increase in energy intake is associated with a 30-40% reduction in mortality risk 34 . Important limitations to this study should be considered.In our retrospective analysis, data quality was dependent on the accuracy of electronic documentation.Given this anticipated limitation, study outcome measures were selected that were objective and were commonly documented as part of standard practice.This study lacked more precise measures of nutritional status, which could not be accurately measured by chart review.Additionally, due to the retrospective nature of the study, we were unable to control all potential sources of bias and effect modi cation.The justi cation of selecting one nutrition intervention versus another was not standardized or documented.Nutrient components also differ between each EN formula, and we could not accurately control for % caloric, protein, fat and carbohydrate needs met in retrospective analysis.Also, those who received EN with immunonutrition including n-3 PUFA supplementation may have received differential management compared to those receiving standard therapy based on the patient characteristics discussed.As with any observational study, correlation does not prove causation.Given the stringent inclusion criteria, the study population was relatively small.A larger study sample would provide a greater power in detecting clinically meaningful differences, especially in subgroups.Future blinded, randomized trials are needed to elucidate the direct impact of immunonutrition formulas on the in ammatory response and short and long-term patient outcomes after moderate-severe TBI.Regarding generalizability, our US-based study population represented a very severe TBI cohort with a large proportion of Latinx and male subjects and may not be fully generalizable to other patient populations and regions of practice.The practicality of the intervention may also be in uenced by the availability of speci c enteral nutrition products in different sites; however, n-3 supplementation products are widely available in many countries and are relatively low-cost. The emerging science in both pre-clinical and human populations of both n-3 PUFA supplementation and other amino acid immunonutrition warrants further study in prospective, pragmatic, randomized clinical e cacy trials.Additional correlations with biomarkers will be helpful in describing the molecular mechanisms as how immunonutrition improves clinical outcomes and to optimize proposed nutritional interventions.If mechanistic activity and positive impact on clinical outcomes can be demonstrated in clinical study, routine incorporation of n-3 PUFA supplementation and immunonutrition with EN would be a novel approach to optimize ICU nutritional status and promote recovery after moderate or severe TBI in thousands of patients annually.Products that provide enriched amounts of n-3 PUFAs and amino acids are already in clinical practice, are widely available, and are relatively low-cost, supporting their rapid translation and implementation into standards of ICU practice if bene t can be shown.Guideline recommendations do provide some support for these interventions in general ICU populations 35,36 , but further rigorous study is needed in patients with TBI 37 . CONCLUSIONS This study identi es patient characteristics of those who received immunonutrition EN with n-3 PUFA supplementation compared to standard, polymeric formulas.The use of immunonutrition was associated with important differences in hospital LOS and ICU LOS; however, the cause of this observed difference cannot be concluded from this study.Together, this suggests that there are potentially meaningful differences in patient characteristics and outcomes associated with the type of enteral feeding provided after severe TBI.A prospective, pragmatic study of the intervention is warranted to con rm the biologic mechanisms of treatment and elucidate their impact on patient outcomes.Our results directly inform the future design of such studies in TBI and other acute neurologic disorders.Given the burden of disease and limited medical treatment options after moderate-to-severe TBI, novel approaches are urgently needed. Declarations Table 2: Unadjusted Outcomes by Study Group Results are displayed as means+SD for continuous variables and % for categorical variables.CRP was analyzed in 63 patients with data available, 38 in the immunonutrition group and 25 in the standard group.WBC: white blood cell count, 10 9 /L; CRP: C-reactive protein, mg/L; NPO: nothing-by-mouth, no enteral nutrition given; Wt: weight; GCS: Glasgow Coma Scale score; SNF: skilled nursing facility; LOS: length-of-stay; ICU: intensive care unit. Table 3: Multivariate Regression Analysis of Outcomes in Both the Whole Study Population and Subgroups Results are displayed as rate ratio (95% con dence interval).LOS: length-of-stay; TBI: traumatic brain injury; ICU: intensive care unit. Figures Figure 1 Flow 1 Figures Funding: RAP is supported by the Southern California Clinical and Translational Science Institute UL1TR001855, with additional funding by R25 NS088248 and U01 NS077352.Details:The following manuscript complies with all instructions set forth by 'Neurocritical care Instructions for Authors', last updated June 1 st , 2023.All authorship requirements have been met and all authors have approved of the nal manuscript version.This manuscript has not been published elsewhere and is not under consideration for another journal.Adherence to ethical guidelines and ethical approvals.This retrospective observational study is IRB exempt.Funding support for this article is reported where indicated.A Con icts of Interest form has been submitted.Relevant funding and disclosures are also listed below.A STROBE Guideline Checklist was used in the preparation of the manuscript.Disclosures:APA reports research support from Microvention, Inc., Medtronic Neurovascular, RapidPulse, Inc., and Valencia Technologies.Author Contributions: Conceptualization, RP; writing, reviewing and editing, RP, JP, GK, FT, PN, SC, SY, SL, ML, MM, AA, NS, GS, and PL.All authors have read and agreed to the published version of the manuscript.Supplementary FilesThis is a list of supplementary les associated with this preprint.Click to download.STROBEchecklistv4NECAD2300673.doc Mortality data on CDC WONDER. 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R A Poblete, S Yaceczko, R Aliakbar, 10.3390/biomedicines11092551Biomedicines. 11925512023 Tables Table 1: Characteristics of the Study Subjects at Baseline Results are displayed as means for continuous variables and % for categorical variables. DM: diabetes mellitus; CAD: coronary artery disease; CKD: chronic renal disease; MVA: motor vehicle accident; Ped: pedestrian; SDH: subdural hemorrhage; EDH: epidural hemorrhage; SAH: subarachnoid hemorrhage, CNS: central nervous system; GCS: Glasgow Coma Scale score; Wt: weight; EN: enteral nutrition; Vital AF: Vital Advance Formula TM ; Vital HP: Vital High Protein TM. CVA/TBI: cerebrovascular accident or traumatic brain injury. Pivot TM

Screening and identification of hub genes of scar physique via weighted gene co-expression network analysis MD a ,Shuxian Ma MDXuze Li MD a ,Wenhao Wu MD bPei Zhang MDYanjie Yang MDLining Huang 0000-0001-5754-2706 MDQian Wan 0000-0001-5754-2706 Hebei Medical University. a Department of Anesthesiology, The Second Hospital of Hebei Medical University, Xinhua District, Shijiazhuang City, Hebei Province, China, Xinhua District, Shijiazhuang City, Hebei Province, China. No. 215 West Heping Road, Xinhua District, Shijiazhuang City 050000, Hebei Province, China Li X, Wu W, Zhang P, Yang Y, Huang L, Wan Q. b Intensive Care Unit of Department of Anesthesiology, The Second Hospital of Hebei Medical University, Department of Anesthesiology, The Second Hospital of Hebei Medical University, Ma S, Screening and identification of hub genes of scar physique via weighted gene co-expression network analysis 5B1F4268CB738F561079AB955EDDB75E10.1097/MD.0000000000036077Received: 25 August 2023 / Received in final form: 23 September 2023 / Accepted: 20 October 2023bioinformatichub genesscar physiqueskinweighted gene co-expression network analysis Scar physique refers to the abnormal repair of skin injury in some people, which may easily lead to keloid or hypertrophic scar.However, the mechanism of scar physique is still unclear.GSE108110 was obtained from the gene expression omnibus database.Differently expression genes (DEGs) between normal skin tissue of non-scar physique individuals and normal skin tissue of scar physique individuals were screened by R package "limma".Weighted gene co-expression network analysis was performed to find highly relevant gene modules.Functional annotation of DEGs was made.Protein-protein interaction network was constructed, and the identification and analysis of hub DEGs were performed, including identification of hub DEGs associated with scar diseases, MiRNA of hub DEGs prediction, and functional annotation of miRNA.A total of 1389 up-regulate DEGs and 1672 down-regulate DEGs were screened.weighted gene co-expression network analysis analysis showed that the dendrogram and heatmap were used to quantify module similarity by correlation.The associations between clinic traits and the modules were identified based on the correlation between module and scar physique.Eight common hub genes were obtained.The comparative toxicogenomics database shows common hub genes associated with scar tissue.Gene ontology and Kyoto encyclopedia of genes and genomes analysis were significantly enriched in "fibroblast growth factor receptor signaling pathway", "epidermal growth factor receptor signaling pathway", "G1/S transition of mitotic cell cycle", protein polyubiquitination", and others.The 8 hub genes might be involved in the development of scarring and used as early diagnosis, prevention and treatment of scar physique.Abbreviations: AMPH = Amphiphysin, CACNA1D = calcium-gated channel subunit alpha1 D, CTD = comparative toxicogenomics database, DEGs = differently expression genes, GO = gene ontology, GSEA = gene set enrichment analysis, KEGG = Kyoto encyclopedia of genes and genomes, PPI = protein-protein interaction, PTPRD = protein tyrosine phosphatase receptor type D, WGCNA = weighted gene co-expression network analysis. Introduction Scar physique refers to the abnormal repair of skin injury in some people, which may easily lead to keloid or hypertrophic scar. [1]Patients with scar physique often have abnormal scars due to minor skin injuries, accompanied by abnormal proliferation of local fibroblasts. [2]In patients with scar physique, scars are most commonly located on the chest, shoulder blade and other regions, with pain and itching. [3]What is more, scars may decreased the appearance and functional status of the body, and decreased quality of life in these patients, and even induce depression and other mental diseases. [4,5]However, the mechanism of scar physique, is still unclear.Genetic factors, the mechanisms of transforming growth factor-beta, vascular endothelial growth factor, fibroblast dysplasia, inflammation, and apoptosis may be involved in scar formation. [6,7]Scar physique may have familial hereditary tendency, major histocompatibility complex genes may play an important role in it. [8]Current treatments include surgical resection, scar skin hormone injection and radiotherapy, but the scar can easily recur after excision. [9,10]herefore, it is of great clinical significance to explore the molecular mechanism and to search for molecular targets that may be used in the early diagnosis and specific treatment of scar formation.Bioinformatics analysis technology is widely used to find molecular changes in the occurrence and development of diseases, and it is a reliable means to find diagnosis and treatment targets. Bioinformatics analysis technology is widely used to find molecular changes in the occurrence and development of diseases, and it is a reliable means to find diagnosis and treatment targets. [11]Liu et al [12] found that many molecules were abnormally expressed in scar tissue by bioinformatics analysis, and functional analysis showed that SFRP1 may participate in the occurrence and development of scar by regulating Wnt/ β-catenin, suggesting that SFRP1 may be used as a therapeutic target for scar.In fact there are still many molecules related to the mechanism of scar formation which are worth exploring.This study screened numerous differentially expressed genes (DEGs) between normal skin tissue of non-scar physique individuals and normal skin tissue of scar physique individuals.Simultaneously, gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analysis were performed.And the research used multiple data algorithm to analyze the role of hub gens on scar physique. Material and Methods Data from the gene expression omnibus database We obtained the transcriptome expression profiles GSE108110 (GPL 570, [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array) from the gene expression omnibus database (Table 1).The GSE108110 contained 9 scar physique samples and 9 normal samples. Screening of DEGs The DEGs were screened by R package "limma," the cutoff criteria were P value < .05. Weighted gene co-expression network analysis (WGCNA) WGCNA was an analysis tool, which could describe the patterns of genes between microarray samples and finds highly relevant gene modules. In our study, WGCNA analysis for DEGs were conducted by R package "WGCNA." Functional annotation of DEGs GO analysis cis an ontology widely used in bioinformatics analysis, which contain 3 aspects of biology, biological processes, cellular components and molecular functions.KEGG analysis could provide specific pathways and linking genomic information with higher-order functional information.Gene set enrichment analysis (GSEA) (http://software.broadinstitute.org/gsea/index.jsp) is a computational method that could execute GO and KEGG analysis with a given gene list.Metascape (http://metascape.org) is an online analysis tool that could provide a comprehensive gene list annotation and analysis resource.In our study, the GO and KEGG analysis of red model DEGs were performed by GSEA and Metascpe (P < .05). Construction and analysis of protein-protein interaction (PPI) network search tool for the retrieval of interacting genes (http://string-db.org), an online database, could predict and provide the PPI network after importing the red model DEGs.Cytoscape is an analysis tool, which could provide biological network analysis and 2-dimensional visualization for biologists.In our study, the PPI network and hub genes were construct and analyzed by SRTING database and Cytoscape. Screening the overlap hub DEGs Funrich (http://www.funrich.org/) is a biological analysis software.The hub DEGs was screened by 4 algorithms.And then, the Venn plots were used to intersect the 4 groups hub DEGs to obtain the common hub DEGs. Identification of hub DEGs associated with diseases Comparative toxicogenomics database (CTD database, http:// ctdbase.org/) is a web-based database.The relationship between gene/protein and disease could be predicted by the CTD database.In our study, the relationships between genes products and diseases were analyzed by this database. MiRNA of hub DEGs prediction TargetScan (www.targetscan.org) is an online database that performs predictive analysis and identifies possible mechanisms for co-regulating the expression of hundreds of genes expressed in different cell types.In our study, TargetScan was used to screen for miRNAs that regulate the hub DEGs. Functional annotation of miRNA DIANA-miRPath v3.0 (http://www.microrna.gr/miRPathv3)was an online analyzes tool suite dedicated to conduct functional and pathway enrichment analysis for miRNA.In our study, GO and KEGG pathway enrichment analysis were performed using miRPath V.3 (P < .05). Statistical analysis The analyses in this study were preform by Perl, R software (version 3.6.1)and SPSS 20.0 (IBM, IBM SPSS Statistics).The hub DEGs and their effect on scar physique based on univariate logistic proportional regression analysis was conducted by SPSS 20.0.Finally, the ROC curves were provided by SPSS 20.0 and MedCalc software. Results Identification of DEGs The merged series contained 1389 up-regulate genes and 1672 down-regulate genes (Fig. 1A). WGCNA In our study, WGCNA analysis were conducted by R package "WGCNA."The cluster of patients was shown in Figure 1B.As shown in Figure 1C, a power value of 8 was the lowest power for which scale independence was below 0.9, and this was selected to produce a hierarchical clustering tree of the 3061 genes (Fig. 1D). In addition, the dendrogram and heatmap were used to quantify module similarity by correlation (Fig. 1E).Interactions between these modules were then analyzed (Fig. 1F).The associations between clinic traits and the modules were identified based on the correlation between module and clinic trait (Fig. 1G). Obtain of module DEGs A heat map of some DEGs expressions in the red model (Fig. 1H). Functional and pathway enrichment analysis of DEGs The enrichment results of GO and KEGG analysis of differently expression genes performed by GSEA were mainly enriched in "regulation_of_multicellular_organismal_development," "regulation_of_cell_proliferation," etc. (Fig. 2A and B) (Tables 2 and 3). The enrichment results of GO and KEGG analysis of red model DEGs performed by Metascape were mainly enriched in very-low-density lipoprotein particle assembly," "transmembrane receptor protein tyrosine phosphatase signaling pathway", regulation of potassium ion transmembrane transport," and others (Fig. 2C and E). Construction and analysis of protein-protein interaction network The PPI network of DEGs was constructed by search tool for the retrieval of interacting genes online database and analyzed by Cytoscape software (Fig. 3A).Four different algorithms were employed to identify hub genes and 8 common hub genes were obtained (Fig. 3B).A summary of common hub genes was shown in Table 4.The PPI network of common hub genes was showed in Figure 3C.The heat map of common hub genes (Fig. 3D). Identification of hub genes The CTD database shows common hub genes associated with scar tissue (Fig. 4). Prediction and functional annotation of miRNA associated with hub genes The miRNA that regulate the hub genes were screened out with TargetScan (Table 5).GO and KEGG analysis of miRNA were performed by DIANA-miRPath.GO and KEGG analysis were significantly enriched in "fibroblast growth factor receptor signaling pathway," "epidermal growth factor receptor signaling pathway," "G1/S transition of mitotic cell cycle", protein polyubiquitination," and others (Fig. 5). Statistical analysis The Roc curves of common hub genes were shown in Figure 6.And the common hub genes and their effect on scar physique based on univariate logistic proportional regression analysis (Table 6). Discussion Hypertrophic scar and keloid often show abnormal proliferation of fibroblasts and abnormal expression of inflammatory factors, which is an overreaction of the body to skin injury. [13]hat is more, slight skin lesions can cause scars in people with physical conditions. [14]Related studies have shown that mesenchymal stromal cells as drivers of inflammation. [15]The cell senescence in the skin microenvironment is related to inflammation.With the occurrence of aging, the body's ability to resolve inflammation is significantly reduced, leading to an imbalance between pro-inflammatory and anti-inflammatory. [16]carring can affect normal function and appearance, which in turn can affect the patient's quality of life. [17]In addition, scars can also be life-threatening in some special cases. [18]However, the molecular mechanism of scar development has not been clarified.Carlavan found abnormal changes of inflammatory cells such as T cells and macrophages by comparing the sequencing data and immunohistochemical analysis of scar-prone and non-scar-prone patients.Further analysis suggests relationship exists between the severity of inflammation and the alteration of sebaceous gland structures. [19]What is more, Saint-Jean et al [20] found abnormal expression of IL-2, IL-10, and TLR-4 in normal skin tissues of acne scar patients, suggesting that inflammation and immune response are involved in the occurrence and development of acne scar.However, the treatment and effect of scar are still far from satisfactory.Therefore, it is of great clinical value to find out the molecular mechanism of scar.Through bioinformatics analysis, multiple abnormal genes were found in normal skin tissues of patients with and without scar tissue.We also found a number of hub genes that were significantly correlated with scar physique and scar occurrence.Among them, Amphiphysin (AMPH), calcium-gated channel subunit alpha1 D (CACNA1D), protein tyrosine phosphatase receptor type D (PTPRD), MTSS1 deserves more attention.Compared with the normal skin tissue of non-scar patients, AMPH, CACNA1D, and PTPRD were highly expressed in patients with scar physique, while MTSS1 was low. AMPH is mainly involved in protein binding, phospholipid binding, endocytosis, synaptic vesicle endocytosis, membrane organization. [21]Abnormal expression of AMPH may be involved in the occurrence and development of a variety of diseases.Pant found that AMPH-1 may affect cell membrane transport by regulating aspartic and glutamic acid, suggesting that AMPH-1 plays an important role in maintaining intracellular circulation. [22]What is more, Chen found that knockout of Table 5 A summary of miRNAs that regulate hub genes. Gene Predicted MiR Gene Predicted MiR amph-1 could regulate cell proliferation and apoptosis.Further analysis showed that AMPH-1 might participate in the regulation of cell cycle by activating ERK pathways. [23]Similarly, Yang proved by gene knockout that AMPH might regulate cell proliferation by regulating apoptosis and ERK pathway. [24]In addition, Jiang demonstrated through experiments that AMPH can regulate cell proliferation and apoptosis, and affecting the prognosis of patients, suggesting that AMPH may be used as a disease treatment target. [25]Similarly with the above study, we found that AMPH is highly expressed in people with scar physique.At the same time, a number of algorithms were used to verify the high expression of AMPH in scar people.We speculate that AMPH participates in the occurrence and development of scar by regulating cell cycle, affecting apoptosis, promoting fibroblast proliferation.AMPH may be used as a target for early diagnosis and specific treatment of scar in people with physique.CACNA1D plays a certain role in regulation of voltage-gated calcium channel activity, calcium channel activity, metal ion binding, alpha-actinin binding, calcium ion transport, adenylate cyclase-modulating G protein-coupled receptor signaling pathway. [26]Abnormal expression of CACNA1D may be involved in the progression of a variety of diseases.Villela suggested that changes in the copy number of CACNA1D participate in the pathogenesis of alzheimer disease by regulating Ca 2+ homeostasis and related pathways. [27]What is more, by sequencing children with congenital heart disease, Flanagan found that CACNA1D might be involved in the occurrence of severe hypotension and cardiac defects by regulating calcium channel function. [28]In addition, Silva showed multiple inflammation-related genes in children with down syndrome, and further analysis revealed CACNA1D might play an important role in down syndrome pathogenesis by regulating inflammatory response. [29]Similarity, we found that CACNA1D was highly expressed in people with scar physique and a large number of algorithms were used to verify the high expression of CACNA1D in scar people.We speculate that CACNA1D participates in the occurrence and development of scar by regulating calcium channels, affecting intercellular signal transduction and cell cycle, and regulating inflammation.CACNA1D may be used as a target for early diagnosis and specific treatment of people with scar physique.PTPRD [30,31] mainly participates in protein tyrosine phosphatase activity, transmembrane receptor protein tyrosine phosphatase activity, cell adhesion molecule binding, synaptic membrane adhesion, which has few studies on scar physique.However, the abnormal expression of PTPRD can affect the normal physiological function of the body, and the mechanism of its involvement in the occurrence and development of the disease is mainly studied in the following aspects.Hsu et al [32] , through sequencing analysis of patients receiving bevacizumab chemotherapy, found that PTPRD may participate in chemotherapeutic drug resistance by regulating JAK/STAT signal, and then affect the prognosis of patients.In addition, Saskin found that PTPRD copy number variants may be involved in the development of Ewing saroma. [33]Furthermore, Bae proved low expression of PTPRD in patients with gastric cancer by microarray technology and immunohistochemistry.Further analysis showed that PTPRD may affect angiogenesis by regulating CXCL8, and then affect the prognosis of patients with gastric cancer, suggesting that related molecules are potential therapeutic targets for gastric cancer. [34]We found that PTPRD was highly expressed in people with scar physique.And a mass of algorithms was used to verify the high expression of PTPRD in people with scar physique.We speculate that PTPRD participates in the development of scar by regulating intercellular signal transduction, affecting cell cycle, promoting proliferation, keratinization and fibrosis of cells. MTSS I-BAR domain containing 1 (MTSS1) mainly participate in actin monomer binding, signaling receptor binding, plasma membrane organization, cell adhesion, negative regulation of epithelial cell proliferation. [35]The abnormal expression of MTSS1 may be involved in the development of many diseases.Taylor proved that MTSS1 can affect cell proliferation and migration by regulating cell cycle and protein phosphorylation and affect the prognosis of patients. [36]What is more, Vadakekolathu found that MTSS1 can regulate intercellular adhesion and affect intercellular signal communication, which in turn affects the prognosis of patients, suggesting that related molecules may be used as therapeutic targets. [37]In addition, Liu found that MTSS1 may affect cell proliferation and migration by regulating cell cycle and protein phosphorylation, and then affect disease progression. [38]Similarly, we found low expression of MTSS1 in people with scar physique.At the same time, a large number of algorithms were used to verify the low expression of MTSS1 in people with scar physique.We speculate that MTSS1 can induce scar by affecting cytoduction, regulating cell proliferation and promoting hyperplasia tissue and skin fibrosis.The molecular mechanism of MTSS1 involved in regulating the development of scar remains further explored. Despite the rigorous bioinformatics analysis in this study, there are still shortcomings.This paper only carries out the big data algorithm verification, a large number of clinical samples and animal experiments should be used for comprehensive verification, so as to better understand the molecule mechanisms of scar. Conclusion In summary, bioinformatics technology could be a useful tool to explore the mechanism of the occurrence and development of scar.In addition, there are DEGs between normal skin tissues of people with scar physique and non-scar physique.These molecules may be involved in the development of scarring and used as early diagnosis, prevention and treatment of scar physique. Table 6 The hub genes and their effect on atrial fibrillation based on univariate logistic proportional regression analysis. GENE Figure 1 . 1 Figure 1.Differential expression analysis and WGCNA analysis of the genes in the merged series.(A) Volcano plots of the genes which are different expression (DEGs) between scar physique group and normal group.(B) The cluster of patients with clinical information, red line represents patients with scar physique.(C) The lowest power for which scale independence.(D) Repeated hierarchical clustering tree of the 3061 genes.(E) The dendrogram and heatmap of genes.(F) Interactions between these modules.(G) The associations between clinic traits and the modules.(H) Heatmaps of the 222 DEGs in the Red model.WGCNA = weighted gene co-expression network analysis. Figure 2 . 2 Figure 2. Gene functional enrichment analysis of the red model DEGs by GSEA and Metascape.(A) Gene ontology (GO) analyses by GSEA.(B) Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of by GSEA.(C) Enrichment_GO-KEGG_ColorByCluster analyses by Metascape.(D) Enrichment_ GO-KEGG_ColorByPValue analyses by Metascape.(E) Enrichment_heatmap_HeatmapSelected GO-KEGG analyses by Metascape.DEGs = differently expression genes, GSEA = gene set enrichment analysis. Figure 3 . 3 Figure 3. Relationship between DEGs.(A) Protein-protein interaction (PPI) network, the more the number of connections, the larger of the protein.(B) The common hub genes identified from different algorithm.(C) The common hub genes of protein-protein interaction network.(D) Heat maps of the common hub genes.DEGs = differently expression genes. membrane depolarization during SA node cell action potential GO:0099703 induction of synaptic vesicle exocytosis by positive regulation of presynaptic cytosolic calcium ion concentration GO:0086015 SA node cell action potential CALML4 Calmodulin like 4 GO:0019722 calcium-mediated signaling GO:0019932 second-messenger-mediated signaling GO:0035556 intracellular signal transduction PTPRD nephron tubule epithelial cell differentiation GO:2001013 epithelial cell proliferation involved in renal tubule morphogenesis GO:0072102 glomerulus morphogenesis SH3GLB2 SH3 domain containing GRB2 like, endophilin B2 GO:0005654 nucleoplasm GO:0031981 nuclear lumen GO:0070013 intracellular organelle lumen AMPH = Amphiphysin, CACNA1D = calcium-gated channel subunit alpha1 D, GO = gene ontology, PTPRD = protein tyrosine phosphatase receptor type D. www.md-journal.com Figure 4 . 4 Figure 4. Relationship to scar physique group and normal group related to DEGs based on the CTD database.CTD = comparative toxicogenomics database, DEGs = differently expression genes. -489-3p hsa-miR-135b-5p hsa-miR-135a-5p 7 MTSS1 hsa-miR-206 hsa-miR-1-3p hsa-miR-613 4 CALML4 hsa-miR-330-3p hsa-miR-33a-5p hsa-miR-33b-5p 8 SH3GLB2 hsa-miR-182-5p hsa-miR-1271-5p hsa-miR-96-5p AMPH = Amphiphysin, CACNA1D = calcium-gated channel subunit alpha1 D, PTPRD = protein tyrosine phosphatase receptor type D. Figure 5 . 5 Figure 5. Functional and pathway enrichment analysis of miRNAs which could regulate hub genes.(A) BP analyses (B) CC analyses.(C) MF analyses.(D) KEGG analyses of the miRNAs.BP = biological processes, CC = cellular components, KEGG = Kyoto encyclopedia of genes and genomes, MF = molecular functions. Figure 6 . 6 Figure 6.ROC curves of hub genes. Table 1 A 1 summary of scar physique microarray datasets from different GEO datasets. SeriesPlatformAffymetrix GeneChipNormalScar1GSE108110GPL 570[HG-U133_Plus_2] Affymetrix human genome U133 plus 2.0 array99 GEO = gene expression omnibus. Table 2 2 GO analysis by GSEA. TERM GO = gene ontology, GSEA = gene set enrichment analysis. Table 3 3 KEGG analysis by GSEA. TERMNESRank at maxLeading edgeUp-regulatedKEGG_JAK_stat_signaling_Pathway−0.66757103tags = 100%, list = 46%, signal = 186%KEGG_cytokine_cytokine_receptor_interaction−0.81086124tags = 100%, list = 56%, signal = 223%KEGG_chemokine_signaling_pathway−0.72824124tags = 100%, list = 56%, signal = 224%KEGG_WNT_signaling_pathway−0.9767272tags = 100%, list = 32%, signal = 147%KEGG_histidine_metabolism−0.9492762tags = 100%, list = 28%, signal = 138%Down-regulatedKEGG_mapk_signaling_pathway1.13356437tags = 100%, list = 17%, signal = 119%KEGG_cell_cycle1.01701242tags = 100%, list = 19%, signal = 123%KEGG_cell_adhesion_molecules_CAMS0.83837895tags = 100%, list = 43%, signal = 174%KEGG_adipocytokine_signaling_pathway1.14044546tags = 100%, list = 21%, signal = 126%KEGG_PPAR_signaling_pathway1.14044546tags = 100%, list = 21%, signal = 126% GSEA = gene set enrichment analysis, KEGG = Kyoto encyclopedia of genes and genomes. Table 4 4 A summary of hub genes. 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University of California San 986CE50FF57C0332DA066CA67464E54A Cell cultureHEK293T, iMEFs (immortalized mouse embryonic fibroblasts), NIH3T3, and U2OS cells were cultured in DMEM (Gibco, 10569044 ) supplemented with 10% fetal bovine serum (FBS) and 1x antibiotic-antimycotic (Gibco, 15240062) in a 37°C incubator with 5% CO2.Cells were treated as indicated in the respective figure legends with the following reagents: DL-β- MEM and 6 µL of RNAiMax (Life Technology) were directly added to the wells.After 10 mins incubation, cell suspensions (4x10 5 cells/well) were added to each well and antibiotic-free medium was added to bring the culture volume to 2 mL.After 24 hours, culture medium was replaced with fresh medium containing antibiotics.24 hours later, cells were harvested and the siRNA transfection was repeated as described above.The next day, cells were treated with BHB at the indicated concentrations in each figure and collected for western blot or qPCR analysis.The following Silencer Select siRNA (Thermo Fisher Scientific) were used: s73 (HDAC1, validated), s6495 (HDAC2, validated), s16876 (HDAC3, validated), s3495 (CBP, validated), s4697 and s534247 (p300), s39121 and s39122 (ACSS1), s31745 and s31746 (ACSS2), and s35911 and s35912 (ACSS3).Each siRNA for p300 and ACSS1-3 were validated by qPCR analysis before the experiments and two siRNAs were mixed and used for efficient knockdown. Plasmids Mouse HDAC2 H141A cDNA was amplified from the pcDNA3.1-HDAC2-H141A(115346, Addgene), and using it as a template, mHDAC2 wild-type (WT) cDNA was generated by over-lapping PCR with primers harboring the wild-type sequence.Using the WT cDNA as a template, other mHDAC2 mutants were generated by overlapping PCR with primers harboring each mutation.These amplicons were inserted into pLenti-CMV-GFP-Puro (658-5) (Addgene #17448) using NEbuilder (NEB).Oligo DNA that contained each gRNA sequence encoding human HDAC1 or HDAC2 was inserted into lentiGuide-Puro vector (52963, Addgene) according to the Addgene resource information.The following gRNA sequences were used: g-hHDAC1#1 (CATCCGTCCAGATAACATGT), g-hHDAC1#2 (GGAGATGTTCCAGCCTAGTG), g-hHDAC2#1 (TCAAAGAGTCCATCAAACAC), g-hHDAC2#2 (CCTCCTCCAAGCATCAGTAA).Lentivirus production vectors, pMD2.G and psPAX2 were obtained from addgene. NAX_CAGGS_CAS9_Blasto is from Addgene (#167856) and the ePiggyBac transposon (PBase) vector was a kind gift from Kirstin Meyer (Orion Weiner lab, UCSF) (1).All plasmids used in this study were confirmed by Sanger sequencing or Nanopore sequencing (Plasmidsaurus). Lentivirus production and spinfection HEK293T cells were seeded at 6.6x10 5 cells per a well of 6-well dish.On day 2, cell culture medium was replaced with antibiotic-free medium.Cells were transfected with pMD2.G (0.80 µg), psPAX2 (0.82 µg) and each lentivirus vector (1.87 µg) using 14 µL of PEI Max (24765, Polysciences) and 170 µL of Opti-MEM, according to the manufacturer's instructions. After 24 hours, cell culture medium was replaced with fresh antibiotic-free medium and 1x ViralBoost reagent (6 µL per 3 mL culture; VB100, ALSTEM).After incubating for 48 hours and medium replacement, virus-containing culture medium was collected and centrifuged at 1,500 rpm for 5 mins to pellet any cells.The supernatant was filtered through a 0.45 µm PES filter, aliquoted into 1.5 mL tubes, and stored at -80°C until use. For infection, cells were harvested by trypsinization, pelleted by centrifugation at 1,500 rpm for 5 mins, and resuspended at 1.5x10 6 cells/mL in medium containing 1 µg/mL hexadimethrine bromide (also known as polybrene; H9268, Sigma-Aldrich).The pre-determined amount of virus-containing culture (approximately 0.75x10 6 transduction unit) was added to each well of 12-well dish.Antibiotic-free medium containing 1 µg/mL polybrene was added to bring the final volume to 1 mL, followed by addition of the cell suspension (1 mL, 1.5x10 6 per well).The 12-well plate was centrifuged at 700 xg for 2 hours at 30°C and incubated at 37°C CO2 incubator overnight.On the following day, infected cells were replated to a 6-well dish with puromycin (1 µg/mL) for drug selection. CRISPR/Cas9-mediated gene knockout in cells HEK293T cells were reverse-transfected with NAX_CAGGS_CAS9_Blasto and PBase vectors using lipofectamine 3000 (Life technology) according to the manufature's protocol. Cas9-integrated populations were selected with blastcidin at 10 µg/mL.Single cells were isolated using limited dilution to isolate Cas9-expressing HEK293T clones.Cas9+ HEK293T cells were reverse-transfected with each gRNA-expressing vector using Lipofectamine 3000 and selected with puromycin at 2 µg/mL and blastcidin at 10 µg/mL.After confirming the efficient reduction of target protein in bulk cells by western blot, single clones for each gRNA were cloned and tested. Western blot analysis Western blot analysis was performed as described previously (2).Cells were harvested after trypsinization and quenching with medium, washed once with PBS, and lysed with RIPA buffer (Thermo Scientific) supplemented with protease and phosphatase inhibitors (Thermo Scientific).The cell lysates were sonicated using a Branson Sonifier 450 until the viscosity of lysates disappeared and then centrifuged at 14,000 rpm (17,968 xg) at 4°C for 5 mins to clarify cell lysates.Protein was quantified with the DC Protein Assay Kit (BioRad Laboratories).For liver and kidney samples, frozen tissues were homogenized in RIPA buffer supplemented with protease and phosphate inhibitors by Bead Mill 24 Homogenizer (Fisher Scientific) with the following parameters: S=4.00, C=04, T=0.30 s, and D=0.10.After centrifugation at 1,500 rpm at 4°C for 5 min, the supernatants were transferred to a new 1.5 mL tube and SDS-PAGE samples for liver and kidney were then prepared as described for cells.SDS-PAGE samples were prepared by mixing clarified lysates wth 4x Laemmli buffer/10% β-mercaptoethanol (BME) (BioRad Laboratories) and then boiled at 95°C for 5 mins. Equivalent amounts of protein were resolved by SDS-PAGE and then transferred to a nitrocellulose membrane using the Trans-Blot Turbo Transfer system (BioRad Laboratories). Membranes were stained with Ponceaus S, blocked with 5% milk/TBS-T (Tris Buffered Saline/0.1% Tween-20) for 30-60 mins, and incubated with primary antibodies, followed by secondary HRP-conjugated antibodies.Blots were developed using a chemiluminescent substrate (SuperSignal™ West Pico PLUS or SuperSignal™ West Femto Maximum Sensitivity Substrate; Thermo Scientific) and imaged with an Azure 300 (Azure Biosystems).Ponceau S staining was used to confirm equal total protein loading across samples. Antibodies The following antibodies were used in this study: anti-Kbhb (monoclonal, PTM Biolabs, PTM-1201RM) which is mainly used in this study, anti-Kbhb (polyclonal, PTM Biolabs, PTM-1201) which is only used in Figure S1, anti-Kac antibody mix (CST, 9814S), anti-Kbu (PTM Biolabs, PTM-301), anti-Kpr (PTM Biolabs, PTM-203), anti-Kla (PTM Biolabs, PTM-1401RM), anti-HDAC1 (CST, 5356), anti-HDAC2 (CST, 5113), anti-HDAC3 (CST, 3949), anti-FLAG (Sigma-aldrich, F3165), Goat anti-Rabbit IgG HRP (Thermo Scientific, 31460), Goat anti-Mouse IgG HRP (Thermo Scientific, 62-6520), Mouse TrueBlot® ULTRA: Anti-Mouse Ig HRP (ROCKLAND, 18-8817-31), and Rabbit TrueBlot®: Anti-Rabbit IgG HRP (ROCKLAND, 18-8816-31). RNA extraction and qPCR analysis Total RNA was isolated using RNeasy micro kit (Qiagen, 74034) according to the manufacturer's protocol.cDNA was generated from equal amounts of RNA using iScript cDNA synthesis kit (Bio-rad, 1708891).Quantitative PCR (qPCR) with Power SYBR™ Green PCR Master Mix (Applied Biosystems™, 4367659) was carried out using CFX384 real-time PCR detection system (Bio-rad).The primer sequences are provided in Table S3. Sample preparation for targeted metabolomic analysis Cells were seeded at a density of 1.0x10 6 cells/well in a 6-well dish.Six wells were used as technical replicates for each condition.After one day of culture, BHB, butyrate, or BHB+butyrate, all at 5 mM, was directly added to the culture medium.After 24 hours, culture medium was removed and 0.9% NaCl was added.Cells were harvested by scraping, and 1.0x10 6 cells were transferred to a collection tube (2 mL polypropylene screw top vials).After centrifugation at 1,500 rpm to remove the supernatant, each cell pellet was resuspended in 0.4 mL ice-cold LC-MS grade methanol, and tubes were immediately placed on ice.After a 5 minute incubation, 0.4 mL ice-cold LC-MS grade water was added to each tube, and samples were stored at -80°C until further processing. Development of Butyryl-CoA and BHB-CoA multiple reaction monitoring signals Multiple reaction monitoring ion pairs were established for butyryl-CoA and BHB-CoA from synthetic standards at method appropriate flow rate and buffer conditions on a Sciex 5500 QTRAP® mass spectrometer.For butyryl-CoA the following daughter ions were used for negative mode detection in combination with the parent mass (836.0):489.0, 426.0, 408.0, 79.0. For BHB-CoA the following daughter ions were used in negative mode in combination with the parent mass (852.0):505.0, 426.0, 408.0, 79.0.Resolution of BHB-CoA from isobaric malonyl-CoA was ensured chromatographically (3). Metabolite and Lipid Sample Preparation For all LCMS methods, LCMS grade solvents (Thermo Fisher Scientific) were used. Samples were collected in 0.4 mL of ice-cold methanol followed by 0.4 mL of water.Cells were scraped to recover insoluble products and transferred to a microtube.To each sample, 0.4 mL of chloroform was added.Samples were agitated for 30 minutes at 4°C and centrifuged at 16,000 xg for 20 min.400 µL of the top (aqueous) layer was collected.A subaliquot of the aqueous layer was taken for O-benzylhydroxylamine (O-BHA) derivatization of carboxylic acids. The aqueous layer was diluted 5x in 50% methanol in water and prepared for LCMS injection. Short Chain Fatty Acid Derivatization To reliably capture butyrate and increase sensitivity for BHB, samples were derivatized O-BHA according to previously established protocols with modifications (4,5).An aqueous pyridine buffer at 1M pyridine and 0.5 M hydrochloric acid was prepared fresh.A volume of 35 µL of the aqueous metabolite extract was sub-aliquoted and 10 µL of 1M O-BHA in reaction buffer and 10 µL of 1M 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide in reaction buffer were added.Samples were shaken at room temperature for 2 hrs.The reaction was quenched with 50 µL of 0.1% formic acid.Derivatized carboxylic acid compounds were extracted via the addition of 400 µL ethyl acetate.Following mixing and centrifugation 16,000 xg for 5 min at 4°C to induce layering, the upper (organic) layer was collected and taken to dryness under vacuum. Samples were resuspended in 300 µL of water for LCMS injection. Liquid Chromatography Mass Spectrometry Tributylamine was purchased from Millipore Sigma.All samples were separated using a Sciex ExionLC™ AC system and measured using a Sciex 5500 QTRAP® mass spectrometer. For detection of acyl-CoA conjugates signals were collected as part of a method looking at a series of central metabolic metabolites based on previously established methods (6). Samples were injected onto a Waters™ Atlantis T3 column (100Å, 3 µm, 3 mm X 100 mm) and eluted using a binary gradient from 5 mM tributylamine, 5 mM acetic acid in 2% isopropanol, 5% methanol, 93% water (v/v) to 100% isopropanol over 15 minutes.Peaks were compared to synthetic standards retention time and ratios between MRMs.O-BHA-derivatized samples were separated with a Waters™ Atlantis dC18 column (100Å, 3 µm, 3 mm X 100 mm) and eluted using a 6 min gradient from 5-80 % B with buffer A as 0.1 % formic acid in water and B as 0.1 % formic acid in methanol.Butyrate and BHB were detected using MRMs from previously established methods and identity was confirmed by comparison to derivatized standards (4,5). All signals were integrated using MultiQuant® Software 3.0.3.Acyl-CoA's were quantified via the 79.0 phosphate MRM after signal confirmation with all four MRMs for each molecule.Signals were normalized to the total signal across all collected metabolite signals to correct for different sample starting abundances prior to statistical comparison between treatments. Immunoprecipitation (IP) for Kbhb proteome HEK293T cells on 10-cm dish (3 dishes) were treated with 10 mM Na-D/L-BHB for 3 hours, and then harvested by trypsinization and quenching with medium, and washed once with PBS.Approximately 1.0x10 7 cells per tube were lysed with 150 µL SDS-lysis buffer (50 mM Tris-HCl at pH7.5, 1% SDS).The cell lysates were sonicated using a Branson Sonifier 450 and protein concentration was measured with DC Protein Assay Kit (BioRad Laboratories).After adding BME at a final concentration of 0.5% to approximately 16 mg/mL lysates, samples were boiled at 95°C for 5 mins to denature proteins.The denatured proteins were diluted 1:10 with NP-40 lysis buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Nonidet P-40) to reduce SDS concentration.The approximately 1.6 mg/mL lysates (700 µL per sample) were incubated with antibody-conjugated ProteinA/G Dynabeads (Invitrogen, 88802) overnight at 4°C.Three independent control (Normal rabbit IgG, 2729S, CST) and three anti-Kbhb (PTM biolabs, PTM-1201RM) samples were processed in parallel.Resin was washed five times with NP-40 lysis buffer, followed by five washes with 50 mM ABC buffer (0.4% Ammonium Bicarbonate/dH2O), and eluted by incubation in 60 µL of 0.2% RapiGest-elution buffer (50 mM ABC, 0.2% RapiGest SF from Waters) at 50°C for 20 mins.A portion of each elution (1/6 th ) was mixed with 4x Laemmli buffer/10% BME (BioRad Laboratories) and boiled at 95°C for 5 minutes for western blot analysis.The remainder of each elution was stored at -80°C until mass spectrometry analysis. Mass spectrometry analysis (Kbhb proteome) Protein was reduced by adding 5 mM DTT and incubating at 60°C for 30 min.After that, samples were treated with 10 mM iodoacetamide at room temperature for 30 min and digested with 500 ng trypsin (mass spectrometry grade, Promega) for 16 h at 37°C temperature.After this, another 500 ng aliquot of the digestion enzyme was added, and the digestion was allowed to continue for 4 h at 37C.Samples were added 5% formic acid and incubated 37°C for 30 min. Digested material was recovered using C18 ZipTips (Waters), eluted in 50% acetonitrile 0.1% formic acid, evaporated and resuspended in 0.1% formic acid for mass spectrometry analysis.Peptide mixtures were loaded onto a 2 um 75um x 50 cm PepMap RSLC C18 EasySpray column (Thermo Scientific).3-hour water/acetonitrile gradients (2-25% in 0.1% formic acid) were used to elute peptides, at a flow rate of 200 nl/min, for analysis in a Orbitrap Lumos Fusion (Thermo Scientific).MS analysis was performed in positive ion mode.MS spectra were acquired between 375 and 1500 m/z with a resolution of 120000.For each MS spectrum, multiply charged ions ions over the selected threshold (2E4) were selected for MSMS in cycles of 3 seconds with an isolation window of 1.6 m/z.Precursor ions were fragmented by HCD using a relative collision energy of 30.MSMS spectra were acquired in centroid mode with resolution 30000 from m/z=110.A dynamic exclusion window was applied which prevented the same m/z from being selected for 30s after its acquisition. For peptide and protein identification, peak lists were generated using PAVA in-house software (7).All generated peak lists were searched against the human subset of the SwissProt database (SwissProt.2019.07.31), using Protein Prospector (8) with the following parameters: Enzyme specificity was set to Trypsin, and up to 2 missed cleavages per peptide were allowed.Carbamidomethylation of cysteine residues, was allowed as fixed modification.N-acetylation of the N-terminus of the protein, loss of protein N-terminal methionine, pyroglutamate formation from of peptide N-terminal glutamines, oxidation of methionine were allowed as variable modifications.Mass tolerance was 10 ppm in MS and 30 ppm in MS/MS.The false positive rate was estimated by searching the data using a concatenated database which contains the original SwissProt database, as well as a version of each original entry where the sequence has been randomized.A 1% FDR was permitted at the protein and peptide level. Downstream analyses for Kbhb proteome Obtained protein (peptide) list was further analyzed by SAINT (Significance Analysis of INTeractome) analysis in MAC-tag PPI (http://proteomics.fi/)(9).We considered proteins with SAINT score > 0.66 and log2(Fold Change; FC, Kbhb vs Mock) >1 as Kbhb-IP-enriched proteins.This SAINT score corresponds to an estimated protein-level Bayesian false discovery rate (FDR) of <0.05.The SAINT score and log2FC plot was generated using "ggplot2" package in R (ver.4.2.2).Using the "clusterProfiler" package in R, we performed redundancy-reduced gene ontology (GO) analysis on the Kbhb-enriched-protein list and used the top 20 enriched GO terms for biological processes and cellular compartment for visualization.Localization information for our Kbhb proteome was obtained from the human protein atlas (https://www.proteinatlas.org/)whenever possible and counted in R. We only considered "Subcellular.main.location"but ignored "Subcellular.additional.location"for this analysis.The nested pie chart for the localizations was generated in Excel (Microsoft) and edited in Affinity Designer 2 (Affinity).Two subcellular localizations "Midbody ring" and "Mitotic spindle" were too few (only 1 protein for each) to be shown on the chart so they are not depicted despite being included in the analysis.We used STRING analysis on our Kbhb proteome, and HDAC1/2realted proteins ("physical direct association") were identified based on the "experimental evidences and database".STRING analysis (https://string-db.org/) for the HDAC1/2-related proteins was re-run, and the network was visualized.The resulting image was slightly edited in Affinity Designer 2 (Affinity) to change font sizes and colors. Sequence alignment and phylogenic tree generation for HDACs All human HDAC sequences were retrieved from Uniprot (https://www.uniprot.org/)and aligned by CLUSTAL W with the default setting in MEGA11 (10).A phylogeny tree was created with the alignment using the Maximum likelihood method with the Bootstrap test (n=500) in MEGA11. In vitro acylation assay with recombinant HDACs Most in vitro reconstitution experiments used recombinant proteins from Cayman: rHDAC1 (10009231), rHDAC2 (36419), rHDAC3/NCOR2 (10009232), rHDAC4 (10009652), rHDAC5 (10009379), rHDAC6 (10009465), rHDAC8 (19380).rHDAC2 protein from Activ Motif (31505) was used for experiments shown in Fig. S6, and rHDAC2 from BPS Bioscience (50002) was used for Fig. 2G-I and Fig. S7.We selected vendors based on the availability of recombinant proteins from each company at that time and observed no difference in rHDAC2 acylation activity among the vendors.Recombinant Histone H3.1 (rH3) was from NEB (M2503S).In most experiments, 1 µg rHDAC and/or 1 µg rHDAC was incubated in reaction buffer (50 mM Tris-HCl pH 7.5) with either (R)-3-Hydroxybutyric acid (R-BHB) (54920, Sigma-Aldrich), DL-β-Hydroxybutyric acid sodium salt (Na-BHB) (H6501, Sigma-Aldrich), sodium butyrate (B5887, Sigma-Aldrich), sodium acetate (S2889, Sigma-Aldrich), sodium propionate (P1880, Sigma-Aldrich) or sodium L-lactate (71718, Sigma-Aldrich) for the indicated time (typically 30-60 mins) at 37°C.In the pH test experiments, 50 mM HEPES-KOH was used as the reaction buffer instead of 50 mM Tris-HCl; no difference in β-hydroxybutyrlyation activity was observed in HEPES versus Tris buffers.Reactions were performed in 8-strip tubes in a thermal cycler (ProFlex PCR system, applied biosystems) in a final reaction volume of 15 µL.Reactions were stopped by adding 4x Laemmli buffer or acetic acid (final.0.15%) for western blot or massspectrometric analysis, respectively. Mass spectrometry analysis for in vitro acylated proteins For propionylation equal amounts of protein were taken across the samples.Samples were dried under vacuum via SpeedVac before being resuspended in 30 µl of 50 mM of tri-ethyl ammonium bicarbonate buffer (TEABC) pH 8.5.To this solution was added 12 µl of propionylation reaction mixture (propionic anhydride and isopropanol in a 1:3 ratio) and the pH adjusted to pH 8 with NH4OH.The reaction was incubated at 37°C for 15 minutes, concentrated to dryness by SpeedVac and repeated twice.Finally, the samples were dried under vacuum via SpeedVac. Histones were digested using Trypsin/Lys-C protease mix in 30:1 enzyme: protein ratio. Vacuum dried samples were resuspended in 30 µl of 50mM (TEABC) pH 8.5, pH was adjusted to 8.5 by 1.5 M Tris buffer.Digestion was performed by incubating samples at 37°C overnight. The reaction was stopped by adding 0.1% formic acid.Digested peptides were concentrated to dryness by SpeedVac, reconstituted in 0.1% formic acid and desalted using C18 Stage-Tips. The C18 cleaned peptides were analyzed on Thermo Scientific Orbitrap Exploris 240 mass spectrometer interfaced with Thermo Scientific UltiMate 3000 HPLC and UHPLC Systems.Peptide digests were reconstituted in 0.1% formic acid and were separated on an analytical column (75 µm × 15 cm) at a flow rate of 300 nL/min using a step gradient of 1-25% solvent B (0.1% formic acid in 100 % acetonitrile) for the first 100 minutes and 25-30% for next min.The mass spectrometer was operated in data-dependent acquisition mode.A survey full scan MS (from m/z 400-1600) was acquired in the Orbitrap with a resolution of 6000.Data were acquired in topN with 13 dependent scans.Fragmented using normalized collision energy with 35 % and detected at a mass resolution of 60,000.Dynamic exclusion was set for 8 s with a 10 ppm mass window. MS/MS searches were carried out using SEQUEST search algorithms against a custom made database for human histones using Proteome Discoverer (Version 3.0, Thermo Fisher Scientific, Bremen, Germany).The workflow included Spectrum files, Spectrum selector, SEQUEST search nodes, Target decoy PSM validator, IMP-ptmRS, peptide validator, event detector, precursor quantifier.Oxidation of methionine, propionylation at lysine, beta-hydroxybutyrylation at lysine and N-terminal protein acetylation were used as dynamic modifications.MS and MS/MS mass tolerances were set to 10 ppm and 0.05 Da, respectively.Trypsin/Lys-C was specified as proteases and a maximum of two missed cleavage was allowed.Target-decoy database searches used for calculation of false discovery rate (FDR) and for peptide identification FDR was set at 1 %.Feature mapper and precursor ion quantifier were used for label-free quantification. FLAG immunoprecipitation followed by In vitro acylation and in vitro deacetylation assay HEK293T cells (80-90% confluent) on a 10-cm dish (1 dish per sample) were harvested by trypsinization and quenching with medium and washed once with PBS.Cells were lysed with 1000 µL High-Salt NP-40 lysis buffer (50 mM Tris-HCl at pH 7.5, 500 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Nonidet P-40, 1x Halt Protease Inhibitors).The cell lysates were sonicated using a Branson Sonifier 450 and clarified by centrifugation at 14,000 rpm for 5 minutes.The clarified lysates were incubated with 15 µL FLAG M2 beads (M8823, Sigma) for 2 hrs at 4°C.The resins were then washed four times with the High-Salt NP-40 lysis buffer, followed by two additional washes with 50 mM Tris-HCl pH 7.5, and resuspended in 300 µL Tris-HCl pH7.5.The 200 µL of IPed samples was used for in vitro lysine β-hydroxybutyrylation reconstitution assay and the remainder (50 µL per well, duplicate) was used for in vitro deacetylation assays. For β-hydroxybutyrylation assays, IP'ed beads containing immobilized HDAC2 were transferred to a 8-strip tube and the buffer was completely removed.All the reagents other than rHDAC2 was added to the 8-strip tubes and in vitro acylation was performed as described above.For in vitro deacetylation assay, IP'ed beads were transferred to a 96-well half area assay plate (3994, CORNING) and the buffer was completely removed.Deacetylation assay with fluorescent molecule-conjugated peptide was performed using HDAC Fluorometric Activity Assay Kit (10011563, Cayman), according to the manufacture's protocol.The reaction volume was 85 µL and deacetylation reaction was performed for 30 mins.The fluorescent signals in duplicates were detected by plate reader at excitation=350nm and emission=460nm (SpectraMax iD5, Molecular Devices).Relative IP'ed 3xFLAG-mHDAC2 amounts were estimated based on in-parallel anti-FLAG western blot analysis and used for the normalization toward both in vitro lysine β-hydroxybutyrylation activity and in vitro deacetylation activity. FLAG immunoprecipitation followed by western blot analysis 3xFLAG-mHDAC2 was immunoprecipitated as desceibed above with the modification of using 10 µL FLAG M2 beads and Normal-Salt NP-40 lysis buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Nonidet P-40, 1x Halt Protease Inhibitors).IPed proteins were eluted in 10 µL SDS-Elution buffer (50 mM Tris-HCl at pH 7.5, 0.2% SDS) by incubating at 60°C for 10 mins.Eluted proteins were further processed by adding 5 µL 4x Laemmli SDS sample buffer and immediately boiling at 95°C for 5 mins.Proteins were analyzed by western blotting. Preparation of inactivated whole cell lysates and high acetylated histones from HEK293T cells for in vitro acylation For whole cell lysates, HEK293T cells were harvested and washed with PBS.Approximately 1.0x10 6 cells were lysed with 100 uL of Buffer 1 (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 5 mM MgCl2, 0.5 mM ZnCl2, 1 mM DTT, 1xHalt Protease inhibitor).Cell lysates were sonicated using a Branson Sonifier 450, incubated at 60°C for 30 mins to inactivate endogenous HDACs, and centrifuged at 14,000 rpm for 5 min to pellet the insoluble proteins.Protein content in the supernatants (soluble protein fraction) were quantified with the DC Protein Assay Kit (BioRad Laboratories), and 5 µL of the inactivated whole lysates were used for in vitro acylation assays. To generate highly acetylated histones, we treated HEK293T cells on a 10-cm dish (approximately 50% confluency) with 2 µM TSA overnight.Cells were harvested and washed with PBS once, and 5.0x10 6 cells were transferred to a new 1.5 mL tube.Cell pellets were lysed in 1 mL of PBS-T (1% Triton-X/PBS/1xHalt protease inhibitor) on ice for 10 mins.After centrifugation at 10,000 rpm for 1 min at 4°C, supernatant was completely removed and the pellet was washed in PBS-T again.Cell pellets were resuspended in 200 µL of extraction buffer (0.5N HCl/10% glycerol in dH2O) and incubated on ice for 30 mins.After centrifugation at 14,000 rpm for 5 min at 4°C, the supernatant was transferred to a new 1.5 mL tube and mixed with 200 uL of 1M Tris Base (pH not adjusted) to neutrize pH.The extraction buffer was replaced with histone storage buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 mM EDTA, 1 mM DTT) using an Amicon filter tube (10 kDa, 0.5 mL; UFC501024, Millipore).Final protein concentration was measured with the DC Protein Assay Kit (BioRad Laboratories) and approximately 2.5 µg extracted histones and 0.25 µg rHDAC2 were used for the assays. In vitro acylation with acyl-CoA For cell lysates, HEK293T cells were harvested and washed with PBS.Approximately 5.0x10 6 cells were lysed with 500 uL of Buffer 2 (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1xHalt Protease inhibitor).Buffer 2 with a pH of 7.5 was also used for BHB-CoA.Cell lysates were sonicated using a Branson Sonifier 450, and centrifuged at 14,000 rpm for 5 min to pellet the insoluble proteins.The lysates were incubated with acetyl-CoA (A2141, Sigma-Aldrich), BHB-CoA (H0261, Sigma-Aldrich), or butyryl-CoA (B1508, Sigma-Aldrich) at 1 mM or 5 mM at 37°C for 2 hours. For purified histone H3.1 (M2503S, NEB) or bovine serum albumin (BSA, A9647, Sigma-Aldrich), each protein was incubated in Buffer 2 (with the indicated pH) with either BHB (200 µM), BHB + CoA (200 µM, C3144, Sigma-Aldrich), or BHB-CoA (500 µM) at 37°C for 2 hours. Stastical analyses Statistical analyses were performed in GraphPad Prism (version 10) unless otherwise indicated.For comparison of two groups, unpaired t-test was used.For comparison of multiple test conditions to a control group, one-way analysis of variance (ANOVA) with post-hoc Dunnett's test was used.For multiple comparisons between all groups, one-way ANOVA with post-hoc Tukey's test was used.For multiple comparisons involving two factors, two-way ANOVA with post-hoc Sidak's test was used.Correlation analysis was performed using linear regression.Analysis information for each figure is indicated in the respective figure legends.For all experiments, p <0.05 was considered significant.*p<0.05,**p<0.01,***p<0.001,****p<0.0001.We performed experiments at least twice independently to confirm reproducibility of the results shown in this study.Hydroxybutyric acid sodium salt (Na-BHB, H6501, Sigma-Aldrich), (R)-3-Hydroxybutyric acid (R-BHB, 54920, Sigma-Aldrich), sodium butyrate (B5887, Sigma-Aldrich), Trichostatin A (TSA, T8552, Sigma-Aldrich), Suberoylanilide Hydroxamic Acid (SAHA, 10009929, Cayman), MS-275 (14043, Active Motif), Triacsin C (ab141888, Abcam), A485 (63875, Tocris).Supplemental FiguresFigure S1. An efficient and reversible transposable system for gene delivery and lineage-specific differentiation in human embryonic stem cells. A Lacoste, F Berenshteyn, A H Brivanlou, Cell Stem Cell. 52009 Non-specific recognition of histone modifications by H3K9bhb antibody. T Tsusaka, 202326107235 Simultaneous quantification of malonyl-CoA and several other short-chain acyl-CoAs in animal tissues by ion-pairing reversed-phase HPLC/MS. L Gao, J Chromatogr B Analyt Technol Biomed Life Sci. 8532007 Fast quantification of short chain fatty acids and ketone bodies by liquid chromatography-tandem mass spectrometry after facile derivatization coupled with liquid-liquid extraction. M Zeng, H Cao, J Chromatogr B Analyt Technol Biomed Life Sci. 10832018 A rapid derivatization based LC-MS/MS method for quantitation of short chain fatty acids in human plasma and urine. A Jaochico, D Sangaraju, S K Shahidi-Latham, Bioanalysis. 112019 A pH and solvent optimized reverse-phase ion-paring-LC-MS/MS method that leverages multiple scan-types for targeted absolute quantification of intracellular metabolites. D Mccloskey, J A Gangoiti, B O Palsson, A M Feist, Metabolomics. 112015 A data processing pipeline for mammalian proteome dynamics studies using stable isotope metabolic labeling. S Guan, J C Price, S B Prusiner, S Ghaemmaghami, A L Burlingame, M111 010728Mol Cell Proteomics. 102011 Role of accurate mass measurement (+/-10 ppm) in protein identification strategies employing MS or MS/MS and database searching. K R Clauser, P Baker, A L Burlingame, Anal Chem. 711999 Analyzing protein-protein interactions from affinity purification-mass spectrometry data with SAINT. H Choi, Curr Protoc Bioinformatics Chapter. 8232012 MEGA11: Molecular Evolutionary Genetics Analysis Version 11. K Tamura, G Stecher, S Kumar, Mol Biol Evol. 382021

An engineered glioblastoma model yields novel macrophage-secreted drivers of invasion Erin A Akins University of California Berkeley University of California San Francisco Graduate Program in Bioengineering BerkeleyCAUSA Department of Bioengineering University of California 94720Berkeley, BerkeleyCAUSA Dana Wilkins University of California Berkeley University of California San Francisco Graduate Program in Bioengineering BerkeleyCAUSA Department of Bioengineering University of California 94720Berkeley, BerkeleyCAUSA Manish K Aghi Department of Neurosurgery University of California San Francisco UCSF Sanjay Kumar skumar@berkeley.edu University of California Berkeley University of California San Francisco Graduate Program in Bioengineering BerkeleyCAUSA Department of Bioengineering University of California 94720Berkeley, BerkeleyCAUSA Department of Chemical and Biomolecular Engineering University of California 94720Berkeley, BerkeleyCAUSA An engineered glioblastoma model yields novel macrophage-secreted drivers of invasion DDB9DCA7E72A92C0BE1D8CBD235FD91610.1101/2023.11.18.567683 Glioblastomas (GBMs) are highly invasive brain tumors replete with brain-and bloodderived macrophages, collectively known as tumor-associated macrophages (TAMs).Targeting TAMs has been proposed as a therapeutic strategy but has thus far yielded limited clinical success in slowing GBM progression, due in part to an incomplete understanding of TAM function in GBM.Here, by using an engineered hyaluronic acidbased 3D invasion platform, patient-derived GBM cells, and multi-omics analysis of GBM tumor microenvironments, we show that M2-polarized macrophages stimulate GBM stem cell (GSC) mesenchymal transition and invasion.We identify TAM-derived transforming growth factor beta induced (TGFβI/BIGH3) as a pro-tumorigenic factor in the GBM microenvironment.In GBM patients, BIGH3 mRNA expression correlates with poor patient prognosis and is highest in the most aggressive GBM molecular subtype.Inhibiting TAM-derived BIGH3 signaling with a blocking antibody or small molecule inhibitor suppresses GSC invasion.Our work highlights the utility of 3D in vitro tumor microenvironment platforms to investigate TAM-cancer cell crosstalk and offers new insights into TAM function to guide novel TAM-targeting therapies. MAIN Glioblastoma (GBM) is the most common, lethal, and aggressive form of primary brain cancer with a medium survival rate of approximately 15 months. 1 The highly invasive nature of GBM complicates surgical resection and promotes recurrence, yet it has been challenging to target invasion therapeutically due to an incomplete understanding of the underlying cellular and molecular mechanisms.Cell invasion is profoundly influenced by interactions between cancer cells and the tumor microenvironment (TME), which consists of non-neoplastic cells as well as extracellular matrix (ECM). 2 Immune cells have emerged as pivotal TME players in tumor development, making them highly attractive targets for therapeutic intervention. 3,4GBMs exhibit a unique immune landscape dominated by tumor-associated macrophages (TAMs), which are composed of multiple subpopulations, including bone marrow-derived (BMD) macrophages and brain-resident microglia. 5,6[12][13][14][15] Macrophage phenotype and function is defined by a polarization state, which has traditionally been described using the M1/M2 polarization model. 16,17TAMs are often associated with the anti-inflammatory M2 polarization state due to their high expression of anti-inflammatory cytokines, scavenger receptors, pro-angiogenetic factors and proteins involved in ECM organization and remodeling. 18The pro-inflammatory M1-like state is more commonly associated with anti-tumor functions such as antigenpresentation and co-stimulation.[21][22] An important and ongoing barrier to understanding TAM regulation of GBM invasion is the lack of in vitro platforms that capture key geometric and mechanical aspects of the GBM TME.Advances in biomaterials and tissue engineering have facilitated the development of 3D TME models. 23,247][28][29][30][31][32] Despite the rapid expansion in 3D brain TME models, integration of macrophages and other immune cells in these platforms remains limited. 33,34re we utilize a 3D HA-based hydrogel platform to test the hypothesis that TAMs establish a pro-invasive microenvironment that facilitates GBM invasion.We first investigate the influence of macrophage polarization state on GBM spheroid invasion across a panel of patient-derived GBM cells.We find that factors secreted by M2polarized macrophages induce GBM invasion.Using a microscale invasion platform that enables regional dissection and characterization of invasive cells, we identify macrophage-induced transcriptional changes in invasive GBM cells.We then characterize the macrophage secretome across polarization states using mass spectrometry, perform a GBM-TAM interaction analysis to identify receptor-ligand pairs driving invasion, and prioritize putative pro-invasive TAM-secreted ligands using singlecell RNA sequencing (scRNA-seq) datasets of human GBM TAMs.We identify two novel TAM-derived secreted factors, BIGH3 and S100A9, that stimulate GSC invasion.We demonstrate that targeting BIGH3 and downstream mammalian target of rapamycin (mTOR) signaling reduces invasion.Overall, our study highlights the utility of engineered 3D platforms to investigate TAM-cancer cell interactions and uncovers novel molecular targets. Results Establishment of a brain-inspired hydrogel platform to study TAM-GBM interactions We engineered a 3D in vitro brain tumor model using our previously published HA-RGD hydrogel formation. 35(Fig. 1a; Ext.Fig. 1a) To study the influence of macrophages on GBM invasion, we performed co-culture tumorsphere invasion assays by encapsulating patient-derived GBM stem cells (GSCs) in HA-RGD hydrogels with THP-1-derived M2polarized macrophages distributed throughout the hydrogel (Fig. 1b-c; Ext.Fig. 1b-e).As monocultures, GSC-295 spheroids are not invasive; however, adding M2-polarized macrophages resulted in a pro-invasive GSC phenotype characterized by long, thin protrusions in the matrix, which we quantified by calculating invasive cell area (See Methods).To test whether M2 macrophages influence invasion through direct or paracrine mechanisms, we performed indirect co-culture assays using M2-polarized macrophage conditioned media (M2 CM).Treatment with M2 CM recapitulated the invasive phenotype seen in direct co-culture, suggesting that GSC invasion did not require macrophage-GSC physical contact (Fig. 1d,e). To examine the effect of polarization state on macrophage-GSC interactions, we treated GSC-295 spheroids with CM collected from monocytes and M1-polarized macrophages.GSC-295 spheroids cultured in monocyte CM (mono.CM) and M1 macrophage CM (M1 CM) were less invasive than GSC-295 spheroids cultured in M2 CM (Fig. 1f,g).These results suggest that in our system, M2 macrophages are the predominant source of proinvasive factors. GBM-Macrophage interaction varies across a panel of patient-derived GBMs To determine whether M2 CM stimulated invasion of additional patient-derived GSCs, we performed tumorsphere invasion assays using a previously characterized panel of patient-derived GSCs spanning three subtype classifications (mesenchymal, proneural, classical). 36The influence of M2 CM on spheroids appeared to be cell line-specific, but the majority of GSC lines (5/6) were highly invasive when treated with M2 CM (Fig. 1h,i; Ext.Fig. 2a-f).Interestingly, GSC-11 spheroids decreased in area when treated with M2 CM (Fig. 1j,k).We confirmed the polarization-specific response and found that like GSC-295, M2 CM drives GSC-268 invasion greater than mono.CM or M1 CM (Ext. Fig. 2g,h). To test whether M2 CM stimulates invasion in patient-derived cells that have not been enriched for GSCs, we performed tumorsphere invasion assays with two GBM patientderived xenograft lines (GBM-43 and GBM-6). 37Interestingly, GBM-43 spheroids decreased invasion when cultured with M2-polarized macrophages or in M2 CM (Ext.Fig. 2i,j).A similar trend was seen in GBM-6 spheroids, although the difference was not statistically different (Ext.Fig. 2k,l). Using GSC-268 spheroids and varied concentrations of M2 CM, we found that M2 CM stimulates invasion in a dose-dependent fashion (Ext.Fig. 2m).To test whether the increased invasion was connected to differences in proliferation, we quantified the percentage of KI-67 positive cells following an invasion assay and found no difference in the percentage of KI-67 cells between GSCs cultured in control GSC media and M2 CM (Ext.Fig. 2n,o). M2-polarized macrophage secreted factors shift GSC transcriptional profiles towards invasion-and mesenchymal-associated signatures To investigate how M2 CM alters the transcriptional profile of GSCs, we performed bulk RNA-sequencing (RNA-seq) on GSCs in hydrogels cultured in GSC maintenance (control) media or M2 CM.We utilized our previously developed microchannel invasion platform, which consists of a cylindrical cell reservoir channel embedded within a 3D hydrogel and enables the physical separation of highly invasive and non-invasive (core) cell fractions from a single device (Fig. 2a). 28,32As expected, GSC-20s in M2 CMtreated devices were more invasive than GSC-20s in control devices (Fig. 2b-c).Following the invasion assay, we micro-dissected the M2 CM-treated devices to isolate the highly invasive GSCs.We performed differential gene expression analysis between cells isolated from control devices, invasive cells isolated from M2 CM-treated devices, and core cells isolated from M2 CM-treated devices.We found invasive and core populations separated along the first principal component (PC1), indicating maximum variance along the invasive phenotype.The second principal component (PC2) generally separated GSCs based on media conditions (M2 CM vs. control media) (Ext.Fig. 3a).We identified ~1000 differentially expressed genes (DEGs) between each sample comparison illustrated in the heat map and volcano plots (Ext.Fig. 3b-f, Supp Table 1). To identify pathways associated with the invasive phenotypes in response to M2 CM, DEGs were used as inputs for pathway enrichment analysis (Fig. 2d-f).M2 CMtreatment led to downregulation of pathways related to Interferon Gamma Response, Interferon Alpha Response, Inflammatory Response and IL-6/JAK/STAT3 Signaling, and upregulation of mesenchymal (MES) pathways including TGF-β Signaling, Apical Junction and Epithelial Mesenchymal Transition (EMT) (Fig. 2d,e).Invasive GSCs were associated with enrichment pathways related to Cholesterol Homeostasis, MYC Targets V1/V2, and Apical Junction (Fig. 2d,f). Interestingly, MES pathways (TGF-β Signaling, Apical Junction and EMT) were upregulated in both the invasive and core populations of M2 CM-treated devices, suggesting that M2 CM treatment induces global upregulation of MES-related genes in GSCs.We further examined bulk RNA-seq expression levels of MES-associated genes across device fractions and culture conditions (Fig. 2g).We also included DEGs associated with the less aggressive proneural (PN) subtype, which has better overall patient survival compared to the MES subtype. 8Compared to GSCs isolated from control devices, GSCs cultured in M2 CM had elevated expression of MES genes and downregulated expression of PN genes.The gene expression changes were seen in both M2 CM-treated core and invasive cells but were larger in the M2 CM-treated invasive cell population.Treatment with M2 CM also led to differential expression of cadherins, including the characteristic MES-associated downregulation of E-cadherin (CDH1) (Ext.Fig. 3g). 38 next explored whether M2 CM induced upregulation of MES genes across multiple patient-derived GSCs.GSC-11 spheroids, which do not invade when cultured in M2 CM, displayed an upregulation of MES-associated genes (CD44, CEBPB, SERPINE, SNAI1) and a downregulation of PN-associated genes (OLIG2, PDGFRA) relative to GSC-11 spheroids in control media (Fig. 2h).GSC-268 spheroids, which do invade when cultured in M2 CM, also displayed an upregulation of MES-associated genes (Fig. 2i).To better understand the association of MES and PN gene signatures with invasion, we compared gene expression levels for PDGFRA (PN marker), SNAI2 (MES marker) and CD44 (MES marker) between GSC-11 and GSC-268.The relative expression of MES marker genes is generally lower in GSC-11 spheroids than GSC-268 spheroids regardless of media condition (Fig. 2j).Additionally, GSC-11 spheroids exhibited higher gene expression levels of the PN marker PDGFRA relative to GSC-268 spheroids, independent of media condition (Fig. 2j). Identification of macrophage-derived pro-invasive proteins using mass spectrometry To directly identify soluble factors secreted by TAMs that may induce MES genes and drive GSC invasion, we characterized macrophage CM with global proteomics.Using size-based exclusion filtration and heat treatment of M2 CM, we first determined that the pro-invasive factor in M2 CM was greater than 10 kDa (Ext.Fig. 4a) and heat-sensitive (Ext.Fig. 4b).From these data, we hypothesized that the pro-invasive factor was a protein and performed mass spectrometry to identify secreted proteins that were more abundant in M2 CM than M1 CM.Mass spectral analysis identified 1093 total proteins, with 207 proteins detected only in M1 CM, 63 proteins detected only in M2 CM, and 823 proteins detected in both M1 and M2 CM (Fig. 3a,b; Ext.Fig. 4d; Dataset 2). As expected, M1 CM had higher abundance of pro-inflammatory proteins (CXCL8, CCL3, CXCL10, CCL5, IFI30) while M2 CM had higher abundance of anti-inflammatory and ECM remodeling proteins (TIMP2, BIGH3, ECM1, ADAM10, CSTHB) (Ext.Fig. 4e).Beyond these conventionally secreted proteins, mass spectrometric analysis identified various unconventionally secreted proteins such as OTUB1, PDIA3, and PGK2 (Fig. 3b).We hypothesized that these detected proteins could have been secreted through extracellular vesicles (EVs).We isolated and purified EVs from M2 CM and performed a tumorsphere invasion assay with full M2 CM, M2 EVs and EV-free M2 CM, referred to as M2 soluble protein fraction (M2 SP).GSC-268 spheroids invaded when treated with M2 SP and M2 CM, but were not invasive when treated with M2 EVs (Fig. 3c-e).GSC-295 spheroids displayed a similar trend and were only invasive when treated with M2 CM or M2 SP (Ext.Fig. 4f,g).From these results, we hypothesized that the pro-invasive factor was not EV-bound and computationally filtered our list of identified proteins to include only conventionally secreted proteins (Fig. 3f). 39 GSC-macrophage interaction analysis identifies putative receptor-ligand pairs driving invasion To identify specific receptor-ligand pairs that may be driving invasion, we created a resource of inferred paracrine crosstalk by mapping the expression of GSC receptors to that of their cognate ligands detected in macrophage CM (Fig. 4a).We identified 97 receptor-ligand pairs of which the receptor is expressed by GSC-20s and the ligand is present in M2 CM (Fig. 4b; Dataset 3).The 97 pairs included 65 unique receptors and 22 unique ligands (Fig. 4c) and although all ligands were detected at higher abundance in M2 CM than M1 CM (Fig. 4d), their relative expression in M2 CM varied (Fig. 4e). Candidate M2 macrophage secreted factors are abundant in human GBM TME and preferentially expressed by TAMs We next investigated the relevance of candidate ligands to human GBM tumors by reanalyzing a publicly available single cell RNA-seq (scRNA-seq) dataset (ref. 40).Since all but one of the 22 ligands identified from our receptor-ligand interaction analysis was a DEG, we focused our analysis on ligands that were predominantly expressed by TAM clusters: ECM1, A2M, APOE, GAS6, BIGH3, S100A9, MMP9, GRN, APOC2, LYZ (Fig. 5a).To predict whether any of these ligands were specific to microglia or BMD TAMs we probed a subclustered version of the same dataset.Every ligand was identified in at least 1 TAM cluster but some ligands were preferentially expressed by either microglia or BMD TAMs (Fig. 5b).Since the majority of TAMs in GBM are of monocytic origin, and CM from polarized human microglia did not drive GSC invasion (Ext.Fig. 5a,b), we prioritized ligands predominantly expressed by BMD TAMs (BIGH3, S100A9 and LYZ).We validated these findings using a second publicly available human GBM scRNA-seq dataset (ref. 41) and found that the top three DEGs upregulated in BMD TAMs were again BIGH3, S100A9 and LYZ (Fig. 5c). We focused further mechanistic investigation on transforming growth factor beta induced (TGFBI/BIGH3), as this secreted extracellular protein was one of the top ligands emerging from the scRNA-seq analysis.We measured BIGH3 mRNA expression levels across a panel of human cells and found that M2-polarized macrophages had the highest BIGH3 mRNA expression levels compared to GBM and microglia lines (Fig. 5d). To determine if BIGH3 mRNA expression varied by glioma grade, GBM subtype, and GBM patient survival, we used GlioVis data portal for visualization and analysis of brain tumor expression datasets. 42In patients with GBM, we found a positive linear correlation between mRNA expression levels of BIGH3 and the monocytic marker CD14 (Fig. 5e).BIGH3 mRNA expression level was highest in GBM tumors compared to lower grade gliomas and non-tumor regions (Fig. 5f) and varied by GBM molecular subtype, with highest expression in patients classified as MES subtype.Furthermore, high BIGH3 gene expression correlated with shorter patient survival (Fig. 5h). TAM-secreted BIGH3 promotes GSC invasion We next tested whether recombinant human BIGH3 (rhBIGH3) was sufficient to drive GSC invasion.We found that while rhBIGH3 induced GSC-268 spheroid invasion (Fig. 6a-b), rhBIGH3 interestingly had no effect on GSC-11 spheroids, which also did not invade with M2 CM (Fig. 6c-d).To test the functional contributions of BIGH3 in M2 CMmediated invasion, we performed invasion assays using a blocking antibody targeting BIGH3 (α-BIGH3).GSC-268 spheroids cultured in M2 CM and α-BIGH3 were less invasive than spheroids cultured in M2 CM with IgG isotype control antibody (Fig. 6e-f). We next explored another top hit from our multi-omics analysis: S100 calcium binding protein A9 (S100A9).GSC-268 spheroids treated with recombinant human S100A9 (rhS100A9) exhibited increased invasion relative to spheroids cultured in control media and this interaction was inhibited using the small molecule S100A9 inhibitor, ABR-238907 (ABR) (Ext.Fig. 5c-d).Interestingly, ABR did not change the invasive capacity of GSC-268 spheroids cultured in M2 CM, so although S100A9 stimulates invasion in our system, it does not appear to be a targetable pro-invasive secreted factor in M2 CM (Ext.Fig. 5e-f). We explored the intracellular signaling pathways activated by BIGH3 that could be driving invasion.BIGH3 is a secreted ECM protein, and its expression is regulated by transforming growth factor beta (TGF-β) signaling. 43According to our bulk RNA-seq analysis, TGF-β signaling and TGFβ1 are both upregulated in highly invasive GSCs (Ext.Fig. 5g).To test the possibility that BIGH3 and M2 CM promotes invasion through TGF-β signaling, we directly induced TGF-β signaling with recombinant human TGFβ1 (rhTGFβ1).The addition of rhTGFβ1 did not change the invasive capacity of GSC-268 spheroids suggesting that the induction of TGF-β signaling with rhTGFβ1 is not sufficient to drive GSC invasion (Ext.Fig. 5h-i). Another pathway upregulated in highly invasive GSCs was the P13K/AKT/mTOR signaling pathway, which is abnormally activated in tumors to promote growth, metastasis and invasion. 44We first asked if mTOR activation was sufficient to drive invasion and found that inducing mTOR signaling with an mTOR activator (MHY1845) induced GSC-268 spheroid invasion (Fig. 6g-h).We next tested whether BIGH3promoted GSC invasion was mediated by mTOR signaling.Indeed, inhibiting mTOR (Temsirolimus) slowed down rhBIGH3-mediated GSC invasion as GSC-268 spheroids treated with rhBIGH3 and Temsirolimus were significantly less invasive than spheroids treated with rhBIGH3 and vehicle control (DMSO) (Fig. 6i-j).We then determined what portion of M2-mediated GSC invasion was driven by mTOR by testing the ability of Temsirolimus to block M2 CM-mediated GSC invasion.We found that GSC-268 spheroids treated with M2 CM and Temsirolimus were significantly less invasive than spheroids treated with M2 CM and vehicle control (DMSO) (Fig. 6k-l).Inhibition of mTOR signaling through Temsirolimus blocked BIGH3-and M2 CM-mediated GSC invasion, suggesting that in our system, M2 CM and BIGH3 stimulate invasion through the mTOR pathway. Discussion Tumor-immune cell interactions within the GBM TME represent a promising and largely untapped set of targets tor limiting disease progression and improving patient outcome.However, progress in identifying these targets is limited by a lack of physiologically mimetic culture platforms that support both tumor cell invasion and incorporation of TAMs.We address this need by developing 3D HA models that enable unbiased multiomics analysis on GBM and TAM populations, which allowed us to identify pro-invasive TAM-secreted factors.We functionally validated two TAM-secreted ligands, BIGH3 and S100A9.BIGH3 has recently been recognized as a key component of the tumor ECM with both tumor suppressor and tumor promoter roles, depending on tumor type and stage. 45While BIGH3 has been proposed as a potential MES subtype signature gene based in part on its ability to promote growth and motility of continuous GBM cell lines, the cellular source of BIGH3 in tumors is unclear. 46,479][50][51][52] Our work provides additional evidence that BIGH3 and S100A9 are active drivers of GBM invasion and suggests that they are primarily derived from the BMD TAM population. 4][55][56] While there have been reports of high M2 TAM accumulation in MES tumors, the mechanism behind this correlation remains unclear, although it has been proposed that MES tumors attract M2 TAMs. 8,57Our work expands on prior studies investigating the relationship between TAM abundance and GBM subtype by proposing the possibility that M2 TAM secreted factors directly induce MES transition in tumor cells.TAMs and GSCs may potentially form a bidirectional feedback loop, where M2 TAMs induce a highly invasive MES state in GSCs, leading to further M2 polarization of TAMs.9][60] In this study, the mTOR pathway emerged as a targetable regulator of GSC-TAM interactions and GSC invasion.Although the exact mechanisms connecting BIGH3, mTOR signaling and MES transition remain unclear, BIGH3 has several known integrin binding motifs. 61,62and mTOR activation has been shown to occur through integrin-dependent mechanisms. 63,64Given the importance of mTOR signaling in cell growth, proliferation, metabolism, and survival, this pathway is potential therapeutic target in many cancers and our data supports ongoing exploration of mTOR pathway inhibition as combination therapy for patients with GBM. 44 exploiting engineered 3D TME models to investigate functional contributions of the TAM secretome on GBM invasion, we show that the effects of TAM secretome on GBM are context-specific and vary by macrophage ontogeny and polarization state, as well as GBM stemness and GSC molecular subtype.BIGH3 and other TAM-secreted factors merit further mechanistic and therapeutic study, including the roles these factors may play in priming the TME for invasion.It is also important to elucidate specific TAM subsets or polarization states beyond the traditional M1/M2 model responsible for the secretion of pro-invasive factors, as well as identifying the TME features driving the presence of these key TAM polarization states and subtypes.Regional variations in TAM-GBM interactions will also be important to study given the microenvironmental differences between the tumor core and edge.Finally, understanding how TAM infiltration and function relates to GBM subtype and tumor recurrence would focus efforts to target TAMs in patients who would most benefit. Methods Me-HA synthesis HA hydrogels were synthesized as previously described. 65Briefly, methacrylic anhydride (Sigma-Aldrich, 94%) was used to functionalize sodium hyaluronate (Lifecore Biomedical, Research Grade, 66 kDa -99 kDa) with methacrylate groups (Me-HA).The extent of methacrylation per disaccharide was quantified by 1 H nuclear magnetic resonance spectroscopy (NMR) and found to be ~85% for materials used in this study.To add integrin-adhesive functionality, Me-HA was conjugated via Michael Addition with the cysteine-containing RGD peptide Ac-GCGYGRGDSPG-NH2 (Anaspec) at a concentration of 0.5 mmol/l. HA hydrogel crosslinking To form hydrogels, 6 wt.% Me-HA conjugated with 0.5 mM integrin binding peptide (RGD) was crosslinked with a protease-cleavable peptide (KKCG-GPQGIWGQ-GCKK, Genscript) in phenol red-free serum-free Dulbecco's Modified Eagle's Medium (DMEM, Thermo Fisher Sci, 21-063-029) containing GBM cell spheroids.[68] Hydrogel rheological characterization Hydrogel stiffness was characterized by shear rheology via a Physica MCR 301 rheometer (Anton Paar) with an 8-mm parallel plate geometry for γ = 0.5% and f = 1 Hz.Frequency was controlled to be between 50 and 1 Hz for the frequency sweep at a constant strain (γ = 0.5%), and the modulus saturation curve with time was obtained under oscillation with constant strain (γ = 0.5%) and frequency (f = 1 Hz).The temperature of the gel solution was controlled (T = 37°C) with a Peltier element (Anton Paar) and the sample remained humidified throughout the experiment. Cell culture GBM-43 and GBM-6 (Mayo Clinic) were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% (vol/vol) fetal bovine serum (Corning, MT 35-010-CV), 1% (vol/vol) penicillin-streptomycin (Thermo Fisher Scientific) and 1% (vol/vol) Glutamax (Thermo Fisher Scientific, 35-050-061).Bulk GBM cell lines (GBM-43, GBM-6) were harvested using 0.25% Trypsin-EDTA (Thermo Fisher Scientific) and passaged less than 30 times.GSC-11, GSC-268, GSC-28, GSC-262, GSC-20 and GSC-295 cells were kindly provided by University of Texas M.D. Anderson Department of Neurosurgery.GSC lines were propagated as neurospheres in DMEM/F12 50/50 1X (Corning, 10-090-CV) supplemented with 2% (vol/vol) B-27 supplement (Gibco), 20 ng/mL EGF (R&D systems, 236-EG), and 20 ng/mL FGF (R&D Systems, 233-FB).All GSC cells were harvested using Accutase cell detachment solution (Innovative Cell Technologies) and passaged less than 20 times.).THP-1 cells were cultured at 3 x 10 5 cells/ml, split when reaching 8 x 10 5 cells/ml, and passaged less than 30 times. Cell line THP-1 cells were differentiated and polarized following previously reported protocols. 70n Brief, cells were differentiated at a concentration of 5 x 10 Cells were seeded at a density of 3x 10 4 cells/cm 2 , split when confluency reached 90%, and passaged less than 20 times. All cells were screened every six months for mycoplasma and validated every year by Short Tandem Repeat (STR) analysis at the University of California Cell Culture Facility. For experiments in hydrogels, 0.5% penicillin and streptomycin (Gibco, 15140122) and 0.125 µg/ml Amphotericin B solution (Sigma-Aldrich, A2942) was added to cell culture media. Conditioned media collection EV purification We used ultracentrifugation and Nanoparticle Tracking Analysis to isolate and characterize M2-polarized macrophage-derived EVs.Conditioned media (CM) was harvested as described above.Microvesicles were removed by centrifugation at 5,000xg for 15 minutes.Supernatant was collected and ultracentrifuged at 140,000xg (28,000 RPM) for 1.5 hours in a SW-28 rotor (Beckman Coulter).The supernatant was collected (soluble protein fraction) and the crude EV pellet was resuspended in 1X PBS and ultracentrifuged at 160,000xg for 2 hours.The supernatant was discarded, and the crude EV pellet was resuspended in 0.02 µm filtered PBS for further characterization. Nanoparticle tracking analysis EV sizes and quantities were estimated using the NanoSight NS300 instrument equipped with a 405-nm laser (Malvern Instruments), analyzed in the scatter mode.Silica 100-nm microspheres (Polysciences) served as a control to check instrument performance.Vesicles collected as described above were diluted 1,000× with 0.02 µm filtered PBS.The samples were introduced into the chamber automatically, at a constant flow rate of 50 during five repeats of 60-s captures at camera level 13 in scatter mode with Nanosight NTA 3.1 software (Malvern Instruments).The size was estimated at detection threshold 5 using Nanosight NTA 3.1, after which "experiment summary" and "particle data" were exported.Particle numbers in each size category were calculated from the particle data, in which "true" particles with track length >3 were pooled, binned, and counted with Excel (Microsoft).M2 CM contained EVs at a concentration of 1.67 x 10 8 ± 7.47 x 10 6 particles/mL with 100-400 nm diameters (mean: 229 ± 89.3 nm, mode: 178.5 nm) according to Nanoparticle Tracking Analysis of concentrated EVs (Fig. 3c). For the invasion assay, EVs were reconstituted in GSC maintenance media at a concentration of 1.67 x 10 8 particles/mL, the mean concentration of EVs in M2 CM. Generation of spheroids Tumorspheres were fabricated using Aggrewell Microwell Plates (Stemcell Technologies).Briefly, 1.2E5 cells were seeded into a single well of the Aggrewell plate to form spheroids consisting of 100 cells after 48 hour incubation at 37˚C and 5% CO 2 . 3D spheroid invasion assay Spheroids were resuspended in phenol red-free serum-free Dulbecco's Modified Eagle's Medium (DMEM, Thermo Fisher Sci, 21-063-029) at a density of 1.5 spheroids/ul and used as solvent for HA hydrogel crosslinking.For direct co-culture experiments, THP-1derived macrophages were added to DMEM solvent for a final concentration of 3.5k cells/µl hydrogel. For invasion analysis in tumorsphere invasion assays, spheroids were imaged every 2 days using Eclipse TE2000 Nikon Microscope with a Plan Fluor Ph1 ×10 objective.Images were acquired using NIS-Elements Software.For each spheroid, total spheroid area was outlined in ImageJ and normalized to total day 0 spheroid area.For experiments with direct co-culture of GBM and THP-1 cells, GSCs were transduced with CAG-GFP lentivirus (Cellomics, PLV-10057-50) and selected with 1 µg/ml puromycin (Invitrogen, A1113803) prior to experiments. Invasion device fabrication Devices were fabricated following a modified version of our previously published invasion device protocol. 28In brief, an acrylic mold was made by laser-cutting and stacking 1.5 mm-thick acrylic posts and acrylic slide (CLAREX Precision Thin Sheet, 1.5 mm, Astra Products) on top of a glass microscope slide (Fisherbrand, 12-550-A3).Next, 0.00695 mm outer diameter cleaning wires (Hamilton, 18302) were inserted into the double channel posts to serve as wire and syringe guides during hydrogel casting and cell seeding.Polydimethylsiloxane (PDMS) was fabricated by mixing a 10:1 mass to mass ratio of Sylgard 184 elastomer with the initiator (Dow Corning) and the mixture was pipetted into the acrylic mold and wire assembly and cured at 80˚C for 2 hours.After curing, the wires, acrylic slide, and acrylic posts were carefully removed.A razor blade with an acrylic guide was used to cut a 5 mm by 75 mm rectangle in the center of the PDMS and the acrylic laser cut lid was attached to the PDMS and glass slide with epoxy.New 0.00695 mm outer diameter cleaning wires were reinserted into the PDMS guides and the entire device was UV-treated for 10 min and stored in cold room prior to use.Laser cutter designs are available upon request. On the day of experiment, devices were brought to room temperature and the HA hydrogel solution was casted within the acrylic molds around the wire and incubated for 1 hour in a humidified 37˚C chamber.After crosslinking, devices with hydrogels and wire were submerged in cell culture media for at least 10 min, before removal of the wire which left an open channel.Afterwards, 100k single cells were seeded into each open channel and the wires were reinserted into the device to plug each end of the cell reservoir.The entire device was placed into a 4-well plate and bathed in 10 ml of medium.Medium was changed every 3 days. For quantification of invasion in devices, cells in devices were imaged every 7 days using Eclipse TE2000 Nikon Microscope with a Plan Fluor Ph1 ×10 objective.Images were acquired and stitched using NIS-Elements Software.For each device, total cell area was outlined in ImageJ and normalized to total day 0 cell area. Device Assembly and RNA extraction For RNA extraction of cells on tissue culture plastic, phenol-free total RNA was extracted from cells directly in cell culture wells using RNeasy Plus Micro Kit with gDNA eliminator columns (Qiagen) following the manufacturer's protocol.For RNA extraction of cells in invasion devices, devices were carefully disassembled, and if applicable, the invasive cells were physically separated from the non-invasive "core" cells based on distance from channel using a scalpel.The invasive cell populations isolated from devices cultured in GSC maintenance media were not large enough to submit for bulk RNA-seq independently and were pooled with the core cell fractions from control devices.For RNA extraction of tumorsphere invasion assays, hydrogels and cells were placed in Eppendorf tubes.All samples were treated with 10k U/ml Hyaluronidase from bovine testes, Type IV-S (Sigma-Aldrich, H3884) for 15 min with agitation until the hydrogel was fully degraded.Afterwards, RNA was extracted using TRIzol Reagent (Invitrogen, 15596018) according to manufacturer's recommendations.In brief, 1M cells were mixed with 100 µl TRIzol Reagent and the solution was vortexed for 30 seconds.20 µL chloroform (Sigma, c2432) was added to the mixture and the solution was placed on ice for 15 minutes.Sample were centrifuged at 14000 rpm for 10 minutes and the clear RNA layer was carefully transferred to a new Eppendorf tube.Each RNA sample was mixed with a 1:1 volume of 2-Propanol (Sigma-Aldrich, 190764-4L) and 0.5 µL Glycogen, RNA grade (Thermo Scientific, R0551).After an overnight incubation at -20˚C, samples were centrifuged at 14000 rpm for 10 minutes at 4˚C and the RNA pellet was rinsed twice by aspirating the supernatant, adding 400 µL 75% Ethanol and centrifuging at 14000 rpm for 1 minute.Following the second rinse, the supernatant was carefully and completely removed, and RNA pellets were incubated at room temperature until visibly dry.RNA was dissolved in 30 µL RNase-free DNase-free distilled H 2 O and RNA concentration and purity was measured with Nanodrop. cDNA synthesis cDNA was synthesized from extracted RNA using iScript cDNA Synthesis Kit (BioRad, #1708891) following the manufacturer's protocol. RNA-sequencing and differential gene expression analysis Isolated RNA was sent to Novogene Corporation Inc. (Sacramento, CA) for library construction, quality control and sequencing.In brief, messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads.After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis.The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification.The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection.Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.Data was filtered to remove low-quality reads and sequences containing adapters and differential expressed genes were identified using DESeq2.Statistically significant differentially expressed genes were chosen based on a statistical cutoff of |Log2FC| ≥ 1 and adjusted p-value < 0.05. Pathway enrichment analysis We used Enrichr [71][72][73] to perform enrichment analysis of gene lists using the Molecular Signatures Database (MSigDB 74 ) hallmark gene set collection.MSigDB Pathways were ranked by Odds Ratio. Receptor-ligand interaction analysis To infer receptor-ligand interactions between GSCs and M1/M2 THP-1 macrophages, we compared our RNA-seq data of GSC-20s and our global proteomics data of M1/M2 macrophage CM to a database of 3631 receptor-ligand interactions 75,76 which was downloaded and analyzed in RStudio (RStudio, Inc., 2022.12.0+353).We first filtered the M1/M2 macrophage CM dataset to identify conventionally secreted proteins using The Human Protein Atlas (Predicted: secreted) database of predicted secreted proteins. 39We then annotated cognate pairs co-expressed by GSC-20s for which the Base Mean transcript expression of the receptor in the GSC-20 + M2 CM (invasive fraction) sample is ≥1.This produced 255 receptor-ligand pairs for which the receptor is expressed by GSC-20s and the ligand is expressed by M1/M2 macrophages. To identify the receptor-ligand interactions dependent on macrophage polarization state, we grouped our inferred receptor-ligand pairs into M2-and M1-specific interactions using Fold Change (FC)=(M2 normalized intensity)/(M1 normalized intensity).FC ≥ 2 indicated M2-specific interactions, 2<FC>0.5 indicated pan-macrophage interactions, and FC≤0.5 indicated M1-specific interactions.This analysis yielded 100 M1-specific interactions, 97 M2-specific interactions, and 58 pan-macrophage interactions (Dataset 3). Acquisition of scRNA-seq datasets Differential expression genes from scRNA-seq analysis of 9 patient GBMs was downloaded from PMID: 34434898 (ref. 77) which obtained 10 X genomics scRNA-seq data from the Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo/)database under GSE131928. 40The sequencing analyses were conducted on samples from nine fresh GBM tumors from adult patients in Massachusetts General Hospital (MGH).Dimensionality reduction, unsupervised clustering, and identification of cell types and marker genes in different clusters were performed as previously described. 77n brief, single-cell transcriptomes for a total of 16,201 cells were retained after initial quality controls and were differentiated into 22 different clusters, which were further sorted into three main cell types by comparing their gene expression characteristics using the following marker genes: malignant cells (GAP43, GPM6B, SEC61G, PTN), tumor-associated macrophages (C1QA, C1QB, C1QC, TYROBP, CD68) and T lymphocytes (CD3G, GZMH, IL2RB, PR1F, and ICOS).Pct.1 represents the percentage of cells where the gene is detected in specific cluster and pct.2 represents the percentage of cells on average in all the other clusters where the gene is detected.Enrichment Score was calculated by dividing pct.1 by pct.2.Clusters 2,3,6,7,9, and 19 represent tumor-associated macrophages, cluster 17 represents T-lymphocytes, and clusters 0,1,4,5,8,10-16,18,20, and 21 represent malignant cells.To comprehensively characterize the signatures of TAMs in GBM, the cells in the macrophage and microglia groups were subclustered and reanalyzed.In total, 5,896 cells were sorted and hierarchically sorted into 14 clusters, as described in ref. 77 .Clusters 4,5,6,7,10 and 11 represent resident microglia TAMs and clusters 0,1,2,3,8,9 and 13 represent bone marrow-derived TAMs. Differential expression genes from human blood-derived and microglial tumorassociated macrophages were downloaded from PMID: 29262845, which performed scRNA-seq on a cohort of 19 total patients comprising 5,455 TAMs (DESeq adj.p value < 1e-3). 41The scRNA-seq datasets were mined for genes encoding proteins that were detected by mass spectrometry in M2 CM and identified in our GSC-macrophage interaction analysis.Data was downloaded and analyzed in RStudio. Acquisition of The Cancer Genome Atlas (TCGA) gene expression TCGA data was queried and downloaded using GlioVis 42 data portal for visualization and analysis of brain tumor expression datasets.Data was graphed and analyzed in Prism GraphPad. Quantitative PCR (qPCR) qPCR was carried out using Applied Biosystems PowerUp SYBR Green Master Mix (Thermo Scientific, A25918) and primers following the manufacturer's recommended protocol in a BioRad CFX Connect Real-Time PCR cycler with a 5 μM final forward and reverse primer concentration.Ct values were calculated using CFX Maestro software accompanying the real-time cycler and relative gene expression was calculated using the 2^(-delta delta Ct) method.Each gene expression was internally normalized by the expression level of housekeeping gene run on the same qPCR batch.Primer sequences were designed using either Primer-BLAST 78 or PrimerBank. 79Primer sequences are listed in Dataset 4. Mass spectrometry sample preparation To prepare Conditioned Media (CM) for mass spectral analysis, M1-and M2-polarized macrophages were cultured in DMEM/F12 50/50 1X (Corning, 10-090-CV) at a density of 1M cells/ml.After 48 hours, CM from M1-and M2-polarized macrophages was collected and centrifuged at 130 g for 5 minutes to remove dead cells and debris.The CM was then added to Amicon Ultra-4 Centrifugal Filter Unit tubes (Millipore, UFC8010-24) and concentrated by centrifugation at 10,000 g for 15 minutes resulting in a 20-fold reduction in volume (V i = 4.0 mL; V f = 0.2 mL). In preparation for mass spectral analysis, samples underwent a trypsin in-solution protein digest followed by sample desalting.Peptides were analyzed in biological triplicate on a ThermoFisher Orbitrap Fusion Lumos Tribid mass spectrometry system equipped with an Easy nLC 1200 ultrahigh-pressure liquid chromatography system interfaced via a Nanospray Flex nanoelectrospray source. Samples were injected on a C18 reverse phase column (25 cm x 75 µm packed with ReprosilPur C18 AQ 1.9 µm particles).Peptides were separated by an organic gradient from 5 to 30% ACN in 0.02% heptafluorobutyric acid over 180 min at a flow rate of 300 nl/min.Spectra were continuously acquired in a data-dependent manner throughout the gradient, acquiring a full scan in the Orbitrap (at 120,000 resolution with an AGC target of 400,000 and a maximum injection time of 50 ms) followed by as many MS/MS scans as could be acquired on the most abundant ions in 3 s in the dual linear ion trap (rapid scan type with an intensity threshold of 5000, HCD collision energy of 32%, AGC target of 10,000, maximum injection time of 30 ms, and isolation width of 0.7 m/z).Singly and unassigned charge states were rejected.Dynamic exclusion was enabled with a repeat count of 2, an exclusion duration of 20 s, and an exclusion mass width of ±10 ppm. Mass Spectrometry analysis Protein identification and quantification were done with Integrated Proteomics Pipeline (IP2, Integrated Proteomics Applications, Inc.San Diego, CA) using ProLuCID/Sequest, DTASelect2 and Census. 80 -83Identified proteins that were detected in only one replicate per sample were filtered out from dataset.Protein fold change was calculated by dividing the average normalized M2 intensity by the average normalized M1 intensity for each protein. Additional reagents Figure 1 : 1 Figure 1: An engineered biomaterials platform to enable investigation of GBMmacrophage crosstalk.a, Schematic of HA-based hydrogel.b, Schematic of mono-or co-culture platforms to study GBM invasion.c, Schematic of THP-1-derived monocyte to macrophage differentiation and M1/M2 polarization protocol.d,e, GSC-295 invasion assay with direct M2 macrophage co-culture and M2 macrophage conditioned media (CM) (d) quantification and (e) representative phase images.f,g, GSC-295 invasion assay with CM from monocytes (mono.),M1 macrophages, and M2 macrophages (f) quantification and (g) representative phase images.h,i, GSC-268 invasion assay with M2 CM (h) quantification and (i) representative phase images.j,k, GSC-11 invasion assay with M2 CM (j) quantification and (k) representative phase images. Figure 2 : 2 Figure 2: GSC transcriptional changes induced by M2 CM-mediated invasion.a, Schematic of HA-based invasion device and microdissection borders (dashed lines). Figure 3 : 3 Figure 3: Mass Spectrometry-based identification of macrophage secreted proteins.a, Venn Diagram illustrating proteins identified by mass spectrometry in M1 and M2 CM. b, Volcano plot of proteins identified by mass spectrometry in both M1 and M2 CM. c, Nanoparticle tracking analysis (NTA) of extracellular vesicles (EV) isolated by ultracentrifugation from M2 CM. d,e, GSC-268 invasion assay with M2 CM, M2 soluble proteins (SP) and M2 EV (d) quantification and (e) representative phase images.f, Volcano plot of conventionally secreted proteins identified by mass spectrometry in both M1 and M2 CM. Figure 4 : 4 Figure 4: Paracrine receptor-ligand interaction analysis of GSCs and M2 macrophages.a, Schematic of Receptor-ligand interaction analysis workflow.b, Quantification of receptor-ligand pair according to ligand expression in M2 CM (x-axis) and receptor expression in GSCs (y-axis).c, Heat map of receptor expression in GSCs and ligand expression in M2 CM for each receptor-ligand pair.d,e, Bar graphs of ligands detected in M2 CM emerging from receptor-ligand analysis graphed to illustrate (d) average protein abundance in M2 CM compared to M1 CM and (e) average relative protein abundance obtained from mass spectrometry. Figure 5 : 5 Figure 5: Integration of M2 macrophage-secreted ligands with published transcriptomic datasets.a,b, Dot plot depicting gene expression profiles of M2 Figure 6 :. 1 :Ext. Fig. 2 :Fig. 3 :Fig. 4 : 61234 Figure 6: M2 macrophage-induced GSC invasion is mediated by secreted BIGH3 and is associated with active mTOR pathway.a,b, GSC-268 invasion assay with 20 ng/ml rhBIGH3 (a) quantification and (b) representative phase images.c,d, GSC-11 invasion assay with 20 ng/ml rhBIGH3 (c) quantification and (d) representative phase images.e,f, GSC-268 invasion assay with M2 CM neutralized with 10 µg/ml IgG (rabbit) or 10 µg/ml anti-BIGH3 (e) quantification and (f) representative phase images.g,h, GSC-268 invasion assay with 10 µM mTOR activator MHY1845 (g) quantification and (h) representative phase images.i,j, GSC-268 invasion assay with 20 ng/ml rhBIGH3 and 10 µM mTOR inhibitor Temsirolimus (i) quantification and (j) representative phase images.k,l, GSC-268 invasion assay with M2 CM and 10 µM mTOR inhibitor Temsirolimus (k) quantification and (l) representative phase images. 6acrophage-secreted ligands identified by receptor-ligand analysis.Human GBM scRNA-seq dataset was obtained from Cui, et al.5Plots include (a) gene expression of all predicted M2 macrophage ligands across all GBM tumor cell type clusters and (b) gene expression of top 10 predicted M2 macrophage ligands across only GBM TAM clusters.c,Differentialgene expression analysis between human GBM bone-marrowderived TAMs and microglia TAMs.Genes with log 2 FC > 1 are expressed higher in microglia TAMs.Hman scRNA-seq dataset was obtained from Müller, et al.6d, Relative BIGH3 mRNA expression obtained from qPCR across a panel of cell lines.e-h, Gene expression data from TCGA database queried and downloaded using GlioVis 7 data portal for visualization and analysis of brain tumor expression datasets.(e) Simple Linear Regression plot and analysis of CD14 and BIGH3 gene expression in GBM tumors. BGH3 mRNA expression (log2) across (f) glioma tumor stage and (g) GBM molecular subtype.(h) Kaplan-Meier survival correlating with high or low levels of BIGH3 mRNA with median expression level used as cutoff.P values determined by logrank Mantel-Cox comparison (n=262 patients in the high expression group, and n=263 patients in the low expression group). AcknowledgementsThis work used the Vincent J. Coates Proteomics/Mass Spectrometry Laboratory at UC Berkeley, supported in part by NIH S10 Instrumentation Grant S10RR025622.Glioma Stem Cell lines were developed at the University of Texas M. D. Anderson Department of Neurosurgery support by grants from the National Cancer Institute (1R01CA214749, 1R01CA247970, P30CA016672 and 2P50CA127001) and the University of Texas M. D. Anderson Moon Shots Program TM .We also thank Dr. Randy Schekman (UC Berkeley) for providing the NanoSight NS300 instrument, Liang Ma for advising on Extracellular Vesicle isolation and Kwasi Amofa for providing qPCR primers.This work was supported by awards from the National Science Foundation (Graduate Research Fellowship to E.A.A.) and the National Institutes of Health (R01CA227136 and R01CA260443 to M.K.A. and S.K.).Author Contributions E.A.A, S.K. and M.K.A. conceived the project.E.A.A. wrote the manuscript, performed most of the experiments and prepared the figures.D.W. contributed to the experimental work.All authors contributed to editing and revising the manuscript.Competing InterestsThe authors declare no competing interests.Supplementary DataDataset 1. Bulk RNA-seq differentially expressed genes Dataset 2. Proteins identified by mass spectrometry in conditioned media isolated from M1-and M2-polarized THP-1 macrophages Dataset 3. 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The K + transporter NPF7.3/ NRT1.5 and the proton pump AHA2 contribute to K + transport in Arabidopsis thaliana under K + and NO 3 -deficiency 10 November 2023 Florencia Sena Institute of Biology/Applied Genetics Dahlem Centre of Plant Sciences Freie Universität Berlin Berlin, Germany Laboratory of Apicomplexan Biology Institut Pasteur Montevideo MontevideoUruguay Facultad de Agronomı ´a Laboratorio de Bioquı ´mica Universidad de la Repu ´blica MontevideoUruguay Reinhard Kunze reinhard.kunze@fu-berlin.de Institute of Biology/Applied Genetics Dahlem Centre of Plant Sciences Freie Universität Berlin Berlin, Germany Jon Pittman Jose M Pardo Edita Tylova The University of Manchester United Kingdom Spanish National Research Council (CSIC) Spain Charles University Czechia The K + transporter NPF7.3/ NRT1.5 and the proton pump AHA2 contribute to K + transport in Arabidopsis thaliana under K + and NO 3 -deficiency 10 November 2023A783DE446488F7F092B85C47E5BC274610.3389/fpls.2023.1287843RECEIVED 02 September 2023 ACCEPTED 12 October 2023Arabidopsis thaliananitrate deficiencypotassium deficiencypotassium transportprotein-protein interactionplasma membraneH + -ATPase Nitrate (NO 3 -) and potassium (K + ) are distributed in plants via short and longdistance transport.These two pathways jointly regulate NO 3 -and K + levels in all higher plants.The Arabidopsis thaliana transporter NPF7.3/NRT1.5 is responsible for loading NO 3 -and K + from root pericycle cells into the xylem vessels, facilitating the long-distance transport of NO 3 -and K + to shoots.In this study, we demonstrate a protein-protein interaction of NPF7.3/NRT1.5 with the proton pump AHA2 in the plasma membrane by split ubiquitin and bimolecular complementation assays, and we show that a conserved glycine residue in a transmembrane domain of NPF7.3/NRT1.5 is crucial for the interaction.We demonstrate that AHA2 together with NRT1.5 affects the K + level in shoots, modulates the root architecture, and alters extracellular pH and the plasma membrane potential.We hypothesize that NRT1.5 and AHA2 interaction plays a role in maintaining the pH gradient and membrane potential across the root pericycle cell plasma membrane during K + and/or NO 3 -transport. Introduction Potassium (K) and nitrogen (N) are essential macronutrients of higher plants.Shortage or excess of these nutrients in soils limit growth and agricultural yield.While potassium is available to plants only as the cation K + , N occurs in soils as inorganic and organic molecules, where nitrate (NO 3 -) is the prevalent form (Xu et al., 2012).For nutrient uptake by the roots and distribution throughout the sporophyte via xylem and phloem, plants have evolved a large number of membrane transporters and channels with different specificities and affinity ranges.In Arabidopsis thaliana NO 3 -transporters and channels are encoded by four gene families (reviewed by Krapp et al., 2014): (1) the NRT1/ PTR (nitrate transporter 1/peptide transporter) family, renamed as NPF family, (2) the NRT2/NNP (nitrate transporter 2/nitrate-nitrite porter) family, (3) the CLC (chloride channel) family, and (4) the SLAC1/SLAHs (slow-type anion channel-associated 1/homologues) family.K + transport is accomplished by the three channel families Shaker, TPK (tandem-pore K + ) and Kir-like (K + inward rectifierlike) (Very and Sentenac, 2003), and the three transporter families KUP/HAK/KT, HKT (high-affinity K + transporters) and CPA (cation-proton antiporters) (Gierth and Maser, 2007;Chanroj et al., 2012). The intersection between K and N physiology in plant cells is not yet fully understood.K + plays an essential role as a counter ion of NO 3 -, facilitating the uptake, translocation, and distribution of these ions between roots and shoots (reviewed in Rodenas et al., 2017 ).In contrast, as plant K + transporters and channels can also transport ammonium ions (NH 4 + ), K + and NH 4 + compete for uptake and translocation (Ten Hoopen et al., 2010).In addition, the expression of several NRT1 members (i.e.NRT1.1, NRT1.4,NRT1.5, NRT1.8) is influenced directly or indirectly by K + nutrition (Drechsler et al., 2015;Li et al., 2017;Fang et al., 2020;Morales de Los Rıós et al., 2021), indicating regulatory interactions between the K + and NO 3 -transport pathways. The nitrate transporter NPF7.3/NRT1.5 (subsequently termed NRT1.5) was initially characterized as a proton-coupled, lowaffinity nitrate transporter involved in long-distance NO 3 transport in A. thaliana (Lin et al., 2008).It is expressed in root pericycle cells close to the xylem vessels and localizes to the plasma membrane (PM), suggesting its involvement in xylem loading of nitrate.Subsequently, the expression of NRT1.5 was found to be dependent both on nitrate and potassium availability.Under low nitrate nutrition, in nrt1.5 mutants the root-to-shoot potassium transport is reduced, suggesting a regulatory loop at the level of xylem transport that maintains the balance between nitrate and potassium (Drechsler et al., 2015).A direct link of NRT1.5 to NO 3 and K + homeostasis is supported by several observations.(1) Both NRT1.5 and the K + transporter SKOR contribute to K + translocation from root to shoot, with SKOR activity dominating under high NO 3 -and low K + supply, and NRT1.5 under low NO 3 availability (Drechsler et al., 2015). (2) NRT1.5 was reported to perceive nitrate starvation-derived signals to prevent leaf senescence by facilitating foliar potassium accumulation (Meng et al., 2016).This might explain why NRT1.5 is highly upregulated during leaf senescence (van der Graaff et al., 2006).( 3) Lateral root formation depends on NRT1.5 under K + deprivation and low supply of NO 3 - (Zheng et al., 2016).(4) NRT1.5 functions as a proton-coupled H + / K + antiporter that facilitates K + release from the root parenchymatous pericycle cells and K + loading into the xylem (Li et al., 2017).( 5) Previous studies have shown that NRT1.5 expression is modulated by changes in K + and NO 3 -levels (Lin et al., 2008;Chen et al., 2012;Drechsler et al., 2015;Meng et al., 2016;Li et al., 2017).The transcription factor MYB59 positively regulates NRT1.5 expression in roots under low K + -stress and thus has a key role in maintaining K + /NO 3 -homeostasis between roots and shoots (Du et al., 2019). Besides transporting nitrate and potassium, NRT1.5 was shown to function as an indole-3-butyric acid (IBA) transporter in A. thaliana root tips, where IBA is converted to the auxin indole-3acetic acid that regulates root gravitropism (Watanabe et al., 2020).NRT1.5 was also reported to be an important component in the regulation and modulation of phosphate deficiency responses (Cui et al., 2019). NRT1.5 is energized by the H + -gradient across the PM, which is established by H + -ATPases that extrude protons from the cytoplasm by ATP hydrolysis.In A. thaliana 11 different members of PM H + -ATPases are encoded by the AHA (Arabidopsis H + -ATPase) multigene family, with AHA1 and AHA2 being the two major isoforms (Falhof et al., 2016).Proton pumps are known to contribute to nutrient assimilation during root uptake and short and long-distance transport (Sze and Chanroj, 2018;Feng et al., 2020). The complex regulation of NRT1.5, its dependence on an electrochemical H + -gradient across the PM, and its ability to transport diverse substrates prompted us to investigate whether NRT1.5 interacts with other PM proteins that might directly or indirectly influence its activity and regulation.In a split-ubiquitinscreen, we discovered the proton pump AHA2 to interact with NRT1.5 and confirmed the interaction by BiFC analysis in tobacco plants.The physiological responses of Arabidopsis wild-type plants and nrt1.5 and aha2 mutant plants under different NO 3 -and K + regimes corroborated an interplay between NRT1.5 and AHA2 to control plant K + and H + homeostasis. 2 Materials and methods Plant material and growth conditions Arabidopsis thaliana T-DNA insertion lines were obtained from the Arabidopsis Biological Resource Center.The knockout nrt1.5 (GABI_347B03) and aha2 (GABI_219D04) mutant lines are in the Col-0 background.The double mutant nrt1.5/aha2was selected from the F2 progeny of the nrt1.5 × aha2 cross.The absence of fulllength NRT1.5 and AHA2 transcripts in the homozygous mutants was verified by RT-PCR (all oligonucleotides used in this work are listed in Supplementary Table 1). For growth assays on plates, Arabidopsis seeds were surface sterilized with 70% EtOH for 2 min, followed by 10% NaClO and 1% SDS for 3 min, and then washed 3 times for 3 min in sterile deionized water.Seeds were placed on 0.5 × MS plates containing 1% sucrose and 0.3% Gelrite and stratified in darkness for 2 days at 4°C, followed by pre-germination under long-day conditions (16 h/ 8 h light/dark, 22°C, light intensity 120 µmol m -2 s -1 , and relative humidity 40%) for 5 days.Modified 0.5 × MS medium lacking potassium and nitrogen ("MS-base": 1.5 mM CaCl 2 , 1 mM MgSO 4 , 0.5 mM NaH 2 PO 4 , 0.05 mM H 3 BO 3 , 0.05 mM MnSO 4 , 0.05 mM FeNa-EDTA, 15 µM ZnSO 4 , 2.5 µM KI, 0.5 µM Na 2 MoO 4, 0.05 µM CuSO 4 , 0.05 µM CoCl 2 , 0.5% MS vitamins, 0.5 g/l MES, 1% sucrose, pH 5.5) was supplemented with KCl and Ca(NO 3 ) 2 to different concentrations (Supplementary Table 2). For growth assays on soil, plants were pre-germinated on a 1:1 mixture of commercial type P soil and unfertilized type 0 soil (Einheitserdewerk Uetersen) under long-day growth conditions (16 h/ 8 h light/dark, 21°C/18°C day/night, light intensity 120 µmol m -2 s -1 , relative humidity 55% to 70%).After 5 days, the seedlings were transferred to either the same soil as control or to unfertilized soil supplemented with different nutrient solutions (Supplementary Table 2). At the end of each fertilization experiment the frequency of chlorotic leaves (chlorophyll degradation/cell death) was determined for each genotype in 20 individual plants. DNA isolation Frozen Arabidopsis thaliana tissue was ground in liquid N 2 , mixed, and homogenized together with 2 metal balls by a mixer mill (Retsch MM 400).Genomic DNA from A. thaliana plants was extracted from frozen plant material according to Chen and Ronald (1999) with modification.600 µl of 2% CTAB buffer (100 mM Tris-Cl pH 8, 1.4 M NaCl, 20 mM EDTA, and 2% CTAB w/v) was added to the ground material and mixed by vortexing, followed by incubation at 65°C for 20 min.The tubes were inverted every 5 min and were centrifuged at room temperature for 10 min at 13000 g.The supernatant was transferred to a fresh tube with one volume chloroform:isoamyl alcohol (24:1) and mixed well by inverting, followed by centrifuging at 13000 g for 5 min.The upper phase was transferred to a fresh tube and mixed with one volume isopropanol, followed by incubating at 4°C for 10 min. Precipitated DNA was obtained by centrifuging at 13000 g for 10 min.The pellet was washed with 70% EtOH, centrifuged at 13000 g for 3 min at room temperature, air-dried, re-suspended in 200 µl of H 2 O with 20 µg/ml RNaseA and incubated at 50°C for 30 min. Split ubiquitin screen Total RNA was extracted from Arabidopsis thaliana Col-0 seedlings 15 days after sowing and from senescing rosette leaves 43 days after sowing as described (Downing et al., 1992), followed by purification of polyadenylated mRNA using oligo (dT)-cellulose Type 7 (Amersham Biosciences).cDNA synthesis, entry cDNA library construction, and cloning into pDONR222 were performed with the CloneMiner ™ Library Construction Kit (ThermoFisher Scientific).The prey library for expression of NubG-cDNA fusion proteins in yeast was generated in pNubG-X (TAIR stock CD3-1737), a modified pNXgate32-3HA vector (Obrdlik et al., 2004).The entry cDNA library was transferred into the attR-sites of pNubG-X using the LR Clonase ™ enzyme mix (ThermoFisher Scientific).Entry cDNA library and prey library plasmid DNA was isolated using the QIAfilter Plasmid Mega Kit (Qiagen). The Cub-NRT1.5 bait vector was constructed by amplifying the NRT1.5 (At1g32450) full-length coding sequence (CDS) including the stop codon on plasmid pcAcNRT1.5-9 (Drechsler et al., 2015) with Phusion High-Fidelity DNA Polymerase (ThermoFisher Scientific) and inserting it into PstI-SacII-digested pBT3-N plasmid (Dualsystems Biotech, Zürich, Switzerland).For the NRT1.5-Cubbait vector, the NRT1.5 CDS lacking the stop codon was amplified and cloned into HindIII-digested pBT3-C plasmid (Dualsystems Biotech, Zürich, Switzerland).Each bait vector was transformed by the LiOAc/ssDNA/PEG method (Gietz and Schiestl, 2007) into yeast strain THY.AP4 [MATa ura3 leu2 lexA::lacZ::trp1 lexA::HIS3 lexA::ADE2], followed by transformation with the prey library plasmids.The split-ubiquitin screen was done following the DUALmembrane Kit 3 protocol (Dualsystems Biotech, Zürich, Switzerland).Colonies appearing after 5 days of growth on selective medium yeast nitrogen base (YNB-trp-leu-his-ade) supplemented with 5 mM 3-AT (3-amino-1,2,4-triazole) were propagated.For confirmation, the prey plasmids were recovered from the yeast, transformed in E. coli DH10B, reisolated, and retransformed into bait vector-carrying THY.AP4 cells.Confirmed prey plasmids expressing NRT1.5-interacting proteins were sequenced to identify the corresponding gene. Split ubiquitin assay pDONR222-NRT1.5 was generated by amplifying the fulllength NRT1.5 CDS lacking the stop codon from A. thaliana Col-0 cDNA using attB1/attB2 Gateway extension primers and Phusion High-Fidelity DNA Polymerase and inserting it into pDONR222.pDONR222-NRT1.5 G209E was generated from pDONR222-NRT1.5 by replacing the G 209 codon GGA with GAA (➔E 209 ) using the Q5site-directed mutagenesis kit (New England Biolabs).pDONR222-AHA2 was constructed by recombining a synthetic attB1-AHA2-attB2 fragment (ThermoFisher/Invitrogen GeneArt Strings DNA Fragments) into pDONR222.The yeast expression vectors for NRT1.5-Cub and NRT1.5 G209E -Cub were generated by LR reactions between pBT3-C and pDONR222-NRT1.5 and pDONR222-NRT1.5 G209E , respectively.The expression vector for Nub-AHA2 was constructed by mobilizing the AHA2 CDS from pDONR222-AHA2 into pNubG-X.Yeast strain THY.AP4 [MATa ura3 leu2 lexA::lacZ::trp1 lexA::HIS3 lexA::ADE2] was cotransformed with bait and prey vectors and positive colonies were selected after 2 days of growth at 30°C on YNB-trp-leu medium.Protein-protein interaction was detected by 4 days of growth on selective YNB-trp-leu-his-ade medium supplemented with 20 mM 3-AT. Bi-molecular fluorescence complementation assay The full-length CDS of NRT1.5 with flanking attB1/attB4 Gateway attachment sites and of AHA2 with flanking attB3/attB2 sites were amplified from A. thaliana Col-0 cDNA and recombined into pDONR221 P1-P4 and pDONR221 P3-P2, respectively.pDONR221p1-p4-NRT1.5 G209E was generated by site-directed mutagenesis of pDONR221p1-p4-NRT1.5.The three CDS cassettes were transferred into destination vector pBiFC-2in1-NN (Grefen and Blatt, 2012) by LR reaction.pBiFC-2in1-NN was a gift from Christopher Grefen (Addgene plasmid # 105111; http:// n2t.net/addgene:105111).The vector pBiFC-2in1-NN includes a gene for expression of the soluble monomeric red fluorescent protein (mRFP1) as a reference marker for transformation and protein expression where the mRFP fluorescence signal visualizes the cytoplasm and the lumen of the nucleus.A pBiFC-2in1-NN construct with unfused nYFP and NRT1.5 fused cYFP was generated as a negative control.Agrobacterium tumefaciens GV3101::pMP90 cells (Koncz and Schell, 1986) were transformed by electroporation at 2.2 kV for 5 ms with the respective plasmid constructs and incubated with rifampicin (50 mg/ml), gentamycin (25 mg/ml), spectinomycin (75 mg/ml), and kanamycin (50 mg/ml) at 28°C for 2 days.Cultures were resuspended at OD 600 nm = 0.05 in infiltration solution (10 mM MES, pH 5.6, 10 mM MgCl 2, and 150 µM acetosyringone), incubated for 2 h at room temperature, and infiltrated into Nicotiana benthamiana abaxial epidermis cells (8week old plants) with a syringe.Protein-protein interactions were detected two days post infiltration (dpi) by confocal microscopy. Yeast growth assays The full-length CDS of AtNRT1.5 and AtAHA2 were amplified with primers containing BamHI/HindIII restriction sites.AHA2 was cloned in the yeast expression vector p425-TEF, and NRT1.5 was cloned in p426-TEF (Mumberg et al., 1995).p426-TEF-NRT1.5 G209E was generated by site-directed mutagenesis of p426-TEF-NRT1.5.The expression plasmids were transformed into the control yeast strain BY4741 [MATa his3D leu2D met15D ura3D] (Brachmann et al., 1998) and its derivative BYT12 [trk1D trk2D] which lacks the two major K + uptake transporters (Petrezselyova et al., 2010).Transformants were precultured overnight in SD-leuura medium supplemented with 0.1 M KCl.The next day, the cells were washed with deionized water and resuspended to an OD 600 nm of 1, and spotted on SD-leu-ura plates.The hygromycin B (HygB) sensitivity test was performed according to Petrezselyova et al. (2010) and Zimmermannova et al. (2021).20 µl of cell suspension (OD 600 nm = 1) were spotted on HygB gradient plates (0 to 0.5 g/l HygB, SD-leu-ura, 0.1 M KCl) and incubated at 30°C for two days. Complementation of nrt1.5 plants with NRT1.5 and NRT1.5 G209E The eGFP coding sequence lacking the initial ATG codon was amplified with overhangs to be cloned by Gibson assembly into pTkan+-PHO1NRT1.5vector (Drechsler et al., 2015).The stop codon TAA of NRT1.5 was removed by PCR.The pTkan+-PHO1NRT1.5 vector was opened by restriction enzyme digestion with PstI and ligated with the eGFP fragment using the NEBuilder HiFi DNA assembly kit (New England Biolabs).The pTkan+-PHO1NRT1.5 G209E eGFP vector was constructed from pTkan+-PHO1NRT1.5eGFP using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs). The constructs were transformed in nrt1.5 plants by the floral dip method (Clough and Bent, 1998).Two independently transformed PHO1p::NRT1.5eGFPand PHO1p::NRT1.5G209E eGFP lines in nrt1.5 background that were homozygous for a single insertion of the transgene were used for this study. Transient expression of NRT1.5eGFP and NRT1.5 G209E eGFP in Nicotiana benthamiana The coding sequences of eGFP, NRT1.5eGFP and NRT1.5 G209E eGFP were amplified by PCR with the restriction enzyme attachment sites of BamHI and HindIII using pTkan+-PHO1NRT1.5eGFPand pTkan+-PHO1NRT1.5 G209E eGFP as templates.The amplified products were ligated into Agrobacterium binary vector pCAMBIA containing the minimal CaMV 35S promoter sequence (Hey and Grimm, 2018).Agrobacterium tumefaciens and Nicotiana benthamiana transformations were performed as described for the BiFC assay. RNA isolation, cDNA synthesis and qPCR Total RNA from frozen Arabidopsis thaliana seedlings was extracted using the citric acid extraction method (Oñate-Sańchez and Vicente-Carbajosa, 2008) and DNase I treated according to the manufacturer's instructions (New England Biolabs).First-strand cDNA was synthesized from 2 µg total RNA using RevertAid H Minus Reverse Transcriptase (ThermoFisher Scientific).Transcription analyses were performed with tissue from separately grown plants in three independent replicates.qPCR reactions were conducted on a CFX Connect real-time PCR detection system (Bio-Rad Laboratories) using the SYBR Green PCR Master Mix (ThermoFisher Scientific) and the thermal profile: 10 min 95°C and 40 times (15 sec 95°C, 1 min 60°C).For the subsequent melting curve analysis, the temperature was increased from 65 °C to 95 °C within 30 min.Expression values for each gene were normalized to the reference gene UBQ10 (At4g05320), and relative expression levels were determined according to (Czechowski et al., 2005). Extracellular acidification assay Extracellular acidification was assayed as described by Haruta et al. (2010) with modifications.Seedlings were pre-germinated in 0.5 × MS medium for five days and then transferred to unbuffered 0.25 × MS-base containing either HK (10 mM K + , 10 mM NO 3 -) or 0K (0 mM K + , 1 mM NO 3 -) fertilizer for another five days.The tenday-old seedlings were incubated in 200 µl modified 0.25 × MS media lacking MES (pH 5.5, unbuffered) with 30 µg/ml fluorescein isothiocyanate-dextran (FITC-dextran) (M r 10,000, Sigma-Aldrich) for 16 h in long-day conditions (16 h/8 h light/dark, 22°C, light intensity 120 µmol m -2 s -1 , relative humidity 40%).Excitation was at 485 nm and fluorescence was detected at 535 nm in a Power-Wave HT microplate spectrophotometer (BioTek).For pH determination, a calibration curve was generated with commercial MS media adjusted to pH values ranging from 4.5 to 7.5. Live-cell imaging using confocal laser scanning microscopy Confocal laser scanning microscopy (CLSM) of abaxial tobacco leaf epidermis cells was performed with a Leica TCS SP5 AOBS Confocal Microscope equipped with 20 x and 63 x water immersion objectives (Leica Microsystems).Simultaneous excitation of YFP (l ex = 488 nm, l em = 520-553 nm), mRFP (l ex = 561 nm, l em = 608-642 nm) and chlorophyll (l ex = 561 nm, l em = 642-702 nm) was achieved.The 514 nm argon laser was used to detect YFP and the 561 nm diode-pumped solid-state laser for mRFP and chlorophyll.Post-acquisition image processing and fluorescence quantification was performed using the Leica LAS AF software. Results 3.1 The K + /NO 3 -transporter NPF7.3/NRT1.5 interacts with the proton pump AHA2 To investigate whether NRT1.5 interacts with other PM proteins, we carried out a split-ubiquitin membrane yeast two-hybrid screen (Stagljar et al., 1998;Thaminy et al., 2003).As AtNRT1.5 is highly expressed in seedling roots and senescing leaves (van der Graaff et al., 2006;Lin et al., 2008;Zheng et al., 2016), we used as prey two A. thaliana cDNA libraries from these tissues and screened them with two bait constructs carrying the Cub-LexA-VP16 reporter module at the Nor C-terminus of NRT1.5, respectively.From the primary NRT1.5-interactorcandidates we selected proteins that are known to be localized in the PM, to be expressed in pericycle cells of Arabidopsis roots, and to be associated with ion-or protein-transport.To confirm the interaction with NRT1.5, the full-length coding sequences of the candidates were amplified from A. thaliana Col-0 cDNA, fused to NubG, and used as prey for the NRT1.5 bait in targeted split-ubiquitin assays.Five putative NRT1.5-interactors were confirmed in this assay (Supplementary Table 3). Among them was the PM proton ATPase 2 (AHA2) whose interaction with NRT1.5 we investigated in more detail in this study.Co-expression of NRT1.5-Cub and NubG-AHA2 in the auxotrophic yeast strain THY.AP4 led to yeast growth in a medium lacking adenine and histidine, indicating that NRT1.5 directly interacts with AHA2 (Figure 1A). To evaluate the protein-protein interaction in plant cells, a bimolecular fluorescence complementation assay was carried out in Nicotiana benthamiana cells.Co-expression of the cYFP-NRT1.5 and AHA2-nYFP fusion proteins reconstituted YFP fluorescence, suggesting the interaction of NRT1.5 and AHA2 at the plant PM (Figure 1B). 3.2 The Gly 209 of NTR1.5 is critical for AHA2 interaction and affects NRT1.5 function but not its cellular localization in plants Li and colleagues (2017) had discovered that the low-K +sensitive A. thaliana mutant lks2 carries a Gly 209 to Glu (G209E) A B FIGURE 1 Protein-protein interaction of NRT1.Frontiers in Plant Science frontiersin.orgsubstitution in NRT1.5 and that the NRT1.5 G209E mutant protein is compromised in K + efflux in Xenopus laevis oocytes and in yeast cells.We observed that the mutant protein does neither interact with AHA2 in the yeast assay (Figure 1A) or in tobacco cells (Figure 1B).This corroborates the specificity of the NRT1.5-AHA2interaction and demonstrates that Gly 209 of NRT1.5 is critical for the interaction with AHA2. Previously it was shown that, as a consequence of an impaired K + root-to-shoot transport, the nrt1.5 mutant displays impaired root architecture and develops an early senescence phenotype when plants grow in media lacking potassium and nitrate (Drechsler et al., 2015;Zheng et al., 2016).Additionally, nrt1.5 seedlings exhibit a reduced Hygromycin B (HygB) sensitivity due to a reduction in the PM potential (Drechsler et al., 2015). The NRT1.5 Gly 209 residue is highly conserved in NRT1.5 homologs in other plant species as well as in the 16 NRT1 family members in A. thaliana (Supplementary Figure 3).To evaluate if NRT1.5 G209E expression in root cells affects NRT1.5 function, we carried out a complementation assay of the nrt1.5 mutant with NRT1.5 G209E .nrt1.5 mutant plants were transformed with a vector containing the promoter of the PHOSPHATE1 (PHO1) gene, and the NRT1.5 or NRT1.5 G209E coding region lacking the stop codon fused to a C-terminal eGFP fluorescent tag. The 1.6 kb PHO1 promoter (PHO1p) served as substitute for the NRT1.5 promoter in nrt1.5 complementation experiments, because expressing the NRT1.5 coding region under the control of the 2.4 kb upstream region of NRT1.5 or the ubiquitous 35S CaMV promoter does not achieve complementation of the nrt1.5 mutant (Drechsler et al., 2015).PHO1 (At3g23430) was identified as a gene whose expression is like that of NRT1.5 primarily directed to the root vasculature in Arabidopsis plants (Hamburger et al., 2002).Transgenic plants with the NRT1.5 coding region fused behind the PHO1 promoter expressed almost wild-type levels of NRT1.5 transcripts in the roots (Drechsler et al., 2015).Homozygous PHO1p::NRT1.5eGFPand PHO1p::NRT1.5eGFPG209E plants were evaluated phenotypically under K + /NO 3 -deficiency and exposure to HygB.The root growth assay indicated that PHO1p::NRT1.5eGFPplants can rescue the reduction in root density (Figures 2A, B) and the early senescence phenotype of nrt1.5 plants under K + and NO 3 -deficiency (Figure 2C), corroborating previous studies of this complementation line without the fluorescent tag (Drechsler et al., 2015).In contrast, the transgenic nrt1.5 PHO1p::NRT1.5 G209E eGFP plants retained the nrt1.5 root and shoot phenotypes, demonstrating that the mutation G209E prevents complementation of the nrt1.5 mutant.Expression of the NRT1.5 and NRT1.5 G209E proteins was confirmed by the detection of GFP signals in the transgenic plant roots and at the PM of tobacco epidermal cells (Supplementary Figure 1).Hygromycin B (HygB) is used as an indicator of the PM potential status in plant and yeast cells (Haruta et al., 2010;Barreto et al., 2011;Haruta and Sussman, 2012).When the PM potential is reduced, the toxic drug is not able to enter the cells and the plants develop an enhanced HygB tolerance in comparison to wild-type, as was observed in nrt1.5 mutant plants (Drechsler et al., 2015).The two independent PHO1p::NRT1.5eGFPlines were sensitive to 5 µg/ml HygB (reduction of root growth and yellow cotyledons), whereas the PHO1p::NRT1.5G209E eGFP lines were more resistant, indicating that the PM potential was restored upon expression of NRT1.5 in the nrt1.5 mutant background (Figure 2A).In contrast, the NRT1.5 G209E mutant protein is not able to complement the nrt1.5 mutant, most likely because of a disturbed ion transport function of mutant protein, consistent with heterologous expression results in Xenopus laevis oocytes (Li et al., 2017). The proton pump AHA2 contributes to K + translocation under limiting K + and NO 3 -supply To investigate if the NRT1.5 interaction with AHA2 is required for nutrient root-to-shoot translocation in Arabidopsis, the nrt1.5/aha2 double mutant was generated.No residual NRT1.5 or AHA2 transcripts were detectable by semi-quantitative RT-PCR in the double mutant (Figure 3A).When grown on unfertilized soil and watered with low-nutrient fertilizer (LK: 1 mM K + , 1 mM NO 3 -), wild-type, single, and double mutant plants showed a similar degree of early shoot chlorosis compared to plants grown under ample nutrient supply (HK: 10 mM K + , 10 mM NO 3 -) (Figure 3B), and no reduction in fresh weight (Figure 3C).Under ample nutrient supply, all plant lines developed chlorosis in less than 10% of leaves, whereas with low nutrient fertilization approximately 40% of the leaves showed leaves chlorosis (Figure 3D). In an earlier study it was shown that nitrate as well as total N concentrations in rosettes of nrt1.5 mutant plants are similar as in Col-0 wild-type plants under both low and high N fertilization regimes, whereas the K concentration in the mutants remained below 30% and 50% of the wild-type levels (Drechsler et al., 2015).We therefore determined here the concentration of mineral nutrients in 30 days old leaves of Col-0, nrt1.5, aha2, and nrt1.5/aha2 plants by inductively coupled plasma optical emission spectrometry (ICP-OES).When fertilized with 10 mM potassium and nitrate (HK), the concentrations of none of the elements K, Ca, Mg, B, Mn, Zn, Al, P, and S differed between wild-type, nrt1.5, aha2, or nrt1.5/aha2leaves (Figure 4).In contrast, under limiting nutrient supply (LK) Ca, Mg, and S concentrations were increased in nrt1.5 and nrt1.5/aha2leaves, indicating that the lack of NRT1.5 disturbs the distribution of these elements between roots and shoots, whereas K levels were significantly decreased in nrt1.5, nrt1.5/aha2 and also aha2 mutant plants.These results suggest that in low nutrient conditions both NRT1.5 and AHA2 are required for efficient K + translocation to shoots, whereas the distribution of Ca, Mg, and S is not dependent on AHA2. The contribution of NRT1.5 to K + translocation from roots to shoots under low K + supply was reported by Li and colleagues before (Li et al., 2017).In addition, under low K + in combination with low nitrate conditions, the nrt1.5 mutant exhibits leaf chlorosis and a reduced lateral root density (Zheng et al., 2016;Li et al., 2017).To investigate whether AHA2 is also involved in nutrient translocation, a seedling growth test was carried out.In the presence of HK root development did not differ in Col-0 wildtype and the three mutant plants, where all plants showed the same lateral root (Figures 5A, B) and root hair density (Figures 5C, D).In 0K medium, nrt1.5 mutant seedlings show a significant reduction in lateral root density.In the nrt1.5/aha2double mutant, the lateral root density is similarly reduced by trend, whereas in aha2 seedlings it is similar to Col-0 (Figure 5B).However, in all three mutant lines and most prominently in the nrt1.5/aha2double mutant plants the density of root hairs is significantly reduced in 0K medium compared to wild-type (Figure 5D) and the cotyledons develop an early chlorosis phenotype (Figure 5E).These observations suggest that in addition to NRT1.5 also AHA2 is involved in nutrient translocation to shoots in young seedlings. 3.4 NRT1.5 and AHA2 expression influence the plasma membrane potential and pH homeostasis under low nutrient supply NRT1.5 exports K + from root parenchyma cells into the xylem vessels at weakly acidic extracellular pH conditions like in the xylem sap (Li et al., 2017).AHA2 is the major PM proton efflux pump in root cells (Harper et al., 1990;Haruta et al., 2010).Both transporters effectuate ion movement across the PM of root cells and thus affect the PM potential.In plants, PM H + -ATPases are responsible for the establishment of the cellular membrane potential (Sondergaard 2) for 40 days.et al., 2004).To investigate if NRT1.5 and AHA2 cooperatively contribute to the maintenance of the PM potential, we indirectly measured this parameter in yeast and plant cells overexpressing or lacking NRT1.5 and AHA2. In yeast cells and plant roots, the uptake of the toxic cationic drug Hygromycin B (HygB) depends on the PM potential.Sensitivity to HygB is often linked to changes in the PM potential, which can be provoked by alterations in K + homeostasis in S. cerevisiae (Barreto et al., 2011) or alterations in H + homeostasis in A. thaliana (Haruta and Sussman, 2012).We observed that the growth of Arabidopsis Col-0 seedling roots was highly sensitive to 5µg/ml HygB.In contrast, root growth of the nrt1.5, aha2, and nrt1.5/aha2mutants is much less sensitive to HygB (Figure 6A), implying that the lack of NRT1.5 and AHA2 leads to membrane depolarization. In a HygB sensitivity assay with the yeast mutant BYT12, which lacks the two main K + -uptake transporters Trk1 and Trk2, the expression of NRT1.5 causes hyperpolarization of the yeast PM, indicating that NRT1.5 is active in yeast cells (Drechsler et al., 2015).To investigate whether NRT1.5 and AHA2 affect the PM potential, both genes were individually or co-expressed in wild-type BY4741 and mutant BYT12 cells.Subsequently, yeast growth was monitored in plates containing high K + concentration (0.1 M), which supports growth of BYT12 cells, and a HygB concentration gradient.Overexpression of the NRT1.5 G209E mutant protein was included in the assay to evaluate the impact of the mutation in yeast.The expression of NRT1.5 in BYT12 cells caused increased sensitivity to high HygB concentrations (Figure 6C), as previously reported by Drechsler et al. (2015).Expression of the mutant NRT1.5 G209E led to partial growth recovery of BYT12 cells, suggesting the mutation G209E affects the NRT1.5 ion translocation function in yeast.The expression of AHA2 in BYT12 cells results in increased HygB sensitivity compared to BYT12 cells expressing NRT1.5, suggesting that the PM potential is strongly influenced by the expression of Phenotyping of wild-type and mutant plants under low (LK) and high nutrient (HK) conditions.(A) RT-PCR analysis of NTR1.5 and AHA2 transcripts in Col-0 and the nrt1.5/aha2double mutant.GAPC1 (At3g04120) was used as reference housekeeping gene (B) Rosette phenotypes, (C) Leaf fresh weight, and (D) Fraction of chlorotic leaves (%) of Col-0, nrt1.5, aha2, and nrt1.5/aha2plants that were pre-germinated in fertilized soil for seven days and transferred to unfertilized soil type for another 30 days while watered with HK or LK medium, respectively.Shown are the means ± SD of the fraction of chlorotic leaves in 20 individual plants.Different letters above columns indicate statistically significant differences (Tukey's test; P <0.05). AHA2.BYT12 cells expressing NRT1.5 and AHA2 exhibited the same sensitivity to HygB as cells expressing AHA2 alone (Figure 6C).Thus, this assay does not indicate a biological interaction between NRT1.5 and AHA2 in yeast cells. Plant PM H + -ATPases use energy from ATP hydrolysis to pump protons from the cytoplasm to the extracellular space, thus creating and maintaining a negative membrane potential at the cytosolic side and a transmembrane pH gradient that acidifies the extracellular space (reviewed by Sondergaard et al., 2004;Falhof et al., 2016) and controls the transport of nutrients across the PM (Cosse and Seidel, 2021).To investigate if NRT1.5 and AHA2 cooperatively influence the extracellular pH, we incubated Col-0, nrt1.5, aha2, and nrt1.5/aha2seedlings in media with different K + and NO 3 -concentrations and determined the pH using the fluorescent dye FITC-dextran.The assay detects pH differences at the Arabidopsis root surface with a sensitivity that is comparable to that of pH-sensitive microelectrodes (Hoffmann and Kosegarten, 1995;Pitann et al., 2009;Haruta et al., 2010;Haruta et al., 2018).At high nutrient supply (HK), the extracellular pH of Col-0 and the nrt1.5 mutant roots were similar (pH ≈5.3), whereas aha2 roots led to an increase to almost pH 5.5 (Figure 6B).An increase in extracellular pH indicates an impaired PM proton-secretion activity and has previously also been observed in two other aha2 mutants (Haruta et al., 2010).The nrt1.5/aha2 double mutants showed an intermediate increase to pH ≈5.4.Wild-type roots showed at 0K supply a reduction of the extracellular pH to ~4.9, supporting that low nutrient availability induces acidification of the extracellular space or rhizosphere (Houmani et al., 2015), presumably due to increased activity of PM H + -ATPases.In contrast, extracellular acidification by nrt1.5, aha2, and nrt1.5/aha2 mutants was reduced under these conditions, where the double mutant showed the highest reduction of acidification to pH ≈5.3.These results are consistent with the hypothesis that in wild-type plants under altered K + and NO 3 -supply NRT1.5 and AHA2 interact and mutually activate each other.As AHA2 exports more protons than NRT1.5 imports, the acidification of the extracellular space is relatively strong.In the mutants the mutual induction of both proteins is absent, resulting in a slightly higher extracellular pH (Figure 6B).The only subtle phenotypic differences of the aha2 and nrt1.5/aha2 double mutants relative to the nrt1.5 mutant prompted us to investigate whether other H + -ATPase family members are upregulated when AHA2 is lacking, we examined the gene expression of all H + -ATPase genes (AHA1-AHA11) in Col-0, nrt1.5, aha2, and nrt1.5/aha2seedlings relative to AHA2 and their response to low nutrient supply.Except for AHA1, which exhibits a similar absolute expression level as AHA2, all other gene family members are expressed at 10-to 10,000-fold lower levels than AHA2 (Supplementary Figure 2A).These results are confirmed by transcriptome data sets in the Arabidopsis eFP Browser (https://bar.utoronto.ca/efp_arabidopsis/cgi-bin/efpWeb.cgi;Winter et al., 2007) which show that AHA1 and AHA2 are the only gene family members that are significantly expressed in seedlings or seedling roots.Under low nutrient supply AHA2 is ~2fold and AHA1 is ~4fold upregulated in the nrt1.5 mutant.Surprisingly though, no upregulation of AHA1 is observed in the nrt1.5/aha2double mutant plants.In the aha2 mutant we observe some upregulation of AHA6 and AHA9 (Supplementary A B C FIGURE 6 pH and plasma membrane potential are influenced by NRT1.5 and AHA2 expression.(A) Root growth phenotypes of Col-0, nrt1.5, aha2, and nrt1.5/aha2 in response to 5 µg/ml HygB.5-day-old seedlings were transferred to 0.5 × MS medium pH 5.5 supplemented with or without 5 µg/ml HygB for fourteen more days.The white bar represents 1 cm.(B) Extracellular pH measurements in Col-0, nrt1.5, aha2, nrt1.5/aha2roots under HK and 0K nutrition supply.Ten days-old seedlings were incubated with 0.25 x MS liquid medium supplemented with 30 µg/ml of FITC-dextran for 16 h.Media pH was determined by a fluorescence standard curve with a pH range from 4.5 to 7.5.Different letters indicate statistically significant differences (Tukey's test) between mutants and Col-0 with P<0.05, (means ± SD, n =12).(C) BY4741 wild-type and BYT12 mutant yeast strains were transformed with empty expression vectors (p425-TEF and p426-TEF) or expression constructs for the indicated proteins.Twelve 20 µl drops of untransformed (negative control) or transformed yeast cell suspension (OD 600 nm =1) were spotted along the HygB gradient (from 0 to 0.5 g/l) on SD-leu-ura pH 6.0, 0.1 M KCl plates and incubated at 30°C for two days.p425-TEF and p426-TEF are the empty expression vectors. Figure 2B), however, due to the extremely low basic expression of these genes in seedlings it is very unlikely that they could compensate for the lack of AHA2 in the aha2 mutant. Discussion 4.1 NRT1.5 and AHA2 coordinately control the H + and K + homeostasis in plants In this study, we demonstrate that the H + /K + -antiporter NRT1.5 interacts in vivo with the plasma membrane H + -ATPase AHA2 and that both proteins contribute to K + transport under K + and NO 3 -deficiency.Our results support the assumption that proton-motive force generated by AHA2 energizes the K + transport across the PM by NRT1.5.AHA2 is expressed in many tissues, most strongly in different root cells, stomatal guard cells, and mesophyll cells (Alexandersson et al., 2004;Sondergaard et al., 2004;Młodzińska et al., 2015;Falhof et al., 2016).In roots it is the major proton pump and, like NRT1.5, it is found in the PM of pericycle cells, which is a prerequisite for a direct interaction between the two proteins (Nicolson, 2014).A close link between proton transport and K + fluxes at the PM has already been concluded by the induction of the K + transporters HAK5 and CHX17 under K + starvation in wild-type (Cellier et al., 2004;Gierth et al., 2005) and aha2 mutant seedlings (Haruta and Sussman, 2012).Moreover, K + uptake under low K + conditions is promoted by the direct interaction between AHA2 and the receptor-like protein kinase BAK1 (Wang et al., 2022).Since the electrical membrane potential and chemical pH gradient generated by the proton-pumping ATPase AHA2 are essential for the function of the majority of plant PM transporters, it is not surprising that AHA2 is regulated by and interacting with many physiological parameters and protein factors (reviewed by Falhof et al., 2016;Michalak et al., 2022).AHA2 expression is affected by changing nitrate supply (Młodzińska et al., 2015) and phosphorus deficiency (Yuan et al., 2017).An additional layer of complexity of AHA2 regulation is added by post-translational control of the AHA2 protein activity.AHA2 can be phosphorylated at its C-terminal end by at least two kinases, which either enables or prevents binding of activating 14-3-3 proteins (Fuglsang et al., 1999;Fuglsang et al., 2007;Haruta et al., 2015).Recently, AHA2 was identified to be a central node in a protein-protein interaction network that undergoes highly dynamic protein-protein interaction assembly and disassembly processes upon nitrogen starvation (Gilbert et al., 2021). We found that under low potassium and nitrate supply the lack of NRT1.5 and/or AHA2 in Arabidopsis seedlings triggered a decrease of K + in leaves (Figure 4A), and a strong reduction in root hair density relative to Col-0 wild-type plants (Figure 5).Modulation of root architecture by nutrient availability is a wellknown and diverse response in plants (Forde and Lorenzo, 2001;Gruber et al., 2013).It is likely that AHA2 indirectly contributes to these root morphology alterations as it facilitates an acidic cell wall environment by extrusion of protons to the apoplast, which is essential for cell wall expansion and cell growth (Pacifici et al., 2018;Sze and Chanroj, 2018). Eliminating or overexpressing NRT1.5 and AHA2 in Arabidopsis and yeast, respectively, caused an alteration of the PM potential (Figures 6A, C).In AHA2-lacking seedlings grown under normal N and K + supply the extracellular pH in the medium is slightly elevated (Figure 6B), as was also observed in other studies (Haruta et al., 2010).When wild-type plants face K + deprivation, the extracellular pH drops sharply, indicating an increased H + export or reduced H + import or both.A reduced H + import could results from downregulation of K + /H + antiporters like NRT1.5.In aha2 and nrt1.5 mutant seedlings the drop in extracellular pH is lower under K + deprivation compared to wild type plants, and in the nrt1.5/aha2double mutant no more pH alteration is observed.This indicates that under these conditions NRT1.5 and AHA2 cooperatively modulate the extracellular pH, which is supposedly accomplished by the direct interaction of the two proteins. In a screen for low-K + -sensitive Arabidopsis mutants Li et al. ( 2017) identified the lks2 mutant, in which a single-nucleotide mutation causes the substitution of Gly 209 to Glu (G209E) in NRT1.5.The NRT1.5 G209E protein is not able to complement the nrt1.5 phenotype in roots or shoots of Arabidopsis (Figure 2), and its expression in yeast does not phenocopy the PM potential status of cells expressing NRT1.5 (Figure 6).As all Arabidopsis NRT1 proteins contain either Gly, Ala or Val at the NRT1.5 G209 homologous position (Supplementary Figure 3B), which is also conserved in NRT1.5 homologs from other plant species (Supplementary Figure 3A), it seems likely that an uncharged amino acid at this position is functionally crucial for NRT1 family transporters.Gly 209 is not exposed at a cytosolic or extracellular loop of the transporter, but is located in the center of transmembrane span 5.It therefore less likely that Gly 209 is directly involved in the interaction with AHA2, but it rather is critical for the structural integrity of NRT1.5. 4.2 Different proton pumps from the H + -ATPases family may contribute to the cellular pH control under low K + and NO 3 -supply In the Arabidopsis thaliana genome 11 PM H + -ATPase (AHA) encoding genes and one pseudogene (AHA12) were identified (reviewed in Gaxiola et al., 2007).AHA1 and AHA2 are the predominantly expressed isoforms in Arabidopsis seedlings, leaves and roots, but also low levels of AHA3, AHA4, and AHA11 were detected by transcriptomics and/or mass spectrometry (Harper et al., 1990;Alexandersson et al., 2004;Haruta et al., 2010).In the root endodermis AHA4 was shown to contribute to ion homeostasis and nutrient transport (Vitart et al., 2001).AHA6 and AHA9 are essential for pollen tube growth and fertility (Hoffmann et al., 2020).Some PM H + -ATPases have been reported to be transcriptionally induced under abiotic stresses including nutrition, light, and salinity (Gevaudant et al., 2007;Caesar et al., 2011;Yuan et al., 2017;Ando and Kinoshita, 2018).However, reports on specific physiological functions of Arabidopsis AHA family members are sparse, presumably because of functional redundancy of the gene family members (Gaxiola et al., 2007;Haruta et al., 2010).For example, AHA1 can functionally complement for AHA2 (Rodrigues et al., 2014). In aha2 and nrt1.5/aha2mutants we observed under K + deprivation conditions up-regulation of AHA4, AHA6 and AHA9 transcription (Supplementary Figure 2).However, as the regulation of these genes under LK-versus HK-conditions was inconsistent in aha2 and nrt1.5/aha2plants and even the induced expression level of these genes remained more than 100 fold lower than that of AHA2, we have no evidence for complementation of the lack of AHA2 in the mutants by other AHA family members. Arabidopsis NRT1.5 is not only a nitrate transporter involved in long-distance NO 3 -transport from root to shoot, but under NO 3 deficiency also a potassium transporter loading K + into the xylem. Here, the identification of the proton pump AHA2 as an interactor partner of NRT1.5 reveals a novel regulatory level in the control of K + and H + homeostasis in Arabidopsis. 5 and AHA2.(A) THY.AP4 cells were transformed with NRT1.5 or NRT1.5 G209E fused to Cub and AHA2 fused to Nub.To detect protein-protein-interaction, 12 µl yeast cell culture with an OD 600 nm =1 and OD 600 nm =2, respectively, were dropped on YNB-Leu-Trp-His-Ade medium supplemented with 20 mM 3-amino-1,2,4-triazole (3-AT) and on YNB-Leu-Trp medium as growth control.The addition of 3-AT reduces nonspecific yeast growth not based on protein-protein interactions.Plates were incubated at 30°C for three days and then photographed.(B) Confirmation of the interaction of NRT1.5 with AHA2 by bimolecular complementation assay.Shown are confocal microscopy images of N. benthamiana epidermis cells two days post-infiltration co-expressing non-fused nYFP, cYFP-NRT1.5 and RFP (top row), nYFP-AHA2, cYFP-NRT1.5 and RFP (center row), and nYFP-AHA2, cYFP-NRT1.5G209E and RFP (bottom row).Column YFP: YFP fluorescence in yellow indicates interaction between proteins.Column RFP, RFP fluorescence in red visualizes the cytoplasm and the lumen of the nucleus as expression control.Column Chl, autofluorescence of chlorophyll (represented in violet).Column BF, bright field image.Column overlay: overlay of all 4 channels.Scale bar = 20 µm.Sena and Kunze 10.3389/fpls.2023.1287843 FIGURE 2Phenotypes of the complementation lines nrt1.5 PHO1p::NRT1.5eGFPand nrt1.5 PHO1p::NRT1.5 G209E eGFP.(A) Root phenotypes.Col-0, nrt1.5, two independent nrt1.5, PHO1p::NRT1.5eGFPand two independent nrt1.5, PHO1p::NRT1.5 G209E eGFP plants were pre-germinated in 0.5 × MS medium for five days and transferred to plates with low nutrient medium (LK: 1 mM K + , 1 mM NO 3 -), high nutrient medium (HK: 10 mM K + , 10 mM NO 3 -) or HK + 5 µg/ml HygB medium for ten more days.The dotted white line represents the maximum root growth of Col-0 in HK + 5 µg/ml HygB.(B) Lateral root density quantification of plants in HK or LK conditions as in panel (A) Different letters indicate statistically significant differences (Tukey's test) between mutants and Col-0 in the different treatments with P <0.05, (means ± SD, n =20).(C) Shoot phenotypes.Plants were pre-germinated on fertilized soil for seven days, transferred to unfertilized type soil, and supplemented with a one-half strength MS-based fertilization solution containing HK or LK (Supplementary Table2) for 40 days. FIGURE 3 FIGURE 4 Elemental analysis by ICP-OES of Arabidopsis Col-0, nrt1.5, aha2, and nrt1.5/aha2plants grown under low (LK) and high (HK) nutrient conditions.(A) Macronutrient and (B) micronutrient concentration in shoots of 30 days-old Col-0, nrt1.5, aha2, and nrt1.5/aha2plants grown in LK and HK medium.Different letters above columns indicate statistically significant differences (Tukey's test; P <0.05) between mutants and Col-0 (means ± SD, n =8). Frontiers in Plant Science frontiersin.org AcknowledgmentsWe thank Dr. Navina Drechsler and Dr. Yue Zheng for contributing experimental results that were the basis for this study and for their support and helpful discussions.We thank Prof. Dr. Nicolaus von Wireń (Leibniz Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany) and his group members for performing the ICP-OES measurements.Data availability statementThe original contributions presented in the study are included in the article/Supplementary Material.Further inquiries can be directed to the corresponding author.Author contributionsFS: Conceptualization, Investigation, Methodology, Writingoriginal draft.RK: Conceptualization, Funding acquisition, Project administration, Supervision, Writingreview & editing.FundingThe author(s) declare financial support was received for the research, authorship, and/or publication of this article.This study was supported by a Graduate School Scholarship to FS from DAAD (German Academic Exchange Service), by the Deutsche Forschungsgemeinschaft (DFG grant KU715/10-2), and by the D a h l e m C e n t r e o f P l a n t S c i e n c e s ( D C P S ) a t F r e i e Universität Berlin.Conflict of interestThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Publisher's noteAll claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.Supplementary materialThe Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2023.1287843/full#supplementary-material Arabidopsis plasma membrane proteomics identifies components of transport, signal transduction and membrane trafficking. 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Utility of Spectral Filtering to Improve the Reliability of Marine Fauna Detections from Drone-Based Monitoring 15 November 2023 Andrew P Colefax colefax.pub@scieye.com.au 0000-0003-4720-0256 Sci-Eye Pty Ltd 2480GoonellabahNSWAustralia Andrew J Walsh 0000-0001-9506-0855 Sci-Eye Pty Ltd 2480GoonellabahNSWAustralia Cormac R Purcell 0000-0002-7491-7386 School of Computer Science and Engineering UNSW Sydney 2052SydneyNSWAustralia Trillium Technologies Pty Ltd 225 Fullarton Road5063EastwoodSAAustralia Paul Butcher paul.butcher@dpi.nsw.gov.au 0000-0001-7338-6037 Department of Primary Industries New South Wales 2450Coffs HarbourNSWAustralia National Marine Science Centre Southern Cross University 2450Coffs HarbourNSWAustralia Utility of Spectral Filtering to Improve the Reliability of Marine Fauna Detections from Drone-Based Monitoring 15 November 20238E166A70DB86B84FF58F42307D7F39FD10.3390/s23229193Received: 26 September 2023 Revised: 1 November 2023 Accepted: 6 November 2023drone-based monitoringmarine fauna detectionspectral filteringmachine learning analysissightability errorshark-bite mitigation Monitoring marine fauna is essential for mitigating the effects of disturbances in the marine environment, as well as reducing the risk of negative interactions between humans and marine life.Drone-based aerial surveys have become popular for detecting and estimating the abundance of large marine fauna.However, sightability errors, which affect detection reliability, are still apparent.This study tested the utility of spectral filtering for improving the reliability of marine fauna detections from drone-based monitoring.A series of drone-based survey flights were conducted using three identical RGB (red-green-blue channel) cameras with treatments: (i) control (RGB), (ii) spectrally filtered with a narrow 'green' bandpass filter (transmission between 525 and 550 nm), and, (iii) spectrally filtered with a polarising filter.Video data from nine flights comprising dolphin groups were analysed using a machine learning approach, whereby ground-truth detections were manually created and compared to AI-generated detections.The results showed that spectral filtering decreased the reliability of detecting submerged fauna compared to standard unfiltered RGB cameras.Although the majority of visible contrast between a submerged marine animal and surrounding seawater (in our study, sites along coastal beaches in eastern Australia) is known to occur between 515-554 nm, isolating the colour input to an RGB sensor does not improve detection reliability due to a decrease in the signal to noise ratio, which affects the reliability of detections. Introduction Due to increasing disturbances, such as effects of climate change, landscape modification, overfishing, and human-wildlife conflict, the importance of monitoring vulnerable marine fauna is intensifying.To this end, aerial survey methods have been a primary means for detecting large marine fauna and estimating their abundance [1,2].However, while traditional methods of using human spotters to record their observations from a crewed aircraft still occur [3], in many cases digital sampling is increasingly preferred [4].This is particularly the case with the relatively recent appearance and development of aerial drones, also referred to as 'UAV', 'UAS', 'RPAS' (see Chabot et al. [5]).Drones are now a common tool in ecology [6][7][8].Furthermore, with the continued advancement of drone technology, as well as associated digital capture technology, it is anticipated that the effective spatial scales that can be efficiently sampled using drone-based methods will expand [9].Therefore, drones are likely to increasingly replace traditional methods of marine aerial survey for monitoring the population health of large marine fauna [10]. Drone-based aerial surveys in the marine environment are perceived as being a relatively efficient and reliable method for detecting and identifying coastal fauna, and have been used to assess animal behaviour [11], abundance [12][13][14], population health [15,16], as well minimising the potential for human-wildlife conflict, such as human-shark interactions along coastal beaches [17,18].Despite the utility, sightability errors that affect reliability of the detections and identifications of marine life, can be apparent and similar to that reported from aerial surveys using crewed aircraft [4,8,17].This can be particularly problematic in marine fauna surveys, where the detection reliability is governed by factors including water clarity, depth, sea state, sun glare, and sea-surface reflection, as well as animal size, behaviour, and its position in the water column [17,19]. Similar to crewed aircraft surveys, the sightability errors associated with detecting marine fauna can be attributed to 'availability errors' or 'perception errors' [1,4].Availability errors occur due to an animal being unavailable for detection at the time of the survey pass.In the marine environment, this occurs when an animal is positioned deeper in the water column than the 'available' portion of surface water that can be seen from above, in the given conditions of water clarity.Perception biases occur when an animal in the water column should have been detected (i.e., it was positioned in the 'available' upper section of the water column), but it was not detected due to an error in human spotting or machine-learning, rather than external factors [1,4,17].In many cases, sampling effort can be constrained to favourable locations and conditions, and methods can be employed (particularly in post-processing) to estimate the errors and biases to adjust the detection data for inferring abundance [8,19,20].Minimising the uncertainty in count data has been, and is, a consistent objective across ecology.However, in situations where detections in real-time are required, such as in drone-based surveys aimed at reducing human-shark interactions, the need to investigate and refine methods to improve the reliability of detections are also apparent. The efficacy of drone-based shark surveys for reducing human-shark interactions currently relies on a drone pilot detecting (and identifying) sharks correctly in real-time from a telemetry screen, and subsequently taking an appropriate course of action based on whether the sighting is a potentially hazardous scenario or not.Despite overwhelming public support for the method [21], a number of scientific research articles are reporting significant error rates in field detections and fauna identifications, which has obvious implications for the efficacy of drone-based shark surveillance for keeping beach-goers safe [17,22,23].As with a number of other types of drone-based animal surveys, machine learning tools are currently being investigated to minimise human-induced errors in the realtime detection of sharks and other marine life from drones [24][25][26][27][28].However, although such methods show initial promise, they are still bound to the same sightability constraints that are imposed due to water clarity and the position of the animal in the water column [17,25].Therefore, despite the utility of machine learning to improve the reliability of detecting and identifying marine animals in real-time (and in post-analysis), potential methods that may improve the contrast of the animal against the background and potentially increase the 'availability' of the animal, such as from using alternative sensor technology, may further reduce error in detections [29][30][31]. The potential for alternative sensors, or wavelength selection, to improve the detectability of submerged fauna has not been thoroughly researched.The overwhelming majority of drone-based surveys in the marine environment use RGB sensors, with some applying polarising filters to these cameras to reduce the effect of sun reflection on collected imagery [32][33][34].Research into applying distortion correction algorithms and augmenting imagery in post-analysis has also demonstrated to have some improvements with regards image clarity, but can involve resource intensive post-analysis [33,35,36].Similarly, the added cost and complexity throughout the survey and analysis, often preclude the use of alternative sensors.However, various sensors such as infrared, are increasingly becoming more compact and turnkey [10]. Unlike terrestrial environments and above-water applications where thermal imagery has shown clear advantages for improving detection rates and abundance estimates of fauna [26,37], infrared radiation is highly attenuated in water and has very limited utility regarding submerged fauna [38].However, research into the use of multi-band sensor technologies, such as multispectral and hyperspectral cameras, to improve the detectability of fauna has indicated potential advantages over standard RGB cameras [29][30][31].Generally, the use of multispectral and hyperspectral cameras in the marine environment have typically used to aid the classification of sessile organisms through analysis of the additional spectral information [39,40].For detecting mobile marine animals from the air, optimising the use of specific wavelength ranges of light being sampled by the sensor (such as from multi-or hyperspectral cameras), is thought to have the potential to improve the depth that fauna can reliably be detected (availability error), as well as improve the contrast of fauna against the background.This would facilitate obtaining a greater confidence with regards detection reliability (perception bias).However, such sensors are expensive, often not intuitive to use, and offer low spatial resolution when compared to normal cameras. Previous research has investigated the contrast of various submerged marine fauna against the surrounding seawater [29] and demonstrated that along coastal beaches of eastern Australia, the greatest spectral difference between fauna and seawater was found to occur in the green colour band for submerged coastal marine fauna (515-554 nm).Therefore, this research aimed to test whether low-cost methods of augmenting typical drone cameras to restrict input to these frequencies can maximise the detection reliability of sharks and improve beach safety.Specifically, we tested whether spectral filtering, and spectral filtering with polarisation, could render improved clarity and reliability in fauna detections over standard unfiltered RGB cameras.This would occur if restricting the passband of the input signal improved the signal to noise ratio by essentially cutting out non-useful information. Materials and Methods Equipment We used a DJI Phantom 4 Pro (1.4 kg drone) due to its portability and versatility in conducting aerial surveys involving multiple flights in remote coastal locations.We attached a small payload to the landing gear of the drone, which enabled comparisons of three camera treatments of identical sensors, including a control, spectrally filtered, and spectrally filtered with polarising filter.Three GoPro Hero8 cameras were used, which we de-cased, minimised, and repackaged with battery-eliminating circuits to be lightweight (~23 g each).They were mounted in a custom housing that attached to flexible vibration dampeners and secured to the landing gear.The GoPro cameras were powered by the drone aircraft battery by a custom auxiliary power plug (Figure 1).A microcontroller with universal asynchronous receiver-transmitter (UART) was used to pair a 915 MHz radio receiver with a transmitter to enable remote start/stop recording of all three GoPros simultaneously.This allowed for a frame-by-frame comparison of video from each of the sensor filter treatments. Specific narrow-green bandpass filters were fitted to aluminium housings that could be applied to the outside of the two camera lenses.One of the filters had an extra layer of neutral density circular polarising glass.The bandpass filtering glass allowed light transmission specifically between 525 and 550 nm, with a peak transmission of >85% (Figure 1), and minimal angular shifting for the focal length of the GoPro.Angular shifting can occur when objects of interest are away from the centre of frame which can cause wavelength transmission to significantly shift. Figure 1. (A) The light transmission (%) for the corresponding wavelength (nm) of the narrow green bandpass filters used for filtering treatments.The near-infrared blocking filters (that come standard) in the CMOS sensor of the GoPro negate transmission in the higher (>850 nm) wavelengths.(B) GoPro sensor array in the custom mounting, attached to the landing gear of the Phantom 4 Pro drone. Survey Survey flights were made between July 2021 and August 2022.In these flights, we employed 'convenient sampling', and found fauna classes: dolphins (Tursiops sp), sharks (Carcharhinus spp.), guitarfish (Rhinobatidae), other rays (Aetobatus narinari, Rhinoptera neglecta), and whales (Megaptera novaeangliae), which have been found along the east Australian coastline in drone-based coastal surveys [18,20,22].However, dolphins were found to be the most appropriate class for the analysis, due to their vertical movement through the water column. Survey flights were conducted in varied winds of up to around 15 knots (7.7 m s −1 ), and in the absence of rain at Ballina, Evans Head, Tuncurry, Forster, Birubi, and Anna Bay on the east coast of Australia (Figure 2).The maximum wind tolerance for sampling was set intentionally lower than the recommended maximum for the aircraft, due to the added payload, which effectively increases the working load on the motors and electronic speed controllers, which require higher amounts of current from the batteries.Care was also taken to fly the aircraft smoothly to avoid unnecessary 'ramp-up' of the motors and Survey Survey flights were made between July 2021 and August 2022.In these flights, we employed 'convenient sampling', and found fauna classes: dolphins (Tursiops sp.), sharks (Carcharhinus spp.), guitarfish (Rhinobatidae), other rays (Aetobatus narinari, Rhinoptera neglecta), and whales (Megaptera novaeangliae), which have been found along the east Australian coastline in drone-based coastal surveys [18,20,22].However, dolphins were found to be the most appropriate class for the analysis, due to their vertical movement through the water column. Survey flights were conducted in varied winds of up to around 15 knots (7.7 m s −1 ), and in the absence of rain at Ballina, Evans Head, Tuncurry, Forster, Birubi, and Anna Bay on the east coast of Australia (Figure 2).The maximum wind tolerance for sampling was set intentionally lower than the recommended maximum for the aircraft, due to the added payload, which effectively increases the working load on the motors and electronic speed controllers, which require higher amounts of current from the batteries.Care was also taken to fly the aircraft smoothly to avoid unnecessary 'ramp-up' of the motors and associated current draw.If wind gusts frequently exceeded ~15 knots, the aircraft was flown back to the ground control station.Flights were made at ~60 m altitude and according to procedures in Colefax et al. [17], using the real-time telemetry of the onboard RGB camera to sight fauna.Flights were made in both directions, following the coastline, just behind the surf break.Once fauna was detected, the drone was lowered to 15-20 m, the camera treatments triggered to record (30 fps at 4k UHD resolution) and the animal/s tracked until it disappeared into the water column, battery was effectively depleted, or weather deteriorated (rainfall or wind >8 m s −1 ).Because the animal was tracked, the trajectory of the drone varied and approximately matched that of the trajectory of the animal.The added payload did impact flight time, with the drone not specifically designed or optimised for the extra weight.Therefore, the maximum wind tolerance for sampling was set intentionally lower than the recommended maximum for the aircraft, due to the added payload, which effectively increases the working load on the motors and electronic speed controllers.This, in turn, draws higher amounts of current from the batteries.Care was also taken to fly the aircraft smoothly to avoid unnecessary 'ramp-up' of the motors and associated current draw.If wind gusts frequently exceeded ~15 knots, the aircraft was flown back to the ground control station.associated current draw.If wind gusts frequently exceeded ~15 knots, the aircraft was flown back to the ground control station.Flights were made at ~60 m altitude and according to procedures in Colefax et al. [17], using the real-time telemetry of the onboard RGB camera to sight fauna.Flights were made in both directions, following the coastline, just behind the surf break.Once fauna was detected, the drone was lowered to 15-20 m, the camera treatments triggered to record (30 fps at 4k UHD resolution) and the animal/s tracked until it disappeared into the water column, battery was effectively depleted, or weather deteriorated (rainfall or wind >8 m s −1 ).Because the animal was tracked, the trajectory of the drone varied and approximately matched that of the trajectory of the animal.The added payload did impact flight time, with the drone not specifically designed or optimised for the extra weight.Therefore, the maximum wind tolerance for sampling was set intentionally lower than the recommended maximum for the aircraft, due to the added payload, which effectively increases the working load on the motors and electronic speed controllers.This, in turn, draws higher amounts of current from the batteries.Care was also taken to fly the aircraft smoothly to avoid unnecessary 'ramp-up' of the motors and associated current draw.If wind gusts frequently exceeded ~15 knots, the aircraft was flown back to the ground control station. Analysis A flight was considered successful when we were able to capture marine fauna across all three sensors, in correct exposure, and with sufficient clarity to reliably identify the fauna as it shifted horizontally and vertically in the water column to encompass a range of 'sightability' conditions [17,25].Initial trials of the three-camera setup determined that the cameras recorded within one frame of each other.This was further verified on each video by carefully assessing frame numbers with short-lived events (e.g., the moment a dolphin breaks the surface for air) that were seen by all sensors and could subsequently be used to verify frame-matching across cameras, which enables direct comparisons of the camera treatments. To empirically contrast the camera treatments of RGB (control), spectral filtering (green filter), and spectral filtering with polarisation, data were analysed using artificial intelligence (AI) deep learning methods.This approach was chosen to eliminate the potential biasing resulting from the subjectivity of using human observation methods to assess the imagery and score for clarity with regards the detectability of submerged fauna.The AI model used as a proxy was an existing RetinaNet single-shot detector (SSD) with a Resnet-50 backbone classifier that was trained on a very large marine fauna dataset (see Purcell et al. [25]).The procedures regarding this previous research for creating the AI model involved carefully annotating (supervised learning) marine faunal datasets that were captured from drone-based shark surveillance trials along coastal beaches of eastern Australia.For each annotation, a set of bounding box coordinates (tightly marking the spatial extent of the animal in the imagery) along with the animal's classification was recorded with reference to the image.The imagery underwent various augmentation steps to normalise the colour profiles and reduce potential data biases with the animal's orientation and size through random rotations and image scaling.The training of the model was conducted in a Tensorflow 1.0 environment (now superseded) for ~50 epochs.Training was deemed finished when the mean average precision (mAP), and loss curves (common observational tools to monitor training success of a machine learning model), were observed to flatten out. The approach for the analysis using this previously developed AI model involved first drawing ground-truth boxes over the video footage from each of the camera treatments, and then comparing the ground-truth to boxes generated by the AI model.To create the ground-truth boxes, we manually annotated a select range of frames for each flight using a custom graphical user-interface (GUI) that supported machine learning labelling operations for video.Through the process of 'boxing', a coordinate system within each image was generated, which defined the location and bounds of an animal.The box represented the smallest rectangle that can be drawn that completely encapsulated the animal.This was conducted across the range of matching frames for each camera treatment, for each video.The frame range was chosen such that there was no ambiguity in the footage about how many dolphins were present and their locations, which were kept to the middle area of the frame to avoid effects of vignetting or phase shift from the spectral filters (Figure 3).We achieved this by following individual dolphins through the footage from the surface, where they are clearly identified, to being deep in the water column.Where there was more than one dolphin in the frame, each individual was separately tracked and boxed.Animals typically surfaced and dived more than once in a video.When an animal disappeared in the water column (i.e., beyond the sightability threshold), we interpolated boxes for the animal for a number of frames to ensure full extent of sightability was achieved for all three camera treatments.Once ground-truth boxes were established, we ran inferencing on the videos with the AI model to create AI-generated boxes (Figure 3).Both the ground-truth boxes and the AI-generated boxes were restricted to correspond with cases where animals in the image were not noticeably impacted by phase shifting or vignetting, which was apparent in some scenarios (Figure 3).The AI-generated boxes across the videos were temporally clipped so that the inferencing only corresponded to the same frame sequences of the groundtruth boxes.An important factor for defining whether an AI-generated box matches a ground-truth box is Intersection over Union (IoU), which is a standard measure of the fraction of the AI box that covers the ground-truth box.For a perfect match, the AI and ground-truth box would be exactly aligned, where the IoU would be 100%.However, a partial overlap can also indicate a correct result.We chose a 50% threshold for the analysis based on the results of data inspection (see results).True positive (TP), false positive (FP), and false negative (FN) detections were defined, based on the IoU threshold.The precision (TP/(TP + FP)), and recall (TP/(TP + FN)) were then calculated for each flight and sensor treatment.Precision represents the fraction of all AI-generated boxes that were correct, whereas recall is the fraction of all ground-truth boxes that the AI-generated boxes replicated.These scores were used to contrast the relative performance of each sensor treatment for detecting submerged fauna.The data was analysed and visualised in python. Results We conducted around 86 flights over 18 separate days, totalling 17 h 18 min of total flight time.Survey efficiency, including the locations and timing of surveys, were impacted by weather constraints and COVID-related restrictions, which led to the general scarcity of fauna across surveys.There was a lack of variation in many animals' vertical Once ground-truth boxes were established, we ran inferencing on the videos with the AI model to create AI-generated boxes (Figure 3).Both the ground-truth boxes and the AI-generated boxes were restricted to correspond with cases where animals in the image were not noticeably impacted by phase shifting or vignetting, which was apparent in some scenarios (Figure 3).The AI-generated boxes across the videos were temporally clipped so that the inferencing only corresponded to the same frame sequences of the groundtruth boxes.An important factor for defining whether an AI-generated box matches a ground-truth box is Intersection over Union (IoU), which is a standard measure of the fraction of the AI box that covers the ground-truth box.For a perfect match, the AI and ground-truth box would be exactly aligned, where the IoU would be 100%.However, a partial overlap can also indicate a correct result.We chose a 50% threshold for the analysis based on the results of data inspection (see results).True positive (TP), false positive (FP), and false negative (FN) detections were defined, based on the IoU threshold.The precision (TP/(TP + FP)), and recall (TP/(TP + FN)) were then calculated for each flight and sensor treatment.Precision represents the fraction of all AI-generated boxes that were correct, whereas recall is the fraction of all ground-truth boxes that the AI-generated boxes replicated.These scores were used to contrast the relative performance of each sensor treatment for detecting submerged fauna.The data was analysed and visualised in python. Results We conducted around 86 flights over 18 separate days, totalling 17 h 18 min of total flight time.Survey efficiency, including the locations and timing of surveys, were impacted by weather constraints and COVID-related restrictions, which led to the general scarcity of fauna across surveys.There was a lack of variation in many animals' vertical position in the water column.This limited the ability to contrast the three camera treatments in many flights.Due to this, we found dolphins to be ideal surrogates for the wider range of fauna classes, as they frequently shift their position in the water column and, in this context, do not spectrally differ from other fauna (see Colefax et al. [29]).Dolphins were also a trained class in the AI model used for the analysis.Due to some technical issues (all three cameras needed to work in sync with the correct exposure), being restricted to specific fauna classes for the analysis, and eliminating cases where the animal in the image may be affected by phase-shifting or vignetting, the dataset was narrowed down to nine successful flights, in a variety of water clarity conditions, on five separate days (Table 1).The assessments of IoU across frames for each flight showed a general normal distribution that highlighted a 50% threshold would be appropriate to contrast the camera treatments for determining the comparative levels of relative detection performance (Figure 4).While the specific value of IoU can be arbitrary, it is not a definitive measure of detection performance, nor does it bias the overall results when the same IoU threshold is used across all treatments.The AI model was used here as a tool to reliably assess the relative performance of each sensor by setting the threshold for the AI to detect fauna, and then count the number of boxes that the AI model correctly predicts. The distribution of IoUs across flights generally peaked at approximately 74% and followed a normal distribution tailing at ~50% and ~90%.During flights where there was more than one dolphin, a second peak in the distribution was generally found, usually peaking around 35% IoU (Figure 4).This was due to dolphins swimming in close proximity to each other, and subsequently creating overlapping boxes in post-analysis for both ground-truth and AI-generated boxes.Therefore, a true positive (TP) was defined as an AI-generated box that overlapped with a ground-truth box by at least 50%.A false positive (FP) was defined as an AI-generated box that did not overlap with a ground-truth box.A false negative (FN) was defined as a ground-truth box that had no corresponding AIgenerated box.The TP, FP, and FN rates were calculated for all flights and camera treatments (Table 2).Because of the nature of the ground-truth boxes, including interpolations that were made for animals beyond the sightability threshold, the precision, recall, and F1 scores relating to the camera treatments are not indicative of AI performance, and reflect relative comparisons between the camera treatments (Figure 5).For more information and an empirical assessment regarding the utility of the AI used in this study for detecting submerged marine fauna along coastal beaches of eastern Australia, see Purcell et al. [25].The figure shows a clear peak of approximately 70%, with a secondary peak of approximately 35%, which was due to a second dolphin in extremely close proximity.From these plots, the arbitrary IoU of 50% was selected. The distribution of IoUs across flights generally peaked at approximately 74% and followed a normal distribution tailing at ~50% and ~90%.During flights where there was more than one dolphin, a second peak in the distribution was generally found, usually peaking around 35% IoU (Figure 4).This was due to dolphins swimming in close proximity to each other, and subsequently creating overlapping boxes in post-analysis for both ground-truth and AI-generated boxes.Therefore, a true positive (TP) was defined as an AI-generated box that overlapped with a ground-truth box by at least 50%.A false positive (FP) was defined as an AI-generated box that did not overlap with a ground-truth box.A false negative (FN) was defined as a ground-truth box that had no corresponding AI-generated box.The TP, FP, and FN rates were calculated for all flights and camera treatments (Table 2).Because of the nature of the ground-truth boxes, including interpolations that were made for animals beyond the sightability threshold, the precision, recall, and F1 scores relating to the camera treatments are not indicative of AI performance, and reflect relative comparisons between the camera treatments (Figure 5).For more information and an empirical assessment regarding the utility of the AI used in this study for detecting submerged marine fauna along coastal beaches of eastern Australia, see Purcell et al. [25]. The comparisons of precision, recall and F1 score between the sensor treatments indicated that the RGB sensor (average F1 score 66.1 ± 5.0%) consistently outperformed the green sensor (average F1 score 35.8 ± 3.6%), which in turn outperformed the green/polarising filter treatment (Table 2, Figure 5 and average F1 score 28.8 ± 23%).The comparisons of precision, recall and F1 score between the sensor treatments indicated that the RGB sensor (average F1 score 66.1 ± 5.0%) consistently outperformed the green sensor (average F1 score 35.8 ± 3.6%), which in turn outperformed the green/polarising filter treatment (Table 2, Figure 5 and average F1 score 28.8 ± 23%). Discussion This study demonstrated that, out of the camera treatments, the best detection reliability results are obtained by using a RGB sensor that is not spectrally restricted, and worst when applying a spectral and polarising filter (in the green band) to an RGB sensor.This is contrary to our initial hypothesis where we expected to see the best performance with the green/polarising filter treatment.The results indicate that the RGB treatment performed better than the green filter treatments.This may suggest that there is useful information captured in the red and blue bands that is lost with applying the green filter.In contrast, while the green/polarising filter was outperformed by the green filter, there is no difference in the wavelength range that the sensors are sensitive to.This may be explained by the fact that only the light of one polarisation is able to pass through to the green/polarised sensor, and the brightness is consequently attenuated by half.Therefore, the signal to noise ratio is also degraded at the sensor, resulting in less information being used to identify a marine animal.This is an unfortunate consequence of light filters, and our results suggest that, whilst narrowing the wavelength range might allow a sensor to 'focus in' on the most information-dense part of the spectrum, there is a much larger loss of light from the filter that means the overall performance is degraded. Detecting submerged marine fauna using drones can be challenging due to various factors such as lighting conditions and the inherent variability of the environment in which the animals reside [1,19,41].This can cause significant sightability errors which affects the reliability of detections and subsequent identification of target fauna [20,42,43].To improve this, hyperspectral research investigating the difference in reflectance between fauna and surrounding seawater along coastal beaches of eastern Australia, across 400-1000 nm wavelengths, suggested the vast majority of contrast for detecting fauna was found consistently within the 515-554 nm range [29].However, the research presented here found that restricting light to these wavelengths entering an RGB sensor worsened the reliability of detections.Furthermore, because the spectral difference of fauna against surrounding seawater in coastal environments does not differ, the results of this study would also apply to other fauna classes.Therefore, if operations are using RGB cameras to spot submerged marine life, then there is no benefit from applying spectral filters.However, other research suggests that polarizing filters can aid in detections by reducing sea-surface reflections [17,42,43]. This study used machine learning to empirically contrast the three camera treatments with arguably less bias than alternative human observation comparison methods.However, as image dimensions are defined as pixels (x, y) as well as layers (one layer for each colour channel), it is evident that the imagery held useful information in the blue and red colour channels.This aided the model in identifying fauna beyond just using the green channel, where the useful information (signal) across all colour channels clearly outweighed the noise [30].Research on the utility of different spectral bands for detecting submerged whales from satellite showed that, in deeper water, the coastal blue band was superior for detecting southern right whales compared to panchromatic or red-edge bands, and provided the greatest contrast of the animal against the surrounding seawater [30].However, other studies have performed spectral processing of spatial information from narrow bands (20 nm bandpass), from the blue (~480 nm), as well as green (~535 nm) and red (~600 nm) and reported enhanced detection of submerged whales over standard RGB imagery [31].This highlights that, although the majority of contrast or ability to detect an animal from surrounding seawater is mostly reliant on a fairly narrow band between the blue and green wavelength range (which would depend on water characteristics of the region of sampling), there is useful information in the broader spectrum (~400-600 nm) beyond the spectral range of the majority of contrast.The greater spectral resolution gained from targeting specific wavelengths of light across the visible spectrum from a multi-or hyper-spectral sensor may offer superior spectral processing and an overall advantage for the detection reliability of submerged fauna than standard RGB sensors for a given spatial resolution.However, research has been proof-of-concept and empirical comparisons between RGB imagery and multispectral cameras are currently scarce [31,44]. It has been recognised that machine learning can reduce the bottleneck that is often encountered during the post-analysis of imagery, and offer further benefits in reducing some sample biases and improving the reliability and consistency of detections [4].Machine learning can also offer valuable real-time decision support, where detection and classification reliability have direct and immediate implications, such as drone-based surveys for reducing shark-bite mitigation [22,24,25,27,28].For the analysis of multiband imagery, it is possible that providing spectral processing to the imagery (i.e., enhancing, augmenting or weighting colour channels in a mixing model) can improve the overall detection response [35].This may also assist as a data pre-processing step for machine learning applications [25].However, if concerning RGB imagery, standard image augmentation processes already adjust the input data and provide weightings in the colour channels through model training.Although it is likely that adding channels from a multi-hyper-spectral array would provide more information and result in a potentially more reliable model for detecting fauna, the implementation of object-detection methods on sensors that provide high numbers of colour bands can be extremely resource intensive.This presents significant challenges, particularly for on edge or real-time applications [22,25,27].This would either result in poor inference time, a need for expensive computing hardware, or a compressed model, which could undermine the benefits of having multiple input channels. The RGB cameras used in this study (GoPro Hero 8 cameras) were not designed for spectral filtering, as applied in the filter treatments.A side effect of this was an effective reduction in the overall light hitting the sensor and an imbalance in the expected colour channel inputs, potentially effecting the colour mixing model applied in the camera.A way to help counteract this was to fix the white balance at 5600 K.However, due to the reduced light, even within the green colour channel, passing through the filter to the sensor, meant that the sensor required the exposure and sensitivity to be increased.This potentially impacted the quality of the image due to exposure compensation of the camera and could bias the results to some degree, particularly with regards to the green polarising filter treatment.Therefore, there may be an improvement in the comparative performance of spectral filtering, such as around 514 and 554 nm for coastal water [29] with sensors more capable of handling wavelength restriction across the colour spectrum (such as panchromatic sensors).However, based on the results of this study, filtering panchromatic lenses (or similar) is unlikely to lead to improvements in detection reliability beyond what can be achieved from standard RGB cameras. This study demonstrated that, although the majority of visible contrast between a submerged marine animal and surrounding seawater occurs in relatively narrow colour bands, such as between 515-554 nm in beach environments in eastern Australia, isolating the colour input to an RGB sensor does not improve detection reliability.Therefore, the signal to noise outside of this range is still high enough to be of benefit to fauna detection.Due to the rapid absorption and scattering of high frequency light, the utility of multispectral cameras likely is restricted to the visible spectrum, and therefore may not provide significant benefits over RGB cameras, unless many colour bands are leveraged, which has implications regarding the utility and application.Further research into spectral processing on imagery to enhance contrast in the image is required, but current technologies in most circumstances are unlikely to increase the range of sightability into the water column beyond what can be achieved by the current range of RGB cameras. Figure 1 . 1 Figure 1.(A) The light transmission (%) for the corresponding wavelength (nm) of the narrow green bandpass filters used for filtering treatments.The near-infrared blocking filters (that come standard) in the CMOS sensor of the GoPro negate transmission in the higher (>850 nm) wavelengths.(B) GoPro sensor array in the custom mounting, attached to the landing gear of the Phantom 4 Pro drone. Figure 2 . 2 Figure 2. Survey locations on the east coast of Australia.Ballina and Tuncurry represented sites with data that were used in the final analysis.Evans Head, Forster, Birubi and Anna Bay were surveyed but not included in the analysis. Figure 2 . 2 Figure 2. Survey locations on the east coast of Australia.Ballina and Tuncurry represented sites with data that were used in the final analysis.Evans Head, Forster, Birubi and Anna Bay were surveyed but not included in the analysis. Figure 3 . 3 Figure 3.An example of a single frame from each sensor treatment taken on Flight 7, 6 October 2021 at Ballina Headland, NSW, Australia.Top (A) shows the unfiltered RGB (red-green-blue channel) sensor, middle (B) shows the sensor with green spectral filter, and bottom (C) shows the sensor with the green and polarising filter.Each sensor frame is matched in time.Yellow boxes are drawn by the AI model and used for direct comparison of sensor performance. Figure 3 . 3 Figure 3.An example of a single frame from each sensor treatment taken on Flight 7, 6 October 2021 at Ballina Headland, NSW, Australia.Top (A) shows the unfiltered RGB (red-green-blue channel) sensor, middle (B) shows the sensor with green spectral filter, and bottom (C) shows the sensor with the green and polarising filter.Each sensor frame is matched in time.Yellow boxes are drawn by the AI model and used for direct comparison of sensor performance. Figure 4 . 4 Figure 4.A plot showing an example of a distribution of intersection over union (IoUs) analysis for the RGB (control) sensor for flight 7 on 6 October 2021.The figure shows a clear peak of approximately 70%, with a secondary peak of approximately 35%, which was due to a second dolphin in extremely close proximity.From these plots, the arbitrary IoU of 50% was selected. Figure 4 .Table 2 . 42 Figure 4.A plot showing an example of a distribution of intersection over union (IoUs) analysis for the RGB (control) sensor for flight 7 on 6 October 2021.The figure shows a clear peak of approximately 70%, with a secondary peak of approximately 35%, which was due to a second dolphin in extremely close proximity.From these plots, the arbitrary IoU of 50% was selected. Figure 5 . 5 Figure 5. Precision and recall histograms for each flight and each sensor treatment, including RGB (red-green-blue channel), GRN (green filtered sensor treatment) and GPL (green and polarising filter treatment).The highest relative precision and recall (detection reliability) are obtained with unfiltered RGB, while the lowest reliability occur from the GPL sensor treatment. Figure 5 . 5 Figure 5. Precision and recall histograms for each flight and each sensor treatment, including RGB (red-green-blue channel), GRN (green filtered sensor treatment) and GPL (green and polarising filter treatment).The highest relative precision and recall (detection reliability) are obtained with unfiltered RGB, while the lowest reliability occur from the GPL sensor treatment. Table 1 . 1 Summary of successful flights where dolphins were captured in all three sensors.The number of frames shown in the last column refers to the frames that were used in direct comparison between the sensors. Flight NumberDateLocationNo. DolphinsNo. Frames131 August 2021Ballina2400231 August 2021Ballina2980331 August 2021Ballina265041 September 2021Ballina145056 October 2021Ballina145066 October 2021Ballina115076 October 2021Ballina637984 October 2021Tuncurry1105098 June 2022Tuncurry3300 Sensors 2023, 23, x FOR PEER REVIEW 5 of 15 Data Availability Statement: Data available on request from the corresponding author with the approval from NSW DPI.Funding: This project was funded by the NSW Government through the NSW Shark Management Program.Institutional Review Board Statement: Drone flights were made under New South Wales Department of Primary Industries (NSW DPI) and Office of Environment & Heritage (OEH) scientific permits (Ref.SL102196) and animal ethics (16/09-SIMS).Author Contributions: Conceptualization, all authors; methodology, A.P.C., A.J.W. and C.R.P.; software, all authors; validation, A.P.C. and A.J.W.; formal analysis, A.J.W.; investigation, A.P.C.; writing-original draft preparation, A.P.C.; writing-review and editing, all authors; funding acquisition, P.B.All authors have read and agreed to the published version of the manuscript.Conflicts of Interest:The authors declare no conflict of interest.A.C. and A.W. are affiliated with Sci-eye Pty Ltd., and C.P. has an affiliation with Trillium Technologies Pty Ltd., and although these are commercial entities, there is no commercial gain for any company from any output of this research. 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Chaga mushroom triterpenoids as adjuncts to minimally invasive cancer therapies: A review 15 November 2023 Selina Plehn Department of Plant, Food, and Environmental Sciences Faculty of Agriculture Dalhousie University B2N 5E3TruroNova ScotiaCanada Sajeev Wagle Department of Plant, Food, and Environmental Sciences Faculty of Agriculture Dalhousie University B2N 5E3TruroNova ScotiaCanada H P Vasantha Rupasinghe Department of Plant, Food, and Environmental Sciences Faculty of Agriculture Dalhousie University B2N 5E3TruroNova ScotiaCanada Department of Pathology Faculty of Medicine Dalhousie University B3H 4H7HalifaxNova ScotiaCanada Chaga mushroom triterpenoids as adjuncts to minimally invasive cancer therapies: A review 15 November 20236A74869FCDC784E789B17A2FA5E4F51310.1016/j.crtox.2023.100137Received 25 June 2023; Received in revised form 13 October 2023; Accepted 14 November 2023Chaga mushroom Triterpenoid Cytotoxicity Cancer IC 50 Cancer has become the second leading cause of death in the world.Integrative cancer therapy management is continuously evolving to enhance treatment outcomes.Chaga mushroom (Inonotus obliquus) is a parasitic fungus acclaimed to contain pharmaceutical and nutraceutical value in the fight against cancer.In particular, triterpenoid constituents derived from Chaga mushrooms have been recognized for their anti-cancer activity after distinguished cytotoxicity was repeatedly observed in cancer cells treated in vitro with lipophilic fractions of extract compared to aqueous ones.Studies that investigate the anti-cancer activity of Chaga mushroom triterpenoids are reviewed in this article to determine which cancer cell lines demonstrate the greatest susceptibility to them while highlighting the structure-activity relationships that are involved.Triterpenoid supplementation as an adjunct to cancer treatment may be a viable option as inotodiol and 3-β-22 α-dihydroxylanosta-8, 25-diene-24-one have been shown to exhibit anti-cancer activity similar to that of conventional drugs.Advances in addressing bioavailability challenges are also included in this review as studies include in vivo components. Introduction Chaga mushroom (Inonotus obliquus) is a parasitic white rot fungus in the Hymenochaetaceae family under the Basidiomycota division (Agnestisia et al., 2019;Lee et al., 2008).Visible sterile conks or sclerotia of densely aggregated mycelia form on the trunks of broad-leaved deciduous angiosperms, namely birch, that contain a vast range of metabolites of nutraceutical and pharmaceutical value (Kim et al., 2011;Campbell and Davidson, 1938;Lee et al., 2008;Jiang et al., 2020;Balandaykin and Zmitrovich, 2015;Kim et al., 2020;Lau and Abdullah, 2016;Willetts and Bullock, 1992).After a suitable living host tree incurs an injury, Chaga mushroom penetrates the wound where decay spreads throughout the heartwood for years during the anamorphic stage (Lee et al., 2008).The spore-forming sexual reproducing resupinate layers of Chaga mushroom initiate underneath the bark of dead wood (Lee et al., 2008) and are rarely detected as they promptly attract mycophagous beetles such as Orchesia cultriformis that may act as vectors to disperse viable spores elsewhere (Bunyard, 2015).Chaga mushroom is widely available in the cold circumboreal regions of the northern hemisphere (Lee et al., 2008), and has been consumed for centuries, especially by Khanty people in Russia, for health maintenance and hygiene (Saar, 1991). Chaga mushroom sclerotium is typically harvested during the winter months for the retrieval of bioactive compounds which have been acclaimed to exhibit protective effects against diabetes (Zhang et al., 2018), inflammation (Kou et al., 2021), hepatoxicity (Alzand et al., 2018), and cancer (Alzand et al., 2018;Zhao et al., 2015;Drąg-Zalesińska et al., 2017;Wold et al., 2020;Ma et al., 2013;Wang et al., 2022;Lee et al., 2021;Tanaka et al., 2011;Li et al., 2010). Trending research has led to the corroboration of traditional claims on the efficacy of Chaga mushroom-derived compounds in exhibiting anti-cancer effects (Zhao and Zheng, 2021).In Japan, the habitual intake of Chaga mushroom infusion is estimated to be around 6 mg/kg/ day, and tumor growth was suppressed in mice administered Chaga mushroom extract at this dose for 3 weeks prior to and 16 days after Lewis lung carcinoma (3LL) cell implantation (Arata et al., 2016).Bioactive constituents found in Chaga mushroom extract include polysaccharides (Jiang et al., 2020), organic acids (Glamočlija et al., 2015), an extensive array of phenolic and isoprenoid compounds (Glamočlija et al., 2015), and most notably, triterpenoids (Zhao et al.,2015).While inhibitory effects against cancer exist for aqueous extracts (Arata et al., 2016;Géry et al., 2018), Chaga mushroom triterpenoids and sterols have taken center stage in current research as studies share similar findings regarding their prominent cytotoxicity toward various cancer cell types without impacting normal cells (Kang et al., 2015;Ma et al., 2013;Kim et al., 2020;Nomura et al., 2008).Zhao and Zheng (2021) reviewed the antitumoral potential of Chaga mushroom extracts and concluded the potential of their bioactive metabolites to reduce the incidence of tumorigenesis in healthy people.These findings are encouraging as cancer remains a serious public health concern (Ferlay et al., 2021), and prognoses may be exacerbated following the use of conventional therapies alone (Schirrmacher, 2018). To address the increasing need for alternative cancer treatments, research has further sought to identify the most effective triterpenoids in Chaga mushroom extract to exhibit anti-cancer activity and associate them with their most susceptible cancer cell lines.To date, the cancer cell lines A549 (Zhao et al., 2015;Wang et al., 2022), 4 T1 (Zhao et al., 2016), MCF-7 (Zhao et al., 2016), HT29-MTX (Li et al., 2010;Wold et al., 2020), Me-45 (Drąg-Zalesińska et al., 2017), KB (Alzand et al., 2018), BEL-7402 (Alzand et al., 2018), L1210 (Tanaka et al., 2011), MDA-MD-231 (Lee et al., 2021), SK-BR 3 (Lee et al., 2021), and PC3 (Ma et al., 2013) have been used to demonstrate significant susceptibility to triterpenoid and steroid derivatives isolated from Chaga mushroom with half maximal inhibitory concentrations (IC 50 ) ≤ 10 µM (Table 1). Due to the diverse nature of triterpenoids, this review takes a closer look at the structure-activity relationships that exist for anticancer, antiinflammatory, and immunomodulatory capabilities.Factors that influence bioavailability and application as a potential adjunct to minimally invasive cancer therapies are also discussed. Nutritional value and bioactive compounds Chaga mushroom sclerotia contain a variety of myco chemicals with medicinal value and nutraceutical properties.Chaga mushroom composition varies, but some extracts have been found to contain trace amounts of oxygenated aliphatic substances like linoleic acid, the presence of fat-soluble vitamin E, approximately 7 % protein, and a fair assortment of minerals including calcium, sodium, magnesium, and boron (Petrović et al., 2019;Ayoub et al., 2009;Abu-Reidah et al., 2021).Extracts of Chaga mushroom have been reported to contain as Table 1 Half maximal inhibitory concentration (IC 50 ≤ 10 µM) results after treatment with Chaga mushroom triterpenoids and steroid derivatives.much as 57-70 % polysaccharides (Jiang et al., 2020), many of which make up insoluble fibers with cross-linked chitin and β-1,3-glucans that support its unyielding cell walls (Rhee et al., 2008).A wide range of phenolic compounds exist in Chaga mushroom sclerotia including but not limited to gallic acid, protocatechuic acid, 3,4-di-hydroxybenzaldehyde, caffeic acid, 2,5-dihydroxy terephthalic acid, syringic acid, and vanillic acids (Abu-Reidah et al., 2021;Hwang et al., 2019;Alzand et al., 2018;Glamočlija et al., 2015).The sterile conks have a rough textured exterior that appears to resemble burnt charcoal (Géry et al., 2018), which can be attributed to the high melanin content of the 1,8-dihydroxynaphthalene (DHN) type (Wold et al., 2020).Melanin deposition normally occurs during sclerotium maturation as seen in other fungal species besides Chaga mushroom (Willetts and Bullock, 1992).Chaga mushroom contains a variety of triterpenoids, mainly of the lanostane type, that have been observed to exhibit anticancer activity.Inotodiol was found to be the most abundant triterpenoid extracted from Chaga mushrooms followed by trametenolic acid (Kim et al., 2020;Zhao et al., 2016).The dark texturized outer layer of Chaga mushroom tends to be discarded during harvest but might prove serviceable in the extraction process as Kim et al. (2020) discovered that it contains higher concentrations of betulinic acid and inotodiol in relation to the interior.More triterpenoid structures isolated from Chaga mushroom extract are being recognized for their biological activity.Kou et al. (2021) isolated seven additional lanostane triterpenes named inonotusols H-N (1-7) with 21, 24-cyclopentanol moiety in the side chain from Chaga mushroom ethanol extract that showed considerable inhibitory activity by Griess reaction of nitric oxide generation in lipopolysaccharide (LPS)induced BV-2 microglial cells with IC 50 ranging from 2.3 to 23.8 µM.Even triterpenoids from other natural sources are being acknowledged for their antineoplastic properties.An oleanane-type triterpenoid saponin, known as raddeanin A, isolated from Anemone raddeana Regal was found to have cytotoxic effects against different cancer cell types (Naz et al., 2020).Garádi et al. (2021) recently studied a lesser-known species closely related to Chaga mushroom, named Inonotus nidus-pici, containing ergostane-type triterpenoids that were found to exhibit cytotoxic activity towards MES-SA, MES-SA/Dx5, and A431 cells with IC 50 between 36.2 and 83.2 µM.Betulin and betulinic acid are lupanetype triterpenoids that developed in Chaga mushrooms through convergent evolution (Safronov, 2022).Chaga mushrooms generally contain more betulinic acid than birch bark and vice versa (Safronov, 2022).Triterpenoid profile and content extracted from Chaga mushrooms and other sources vary significantly depending on environmental factors such as the geographical location of harvest (Géry et al., 2018;Zhou et al., 2021).For instance, Chaga mushroom from Normandy (northern France) was found to contain more betulin and betulinic acid, but less inotodiol when compared to their Canadian counterparts (Géry et al., 2018).Harsh growth conditions and environmental stress were shown to increase triterpenoid concentration and diversity while cultured Chaga mushroom mycelia in a medium consisted of fewer bioactive compounds (Zheng et al., 2007;Razumov et al., 2019). While inhibitory effects against cancer exist for aqueous extracts (Arata et al., 2016;Géry et al., 2018), Chaga mushroom triterpenoids have been recognized for their anti-cancer activity as distinguished cytotoxicity was repeatedly observed in cancerous cells treated with lipophilic fractions of hexane, petroleum ether, chloroform, and dichloromethane, displaying IC 50 compared to aqueous fractions (Kang et al., 2015;Kim et al., 2020;Baek et al., 2018;Nomura et al., 2008).Triterpenes are routinely identified as being the main constituents in these fractions due to their relatively low water solubility (Glamočlija et al., 2015).Quantification of triterpenoids is generally completed with the use of liquid chromatography-mass spectrometry (HPLC-MS), and 1 H nuclear magnetic resonance (NMR) data are often referred to for structural elucidation.Triterpenoid compounds continue to be discovered in Chaga mushroom extracts as studies optimize fractionation procedures, employ advanced analytical techniques, and maximize extraction efficiency (Alzand et al., 2018). Formation of triterpenes and their classification Isoprenes are the basic constituents that form terpenoids, a diverse group of ubiquitous organic compounds involved in metabolic processes, biological activities, and the formation of structures in cellular membranes.A monoterpene is an isoprene dimer, and the number of terpenes that exist in an organic compound is denoted by a prefix.Hence, triterpenes contain six isoprene units and a total of 30 carbons.Terpenoids are derivatives of organic terpene hydrocarbons that contain characteristic functional groups, while triterpenoid saponins refer to triterpene glycosides (Zhao et al., 2010). The pattern of isoprene head-to-tail addition in the formation of higher molecular terpenoid structures, discovered by Leopold Ruzika, is known as the isoprene rule (Hillier and Lathe, 2019).The active isoprene building block, isopentyl pyrophosphate, is first synthesized in the mitochondria via the mevalonate pathway or the cytoplasm by the non-mevalonate pathway depending on the organism or physiological conditions (Zhao et al., 2010;Kushiro and Ebizuka, 2010).Pacbio genome sequencing of Chaga mushroom and associated birch from the Merikarvia region in Finland revealed CYP450 monooxygenase enzymes involved in mevalonate pathways for betulinate biosynthesis (Safronov et al., 2021).Cytochrome CYP505 monooxygenases are responsible for encoding enzymes that facilitate the formation of terpenoid metabolites in Chaga mushrooms as well as other fungi (Safronov, 2022).Terpene and terpenoid biosynthesis occur through a series of enzymatic reactions that are initiated when isopentyl diphosphate and its isomer dimethylallyl diphosphate are joined through condensation to form the monoterpene or isoprene dimer (C 10 ), geranyl diphosphate (Faylo et al., 2021;Gill and Kumar, 2016).The addition of another active isopentyl pyrophosphate generates farnesyl diphosphate (C 15 ), which can act solely as a precursor for sesquiterpenes (C 15 ) or create squalene (C 30 ) by dimerization (Gill and Kumar, 2016;Brodelius et al., 2002).Squalene is a triterpenoid precursor that undergoes cyclization to form a variety of ring systems with a vast combination of terpene skeletal diversity (Brodelius et al., 2002;Kushiro and Ebizuka, 2010;Xu et al., 2004).Triterpenes are subdivided by the number of rings present in their structure and further differentiated by their stereochemical configuration.Tetracyclic and pentacyclic triterpenoids (4 and 5 ring containing triterpenoids) are abundantly found in nature (Xu et al., 2004) and are studied extensively for their anti-cancer activity (Gill and Kumar, 2016).While many dicotyledonous plants synthesize triterpenoids in response to infection for host defense (Osbourn, 1996), parasitic fungi metabolize triterpenoids from their host and synthesize others to modulate plant growth and provide protection against environmental stressors (Jan et al., 2021). Anti-cancer activity of triterpenoids It has been deduced that triterpenoids are responsible for the antineoplastic properties of Chaga mushroom extracts as significant dosedependent cytotoxicity was repeatedly observed in cancerous cells treated with lipophilic fractions of hexane, petroleum ether, chloroform, ethyl acetate, and dichloromethane, displaying IC 50 compared to aqueous fractions (Kang et al., 2015;Kim et al., 2020;Baek et al., 2018;Ma et al., 2013).Methanol and ethanol extracts are generally the primary solvents used for crude extraction prior to the fractionization of Chaga mushroom triterpenoid constituents.Solvent-partitioned fractions of hexane and dichloromethane featured greater cytotoxicity on cancer cells than crude methanol extract (Kahlos et al., 1987;Baek et al., 2018).Ma et al. (2013) recognized the increased cytotoxicity of petroleum ether fractions against PC3 and MDA-MB-231 cancer cells tested in vitro.Compared to hexane and ethyl acetate, dichloromethane fractions were found to be superior at inhibiting proliferation through cell cycle arrest at the G 1 phase in HT-29 cells (Lee et al., 2015).Cytotoxic concentrations of chloroform-extracted fractions were found to be similar to inotodiol (Nomura et al., 2008).The anti-cancer activity of Chaga mushroom-extracted triterpenoids has been routinely observed through in vitro growth inhibition assays, usually by MTT colorimetric method (Ni et al., 2009).Half maximal inhibitory concentrations reported to be less than 10 µM in cancer cells treated with triterpenoids are outlined in Table 1.Chaga mushroom-derived triterpenoids have been widely studied for their inhibitory effect on cancer cell types such as lung adenocarcinoma cells (Baek et al., 2018;Chung et al., 2010;Wold et al., 2020;Li et al., 2010;Zhao et al., 2020;Wang et al., 2022), colorectal adenocarcinoma cells (Wold et al., 2020;Zhao et al., 2015;Kim et al., 2020;Li et al., 2010;Tsai et al., 2017), hepatoma cell lines (Zhuo et al., 2018;Li et al., 2010;Alzand et al., 2018), human epithelial carcinoma cells (Alzand et al., 2018), melanoma cancer cells (Drąg-Zalesińska et al., 2017), cervical cancer cells (Chung et al., 2010;Wang et al., 2022;Zhang et al., 2019;Li et al., 2010), breast cancer cells (Chung et al., 2010;Lee et al., 2021;Li et al., 2010;Wang et al., 2022), prostate cancer cells (Li et al., 2010;Kim et al., 2020), leukemia cells (Nomura et al., 2008;Li et al., 2010;Zhuo et al., 2018;Handa et al., 2012), and gastric cancer cells (Chung et al., 2010;Wang et al., 2022;Kim et al., 2020). In some cases, triterpenoids have demonstrated inhibitory concentrations similar to conventional drugs alone (Alzand et al., 2018;Zhang et al., 2019).HeLa cells treated with inotodiol were inhibited almost to the same extent as those treated with 5-fluorouracil, a chemotherapy drug (Zhang et al., 2019).Similarly, 3-β-22 α-dihydroxylanosta-8, 25diene-24-one and 5-fluorouracil displayed cytotoxic activity against KB epithelial cell types producing IC 50 of 9.6 µM and 8.1 µM, respectively (Alzand et al., 2018).The effects of standard breast cancer treatment drugs tamoxifen, lapatinib, and doxorubicin combined with inotodiol-and trametenolic acid-enriched fractions displayed synergistic or additive cytotoxic effects (Lee et al., 2021).An additional lanostane triterpenoid was recently found to have a cytostatic effect on gastric adenocarcinoma cell line BCG-823 with an average IC 50 of 12.0 µM, which is lower than that of suberoylanilide hydroxamic acid at 12.4 µM (Wang et al., 2022). Research has progressed in identifying bioactive constituents that contain anti-cancer activity by producing consistent reports on similar cancer cell types.For instance, there are numerous reports on the antiproliferative effects of Chaga mushroom triterpenoids on breast cancer MCF-7 cells.Cytotoxic effects were observed on MCF-7 cell lines at 300 µg/mL from a triterpenoid fraction consisting of betulin, betulinic acid, trametenolic acid, and inotodiol (Kim et al., 2020).An even greater reduction in cell viability was observed by Lee et al. (2021) in MCF-7 cell lines treated with fractions of inotodiol and trametemolic acid at concentrations lower than 10 µg/mL.Inotodiol-enriched fractions were three times more cytotoxic than trametenolic acid against this cancer cell lines with IC 50 between 1.8 and 2.1 µg/mL and 6-7 µg/mL, respectively (Lee et al., 2021).Silica gel CC eluted with chloroform, methanol, and water (13:0.95:0.05)combined with RP-HPLC with methanol and water (83:17) by Zhao et al. (2016) yielded a fraction containing saponaceoic acid I that inhibited MCF-7 cells with an IC 50 concentration of 8.35 µM (Table 1).The cytotoxicity of chaga mushroom-derived triterpenoids differs in magnitude and specificity.Although saponaceoic acid I may demonstrate significant cytotoxicity towards MCF-7 cells, no effect was observed in lung cancer cell lines Calu-6, H1299, A549, and H1264 (Baek et al., 2018). Chaga mushroom triterpenoids are further described here in relation to one another in terms of their cytotoxicity starting with the tetracyclic triterpenoid 3b-hydroxylanosta-8,24 -dien-21-al that has exclusively been attributed to chaga mushrooms since initial reports in 1984 (Kahlos et al., 1984;Wold et al., 2020).It demonstrates greater water solubility compared to other Chaga mushroom-extracted triterpenoids and was found to exhibit stronger inhibitory activity against cancer cell lines A549, AGS, MCF-7, and HeLa than subfractions containing inotodiol and lanosterol (Chung et al., 2010;Kahlos et al., 1984).In vivo observations from the same study conducted by Chung et al. (2010) coincided with in vitro findings where 3b-hydroxylanosta-8, 24-dien-21al inhibited tumor growth to a greater extent than inotodiol and lanosterol with a 33.7 % reduction in tumor weight compared to the control group in Balbc/c mice bearing Sarcoma-180 cells after oral administration at 0.2 mg per mouse per day for 20 days.Further, inotodiol was not detected to have any effect against lung adenocarcinoma cells compared to 3b-hydroxylanosta-8, 24-dien-21-al (IC 50 range of 75-128 µM), trametenolic acid (IC 50 range of 184-227 µM), and chagabusone A (IC 50 range of 82-141 µM) (Baek et al., 2018).Lung cancer cell lines H1264, Calu 6, and A549 were observed to be highly susceptible to 3bhydroxylanosta-8, 24-dien-21-al whereas H1299 was significantly inhibited by the triterpenoid identified as chagabusone A to a greater extent in comparison (Baek et al., 2018).This could be due to the fact that H1299 cells are derived from lymph tissue as opposed to lung tissue, and they have been found to express the tumor-modulating ubiquitin Cterminal hydrolase (UCH-L1) protein (Liu et al., 2014).Future studies should investigate the anti-cancer effects of 3b-hydroxylanosta-8, 24dien-21al due to its enhanced water solubility for in vivo applications. As studies optimize fractionation procedures with different combinations of solvent eluent ratios, a number of inonotsutriols (A-E) have been extracted from Chaga mushroom (Baek et al., 2018;Wang et al., 2022;Taji et al., 2008;Zhao et al., 2015).In a study conducted by Tanaka et al. (2011), inonotsutriol D was shown to have a broad cytotoxic effect against three leukemia cell lines (P388, HL-60, L1210) and an epidermoid carcinoma cell line (KB) with IC 50 between 10 and 24 µM.Murine leukemia cell line L1210 was more vulnerable to Chaga mushroom triterpenoids when compared to P388, HL-60, and KB cells (Tanaka et al., 2011).Inonotsutriol E, D and A were found to strongly inhibit A549 cells (IC 50 1.6, 8.4, and 2.3 µM, respectively) by Zhao et al., 2015.However, in another study conducted by Baek et al. (2018), inhibition was not detected in A549 cells treated with inonotsutriol E or A. Inonotsutriols should be further studied to confirm the cytotoxic effects, especially against A549 lung cancer cell lines. The biological mechanisms involved in the antiproliferative effects of chaga mushroom triterpenoids have been investigated alongside reports of cytotoxicity. One of the hallmarks of cancerous cells is the ability to evade apoptosis and proliferate uncontrollably (Matsuura et al., 2016;Merlin et al., 2021).Using western blot analysis, several apoptotic regulatory protein expression observations confirm the cascading effects involved in the mitochondrial pathway of induced apoptosis in cancer cells treated with Chaga mushroom-derived triterpenoids (Li et al., 2010;Hordyjewska et al., 2019).As such, protein expression and the nature of cancer cells can be strong indicators for targeted triterpenoid susceptibility.It is becoming more established that many Chaga mushroom triterpenoids induce caspase 3-mediated mitochondrial apoptosis through p53 expression in susceptible cancer cell lines (Nomura et al., 2008;Baek et al., 2018;Chung et al., 2010;Li et al., 2010).In addition to apoptosis, Chaga mushroom triterpenoids have been shown to disrupt the cell cycle by eliciting inhibitory signals at certain phases (Lee et al., 2015;Zhuo et al., 2018).Colorectal carcinoma (HCT-116), lung cancer (HKULC2), and glioblastoma (U87, A172) cell lines treated with Chaga mushroom extracts were found to exit the cell cycle at G 0 /G 1 (Li et al., 2022;Tsai et al., 2017) and in G 1 phase (Zhao et al., 2020).The derivative 3,28-di-(2-nitroxy-acetyl)-oxy-betulin was found to inhibit the transition from G 2 /M phase in Huh7 cells (Zhuo et al., 2018).Some triterpenoids have immunomodulatory effects that may target tumor cells aggressively.Intensified dendritic cell maturity, a greater expression of major histocompatibility (MHC)-II and costimulatory molecules, was observed after treatment with inotodiol through phosphatidylinositol-3-kinase activation without a significant increase of inflammatory cytokine promotion (Maza et al., 2021). Overall, lung adenocarcinoma, murine breast cancer, human breast cancer, methotrexate-resistant colorectal adenocarcinoma, melanoma, epidermal carcinoma of nasopharynx, hepatocellular carcinoma, murine S. Plehn et al. lymphocytic leukemia, and prostate cancer cells have demonstrated significant susceptibility to triterpenoid treatment at (IC 50 ) ≤ 10 µM (Table 1) Additional research is needed to confirm the level of cytotoxicity in the listed triterpenoids, especially using the MTT colorimetric method with multiple incubation times.Highlighting the structure-function relationships that appear as an indication of anti-cancer activity could aid in the prediction of other strongly associated triterpenoids with cancer cell lines that are more prone to susceptibility. Structure-function relationships of triterpenoids and biological significance Triterpenoids, while classified broadly, interact uniquely depending on their structure, stereochemical configuration, and polarity (Gill and Kumar, 2016;Hillier and Lathe, 2019).To illustrate, the absence of a hydroxyl group in lanosterol at carbon 22 compared to inotodiol appears to eliminate the capacity of lanosterol to influence dendritic cell maturity (Fig. 1) (Maza et al., 2021).In terms of stereochemistry, the antiproliferative activity between epimeric triterpenoids inonotsutriol E and inonotsutriol D demonstrated IC 50 1.6 and 8.7 µM, respectively, against A549 cells (Zhao et al., 2015).Due to the lack of functional groups in lanosterol (Fig. 1), the anticancer activity is hardly reported to the same extent as other Chaga mushroom-derived triterpenoids against cells tested in vitro.Aside from murine leukemia L1210 cell line IC 50 37.2 (Zhao et al., 2015), most cancer cell lines fail to exhibit susceptibility to lanosterol.Triterpenoids that share similar polarities such as 3b-hydroxylanosta-8, 24-dien-2-al and inotodiol were found to activate human complement thereby inhibiting serum-induced hemolysis of sheep erythrocytes, whereas trametenolic acid failed to initiate the same cascading effect (Wold et al., 2020).Oncogenic capabilities of the complement system may exist through the facilitation of cellular proliferation and regeneration by dysregulating mitogenic signaling pathways and preventing apoptosis (Rutkowski et al., 2010). Under certain conditions, characteristic patterns of moiety side chains in triterpenoids can be a strong indicator of biological activity (Yang et al., 2015).In two separate studies, betulin-3-O-caffeate, morolic acid 3-O-caffeate, and betulinic acid 3-O-caffeate were observed to have significant anti-inflammatory effects by reducing nitric oxide levels in murine primary macrophages and LPS-stimulated RAW264.7 cells without impacting cell viability, while betulin alone did not (Jeong et al., 2009;Wold et al., 2020).Moreover, C-21 aldehyde groups existing in lanostane-type triterpenoids (Fig. 1) have been observed to enhance inhibitory effects on the formation of Epstein-Barr virus early antigens activated by tumor promotors (Nakata et al., 2007).Inonotusols with a hydroxyl group at C-28 had a less inhibitory activity of nitric oxide production except when accompanied by a conjugated diene system (Kou et al., 2021).In some cases, biological activities exhibited by triterpenoids may be inversely related.Inotodiol, betulinic acid, and betulin failed to exhibit anti-inflammatory activity, yet demonstrated the greatest cytotoxicity towards colorectal cell line HT29-MTX and lung cancer cell line NCI-460, contrary to 3 beta-22-alpha-dihydroxylanosta-8, 25-diene-24-one and trametenolic acid (Wold et al., 2020).Pentacyclic triterpenoids with polar constituents tend to have higher pharmacological activity.Modifications made to C-3 and C-28 carbon positions in betulin, especially with an added piperidine group at C-28, enhanced cytotoxicity up to four times as much as betulin toward cancer cell lines MGC-803, PC3, Bcap-37, A375, and MCF-7 (Yang et al., 2015).A semisynthetic derivative of betulin (3, 28 -di-(2-nitroxy-acetly)-oxy--betulin) demonstrated enhanced cytotoxicity toward hepatocellular carcinoma Huh7 cells with an IC 50 of 13 ± 1.4 µM (Zhuo et al., 2018).Unlike inotodiol (IC 50 of 14 µM), the lanostane triterpenoids trametenolic acid and 3β-hydroxylanosta-8,24-dien-21-al were suggested not to have noticeable anti-proliferative effects for P388 cell lines due to oxygen substitution at C-21 and the lack of a hydroxyl group at C-22 (Nomura et al., 2008).Interestingly, inotodiol with an additional OH group at C-24 (inonotsutriol D, IC 50 10 µM) had greater antiproliferative than insotodiol for P388 cells (Tanaka et al., 2011;Nomura et al., 2008).However, in the study by Tanaka et al. (2011), inotodiol, trametenolic acid, and 3β-hydroxylanosta-8,24-dien-21-al displayed similar cytotoxicity for P388 cells (IC 50 to 51 µM). Chaga mushroom sclerotium contains bioactive secondary metabolites that signify host-pathogen associations as well as convergent evolution (Safronov, 2022;Géry et al., 2018).Betulin and betulinic acid are highly characteristic lupane-type pentacyclic triterpenoids of Chaga mushrooms since they are naturally present in birch bark (Géry et al., 2018;Hordyjewska et al., 2019).Betulin was previously thought to be a relatively inactive constituent.However, betulin and betulinic acid both inhibit cancer cell growth via separate mechanisms (Li et al., 2010).The anticancer effect of betulinic acid has been observed over a wide range of cancer cell types due to the direct effect it has on mitochondrial membrane permeability transition pore (MPTP), whereas betulin cytotoxicity depends on the expression patterns of apoptosis regulating proteins (Li et al., 2010).Compounds that negatively impact MPTP may exhibit toxicity raising safety concerns; thus betulinic acid requires further investigation.Treatment of isolated mitochondria with betulin and betulinic acid confirms this as only betulinic acid-treated mitochondria result in cytochrome c release (Li et al., 2010).Cytotoxic cancer drugs have been reported to facilitate apoptosis by initiating the cytochrome c/Apaf-1/caspase-9-dependent pathway (Druškovič et al., 2006).Enhanced expression of caspase-9 in cancer cell lines such as hepatocellular carcinoma HepG2 was shown to increase their susceptibility to treatment with betulin (Li et al., 2010).In the same study, SK-HEP-1 cells constructed with overexpression of caspase-9 similar to HepG2 cells had enhanced susceptibility to betulin treatment compared to original SK-HEP-1 cells.Li et al. (2022) found betulinic acid to exhibit antiproliferative effects against U87 cells in a dose-dependent manner with IC 50 of 12 µg/mL or 26 µM.Furthermore, betulinic acid treatment at 13 µM resulted in approximately 80 % PC3 cell inhibition (Shin et al., 2011).The same study by Shin et al. (2011) found that treatment with 10 µM betulinic acid prevented hypoxia-induced angiogenesis in PC3 cells by ameliorating the signal transducer and activator of transcription-3 (STAT3) and hypoxia-inducible factor (HIF)-1α interaction with vascular endothelial growth factor (VEGF) promotor, thereby reducing VEGF production.Methotrexate-treated (MTX) HT29 colorectal cell lines tend to produce more mucus and inhibit the permeability of lipophilic compounds, but this was not the case for betulinic acid (Behrens et al., 2001;Wold et al., 2020).In fact, betulinic acid treatment of colorectal HT29-MTX resulted in an IC 50 of 0.8 µM, which makes this triterpenoid-cancer cell type association the most inhibitory one reported to date. Betulinic acid is formed through betulin oxidation where the Fig. 1.Four lanostane-type triterpenoids commonly isolated from Chaga mushrooms (Kim et al., 2020;Zhao et al., 2016).Functional groups present at C-20 and C-22 have been shown to influence biological activity (Maza et al., 2021;Nakata et al., 2007). S. Plehn et al. hydroxyl at C-28 is replaced with a carboxyl group as illustrated in Fig. 2. Comparative analysis of betulin and betulinic acid highlights the significance of functional groups and substituents in addition to overall structure when it comes to modulating anti-cancer activity.As noted previously, treatment with inotodiol (tetracyclic), betulin, and betulinic acid (pentacyclic) on colorectal cell line HT29-MTX and lung cancer cell line NCI-460 shared cytotoxic characteristics despite having different skeletal backbones (Wold et al., 2020).In some cases, generalized characteristics of moiety side chains in triterpenoids as an indicator of biological activity could lead to false assumptions.In general, inonotusols that include 21,24-cyclopental moiety in their structure have shown reduced cytotoxicity towards lung cancer cells in vitro compared to triterpenoids that do not, such as inonotsutriol G (Kou et al., 2021;Liu et al., 2014).However, this was not the case for inonotsutriol E, which has demonstrated prominent cytotoxicity toward A549 cells (Zhao et al., 2015). Biochemical mechanism(s) of anti-cancer activity The biochemical mechanism(s) of triterpenoids of Chaga mushroom is dependent on the specific compounds and their various interactions with cell signalling associated with proliferation, apoptosis, and autophagy (Table 2).For example, betulin and betulinic acid derivatives could exert their anticancer effects through different mechanisms, inclusive of induction of apoptosis and autophagy, antiangiogenesis, inhibition of invasion and migration, cell cycle arrest and multidrug resistance reversal (Li et al., 2010;Shin et al., 2011;Wold et al., 2020).However.current understanding of the toxicological mechanism of various triterpenoids and their mixtures exhibit their selective cytotoxicity to various cancer cells is very limited, thus in-depth investigations are required. Bioavailability of triterpenoids While numerous in vitro studies report the anti-proliferative activity of Chaga mushrooms extracted triterpenoid compounds on various cancer cells, there are some concerns over their bioavailability for effective cancer treatment (Wold et al., 2020;Kim et al., 2021).During LC-MS/MS method validation for the analysis of inotodiol levels in Fig. 2. Characteristic lupane-type Chaga mushrooms derived triterpenoids (Safronov et al., 2021;Safronov, 2022;Géry et al., 2018).Esterified functional amine groups at C-28 were found to enhance the cytotoxicity of betulin (Yang et al., 2015).The presence of moiety 3-O-caffeate may increase anti-inflammatory effects by reducing nitric oxide levels (Wold et al., 2020;Jeong et al., 2009).Modifications at C-3 and C-28 have also been made to improve aqueous solubility (Zhao et al., 2020).A549, human lung cancer cells, 4T1, mouse breast cancer cells; HeLa, human cervical cancer cells; PC3, human prostate cancer cells; HT29-MTX, methotrexate-resistant colon adenocarcinoma cells, bcl2, Antiapoptotic B cell leukemia/lymphoma 2; JAK, Janus kinase; STAT3, signal transducer and activator of transcription 3; HIF-1α, hypoxia-inducible factor-1α; VEGF, vascular endothelial growth factor; AOM/DSS, azoxymethane/dextran sodium sulphate. mouse plasma, Kim et al. (2021) found that oral administration is not an efficient delivery approach.Even after intravenous administration, a low volume distribution of inotodiol was observed within tissues and organs (Kim et al., 2021).In addressing this, a study by Lu et al. (2021) utilized folic acid as a potential targeting ligand in liposomal formulation to increase the bioavailability of inotodiol for its use in treating various types of epithelial-originating cancers.Interestingly, P388bearing mice intraperitoneally administered inotodiol at 10 mg/kg had extended survival without observed side effects compared to the control group (Nomura et al., 2008).Furthermore, mice bearing STZ-DMBA-induced mammary tumors intravenously treated with inotodiol experienced improved survival and limited tumorigenesis through reduced total and phosphorylated β-catenin signaling (Zhang et al., 2018).In addition to inonotiol, the low absorption following oral administration of betulin and betulinic acid also presents a challenge.Derivatives of betulinic acid and betulin have been synthesized for enhanced bioavailability and cytotoxicity.A study found that synthesized betulinic acid nanoparticles were able to significantly inhibit HKULC2, H1299, and H23 by 67 %, 72 %, and 76 %, respectively after treatment with 10 µM (Zhao et al., 2020).Moreover, betulinic acid nanoparticles developed by Li et al. (2022) were able to successfully cross the blood-brain barrier to increase their cellular uptake in central nervous system glioblastoma tumors.In 2017, Drag-Zalesinska et al. developed amino acid ester derivatives of betulin to increase their aqueous solubility while maintaining cytotoxicity toward melanoma Me-45 cells after 72 h of incubation.The greatest anti-cancer activity remained in the lysine and ornithine esters of betulin (Drąg-Zalesińska et al., 2017).An increasing number of in vivo studies are investigating the antitumor activity of Chaga mushroom triterpenoids that show consistent cytotoxicity toward certain cancer cell lines.A study by Chung et al. (2010) with an in vivo component found 3b-hydroxylanosta-8,24-dien-21-al to inhibit tumor growth in Balb/c mice bearing sarcoma-180 cells more than lanosterol and inotodiol.Because aqueous solubility is important for clinical application, 3b-hydroxylanosta-8, 24-dien-21-al should be further studied in vivo in relation to conventional anti-cancer drugs (Chung et al., 2010;Kahlos et al., 1984).Successful encapsulation and modified release of 80 % Chaga mushroom extract in calcium alginate beads was achieved, where the extract was mostly retained at a gastric pH of 1.75 and heavily released at alkaline pH of 8.5 (Petrović et al., 2019).As more cancer studies involving Chaga mushrooms expand to include in vivo components, developments aimed at optimizing the bioavailability and pharmacokinetics of triterpenoids are essential to ensure they encounter target cells. Conclusions Cancer remains a global public health concern that needs to be addressed using multiple approaches.Treatment options and preventative measures that are non-invasive, successful, and easy to administer could promote the global healthcare system, economic recovery, and sustainable work productivity in response to the increasing incidences of cancer worldwide.Additional research is needed to develop safe and effective targeted cancer therapies to mitigate the increasing incidences of mortality and disability-adjusted life years as a result of cancer.In some cases, conventional therapies available to combat advanced stages of cancer have deleterious side effects that require extensive risk-benefit analysis prior to implementation.Among Chaga mushroom bioactives, the strongest anti-cancer activity associations were reported for betulinic acid (IC 50 0.8 µM) against HT29-MTX and inonotsutriol E against A549 (IC 50 1.6 µM).However, in vitro studies should be performed to provide more robust and reproducible data by expanding cell viability tests using multiple assays under different incubation times as well as assessing specificity to cancer cells by comparing the cytotoxicity to the counterpart normal cells.Future research should also focus on susceptible types of cancers using pre-clinical experimental animal models to assess Chaga mushroom triterpenoids and steroid derivatives.These studies can also aim at the biochemical mechanism(s) that can promote anti-cancer drug discovery using Chaga triterpenoids as lead molecules.Structural indicators of oncogenic capabilities largely depend on carbon positions C-3/C-28 in lupane-type and C-21/C-22 in lanostane-type triterpenoids.As aqueous solubility is important for bioavailability, functional constituents such as amino acid esters have been strategically incorporated at C-28 in betulin to improve solubility without sacrificing cytotoxic effects.The next step in the application of triterpenoids as an adjunct to minimally invasive cancer treatment is optimizing pharmacokinetics for effective targeted therapy.Introducing integrative cancer therapies that include triterpenoid supplementation as an adjuvant could enhance efficacy and efficiency to improve cancer treatment outcomes. CRediT authorship contribution statement 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. Selina Plehn : Plehn Conceptualization, Writingoriginal draft.Sajeev Wagle: Conceptualization, Writingoriginal draft.H.P. Vasantha Rupasinghe: Conceptualization, Writingreview & editing, Supervision. Table 2 2 Anti-cancer and pharmacological mechanisms of triterpenoid of Chaga mushroom. Triterpenoid orExperimental modelProposed anticancerReferencesteroid derivativemechanismInotodiol andBreast cancer cellsInduced autophagy byLee et al.,tramatanolicin vitro and 4T1-activating AMPK and2021acid-richtumor-bearing miceinhibiting the mTORextractsignaling pathwayInotodiolHeLa cells in vitroInhibition of cyclin EZhao et al.,and bcl-22014Inotodiol extractA549 cells in vitroArresting cell cycle inZhongthe S phase andet al.,inhibition of bcl-22011Inonotsutriol EVarious breastInhibition of JAK2/Shan et al.,cancer cell linesSTAT3 signaling2023pathwayBetulinHeLa cells in vitroMitochondrialLi et al.,cytochrome c release2010through expression ofapoptosis regulatingproteins such ascaspasesBetulinic acidHypoxic PC3 cellAnti-angiogenic effectShin et al.,modelthrough disturbing the2011binding of STAT3 andHIF-1α to the VEGFpromotor, therebyreducing VEGFproduction.Betulin andHT29-MTX in vitroAnti-proliferativeWoldbetulinic acidactivityet al.,2020ErgosterolAOM/DSS-treatedTumor suppression andKang et al.,peroxidemice and humananti-proliferative2015colorectal canceractivity through down-cell linesregulation of β-cateninsignaling S.Plehn et al. Data availabilityNo data was used for the research described in the article. 22 α-dihydroxylanosta-8, 25-diene-24-one A549 KB Human lung cancer Human squamous cell carcinoma (SCC). 96 Zhao, Betulinic acid HT29-MTX NCI-H460. 2015. 2018 . Li, 2010. 2020 Betulin NCI-H460 HT29-MTX Human lung cancer Human colon cancer 24 24 2.8 1.6. Wold, 2020 . A A549 ; Inonotsutriol, Zhao, 2015 Inonotsutriol D A549 L1210 Human lung cancer Mouse lymphocytic leukemia 96 72 8. Zhao, 2015. 2011Tanaka et al3910 Inonotsutriol E A549 Human lung cancer 96 1.63. Zhao, 2015 MCF-7 Human colon cancer Human lung cancer Human breast cancer Human breast cancer Human breast cancer 24 24 48. Ht29-Mtx Nci-H460 Sk-Br3 Inotodiol, Mda-Md-231 ; Lee, 2020. 2021488 4.7 4.06 4.74 Wold et al. Inonotusane D 4 T1 Mouse breast cancer 48 9. Zhao, 2016 ,4 dihydroxyphenyl) but-3-en-2-one BEL-7402 Human liver cancer 96 4. Alzand, 2018 Ornithine ester of betulin Me-45 Human melanoma 72 2.47. Drąg-Zalesińska, 2017 Lysine ester of betulin Me-45 Human melanoma 72 2.46 Drąg-Zalesińska. 2017 amine group betulin at C-28 MGC-803 PC3 Bcap-37 MCF-7 Human gastric cancer Human prostate cancer Human breast cancer Human breast cancer. 72 72 72 72 . Yang , 2015 Pyrrolidine group betulin C-28 MGC-803 PC3 Bcap-37 Human gastric cancer Human prostate cancer Human breast cancer. 72 . Yang , 2015 Piperidine group betulin at C-28 MGC-803 PC3 Bcap-37 A375 MCF-7 Human gastric cancer Human prostate cancer Human breast cancer Human melanoma Human. breast cancer 72 72 72 72 72 . Yang , 2015 Heterocyclic group betulin at C-28 MGC-803 Bcap-37 Human gastric cancer. Human breast cancer. 7272 Effects of PH and temperature on water under pressurized conditions in the extraction of nutraceuticals from chaga (inonotus obliquus) mushroom. 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Maintaining Excellent Mechanical Properties via Additive Manufacturing of Low-N 25Cr-Type Duplex Stainless Steel 10 November 2023 Jianguo He hejianguo@nercast.com Research Institute of Special Steels Central Iron & Steel Research Institute Co., Ltd 100081BeijingChina Jiesheng Lv Research Institute of Special Steels Central Iron & Steel Research Institute Co., Ltd 100081BeijingChina Zhigang Song songzhigang@nercast.com Research Institute of Special Steels Central Iron & Steel Research Institute Co., Ltd 100081BeijingChina Changjun Wang wangchangjun@nercast.com Research Institute of Special Steels Central Iron & Steel Research Institute Co., Ltd 100081BeijingChina Han Feng fenghan@nercast.com Research Institute of Special Steels Central Iron & Steel Research Institute Co., Ltd 100081BeijingChina Xiaohan Wu wuxiaohan@nercast.com Research Institute of Special Steels Central Iron & Steel Research Institute Co., Ltd 100081BeijingChina Yuliang Zhu zhuyuliang@nercast.com Research Institute of Special Steels Central Iron & Steel Research Institute Co., Ltd 100081BeijingChina Wenjie Zheng zhengwenjie@nercast.com Research Institute of Special Steels Central Iron & Steel Research Institute Co., Ltd 100081BeijingChina Maintaining Excellent Mechanical Properties via Additive Manufacturing of Low-N 25Cr-Type Duplex Stainless Steel 10 November 20232208D25F6881490C435D4F6060FB8E0810.3390/ma16227125Received: 29 August 2023 Revised: 18 September 2023 Accepted: 21 September 2023selective laser meltingduplex stainless steelsnano-inclusionmicrostructuremechanical properties Duplex stainless steel (DSS) exhibits good mechanical properties and corrosion resistance, and has attracted more and more attention within the fields of both science and technology.However, the increasing levels of N and of Cr, Mo, etc., as alloying elements in DSS increase production difficulty.In particular, the N element increases the risk of Cr 2 N precipitation, which can seriously deteriorate the thermal plasticity of DSS, while increasing its strength.For this reason, a low-N-content 25Crtype DSS was designed in order to adapt additive manufacturing processes.With regard to the nano-inclusions of oxide precipitation and effective grain refinement, and considering the benefits of selective laser melting fabrication, a low-N 25Cr-type duplex stainless steel with a 0.09 wt.% N content achieved high mechanical properties, with a yield strength of 712 MPa and an elongation of 27.5%, while the V-notch impact toughness was 160 J/cm 2 .The microstructure evolution and the reasons behind the improvement in mechanical properties will be discussed in detail. Introduction Duplex stainless steel (DSS) is composed of ferrite and austenite phases that exhibit a higher strength than conventional austenitic stainless steel, while the higher Cr, Mo, and N concentrations in DSS contribute to an increased and outstanding corrosion resistance compared to that of conventional austenitic stainless steel [1][2][3][4][5][6][7].DSS also exhibits a unique resistance to intergranular corrosion in certain environments, making it irreplaceable in the petroleum, chemical, and marine [8][9][10][11] industries, among others.Compared to the 300-series austenitic stainless steels, duplex stainless steel can achieve higher strength and PREN values at similar or lower costs. Although duplex stainless steel has certain advantages over austenitic stainless steel in terms of performance, there are still challenges facing the complete replacement of austenitic stainless steel by duplex stainless steel on the market when it comes to widely used application.The American Society for Testing Materials (ASTM) only includes seven types of cast duplex stainless steels in its standards, while there are hundreds of types of forged duplex stainless steels [8].The slow cooling process during the cooling process after solidification leads to the formation of harmful secondary precipitates, such as CrN, Cr 2 N, sigma (σ) phase, or chi(χ) phase, which significantly affect both the mechanical properties and the corrosion resistance [11][12][13].This makes it challenging to maintain the performance of duplex stainless steel in complex structural applications using traditional casting processes, which is an important reason behind the limited scope of application for duplex stainless steel. In recent years, the development of additive manufacturing technology has introduced a new metal-forming process with extremely fast cooling rates [14][15][16][17][18][19][20][21].The one-time Materials 2023, 16, 7125 2 of 11 forming method reduces spending on molds and allows for the manufacturing of more complex structural components, while avoiding issues like σ phase precipitation and grain coarsening during casting cooling [19,[22][23][24][25][26][27][28].The characteristics of additive manufacturing effectively address the issues of casting formation and the diversification of product forms in duplex stainless steel. Based on the aforementioned research background, this work focuses on 2507-type duplex stainless steel with a lower N content (≤0.1 wt.%) and utilizes the laser powder bed fusion (L-PBF) method to fabricating 25Cr-type DSS with a high strength and plasticity and excellent toughness.The evolution of its microstructure at different stages is analyzed, and precipitation-strengthening and grain-refinement advantages are achieved through control of the composition and printing methods.This method is expected to provide an alternative composition optimization strategy for additive manufacturing processes for the application of duplex stainless steel. Materials and Methods Argon-gas atomization was used to prepare 25Cr-type DSS powder with the size distribution of 15-50 µm.The main chemical composition of the powder was measured via inductively coupled plasma atomic emission spectrometry (ICP-AES), and the results are shown in Table 1. Figure 1a shows the morphology observed under scanning electron microscopy (SEM); the powder exhibits a smooth and round shape without satellite particles.The X-ray diffraction (XRD) analysis results for the powder are shown in Figure 1b, revealing a phase composition of 99% ferrite and 1% austenite, with no harmful phases detected.In recent years, the development of additive manufacturing technology has introduced a new metal-forming process with extremely fast cooling rates [14][15][16][17][18][19][20][21].The onetime forming method reduces spending on molds and allows for the manufacturing of more complex structural components, while avoiding issues like σ phase precipitation and grain coarsening during casting cooling [19,[22][23][24][25][26][27][28].The characteristics of additive manufacturing effectively address the issues of casting formation and the diversification of product forms in duplex stainless steel. Based on the aforementioned research background, this work focuses on 2507-type duplex stainless steel with a lower N content (≤0.1 wt.%) and utilizes the laser powder bed fusion (L-PBF) method to fabricating 25Cr-type DSS with a high strength and plasticity and excellent toughness.The evolution of its microstructure at different stages is analyzed, and precipitation-strengthening and grain-refinement advantages are achieved through control of the composition and printing methods.This method is expected to provide an alternative composition optimization strategy for additive manufacturing processes for the application of duplex stainless steel. Materials and Methods Argon-gas atomization was used to prepare 25Cr-type DSS powder with the size distribution of 15-50 µm.The main chemical composition of the powder was measured via inductively coupled plasma atomic emission spectrometry (ICP-AES), and the results are shown in Table 1. Figure 1a shows the morphology observed under scanning electron microscopy (SEM); the powder exhibits a smooth and round shape without satellite particles.The X-ray diffraction (XRD) analysis results for the powder are shown in Figure 1b, revealing a phase composition of 99% ferrite and 1% austenite, with no harmful phases detected.The aforementioned powder was used for selective laser melting (SLM) on a DLM-280 metal 3D printer.The build process was performed on a 316 stainless steel substrate in a protective atmosphere of high-purity argon gas (99.9%).The specific sintering parameters were as follows: a laser input power (P) of 190 W, a laser spot diameter of 0.1 mm, a powder layer thickness (h) of 0.02 mm, line spacing (t) of 0.1 mm, a scanning speed (v) of 850 mm/s, and a bidirectional scanning pattern with a 45° angle between each layer.The calculated energy density, using Formula (1), was 117.65 J/cm³.The aforementioned powder was used for selective laser melting (SLM) on a DLM-280 metal 3D printer.The build process was performed on a 316 stainless steel substrate in a protective atmosphere of high-purity argon gas (99.9%).The specific sintering parameters were as follows: a laser input power (P) of 190 W, a laser spot diameter of 0.1 mm, a powder layer thickness (h) of 0.02 mm, line spacing (t) of 0.1 mm, a scanning speed (v) of 850 mm/s, and a bidirectional scanning pattern with a 45 • angle between each layer.The calculated energy density, using Formula (1), was 117.65 J/cm 3 . E = P v•h•t (1) The built specimens were subjected to solid solution treatment at temperatures of 1100, 1150, and 1200 • C for 1 h each.Cube samples measuring 10 × 10 × 5 mm were used to characterize the microstructure, while rough impact samples with dimensions of 10 × 60 × 6 mm and rough tensile samples with dimensions of 20 × 70 × 6 mm were used to test the mechanical properties.The final dimensions of the impact and tensile specimens are shown in Figure 2a,b, respectively.The relative density of the specimens, using the Archimedes drainage method, was 99.87%. 𝐸 𝑃 𝑣 ℎ 𝑡(1) The built specimens were subjected to solid solution treatment at temperatures of 1100, 1150, and 1200 °C for 1 h each.Cube samples measuring 10 × 10 × 5 mm were used to characterize the microstructure, while rough impact samples with dimensions of 10 × 60 × 6 mm and rough tensile samples with dimensions of 20 × 70 × 6 mm were used to test the mechanical properties.The final dimensions of the impact and tensile specimens are shown in Figure 2a,b, respectively.The relative density of the specimens, using the Archimedes drainage method, was 99.87%.Tensile testing at room temperature was performed using a WE300B tensile testing machine (supplied by Marxtest Technology Co. Ltd., Jinan, China) with a strain rate of 1 × 10 −3 s −1 .Impact testing at room temperature was conducted using a NI750 metal pendulum impact testing machine (supplied by NCS Testing Technology Co. Ltd., Beijing, China).After mechanical grinding and polishing, the square samples were immersed in a potassium permanganate solution at 50 °C for 3 h, and their microstructure was observed using a LEICA MEF4M optical microscope (supplied by Leica Microsystems Shanghai Ltd., Shanghai, China).The backscattered electron diffraction (EBSD) samples were immersed in a 10% alcoholic hydrochloric acid solution and subjected to electrolytic polishing at a voltage of 25 V for 30 s. EBSD characterization was carried out using a FEI Quanta650 field-emission scanning electron microscope (supplied by FEI Co. Ltd., Hillsboro, American), and the data were processed using Channel 5 software.Transmission electron microscope (TEM) samples were mechanically thinned to a thickness of 40 µm using silicon carbide paper and then further thinned using a dual-jet electropolisher at a voltage of −28 V and a temperature of −20 °C.The electrolyte consisted of 10% hydrochloric acid and 90% anhydrous ethanol.Observation was performed using a FEI TECNAI G2 F20 transmission electron microscope (supplied by FEI Co. Ltd., Hillsboro, American) operating at an accelerating voltage of 200 kV.X-ray diffraction experiments on the samples were conducted using a Bruker D8 Advance X-ray diffractometer (supplied by Bruker Co. Ltd., Billerica, American) with a tube voltage of 35 kV, a tube current of 40 mA, an incident wavelength of λ = 0.179 nm, and a scanning speed of 2°/min.The nitrogen content in the printed specimens was measured using an NCS ONH-5500 analyzer (supplied by NCS Testing Technology Co. Ltd., Beijing, China).Tensile testing at room temperature was performed using a WE300B tensile testing machine (supplied by Marxtest Technology Co., Ltd., Jinan, China) with a strain rate of 1 × 10 −3 s −1 .Impact testing at room temperature was conducted using a NI750 metal pendulum impact testing machine (supplied by NCS Testing Technology Co., Ltd., Beijing, China).After mechanical grinding and polishing, the square samples were immersed in a potassium permanganate solution at 50 • C for 3 h, and their microstructure was observed using a LEICA MEF4M optical microscope (supplied by Leica Microsystems Shanghai Ltd., Shanghai, China).The backscattered electron diffraction (EBSD) samples were immersed in a 10% alcoholic hydrochloric acid solution and subjected to electrolytic polishing at a voltage of 25 V for 30 s. EBSD characterization was carried out using a FEI Quanta650 fieldemission scanning electron microscope (supplied by FEI Co., Ltd., Hillsboro, OR, USA), and the data were processed using Channel 5 software.Transmission electron microscope (TEM) samples were mechanically thinned to a thickness of 40 µm using silicon carbide paper and then further thinned using a dual-jet electropolisher at a voltage of −28 V and a temperature of −20 • C. The electrolyte consisted of 10% hydrochloric acid and 90% anhydrous ethanol.Observation was performed using a FEI TECNAI G2 F20 transmission electron microscope (supplied by FEI Co., Ltd., Hillsboro, American) operating at an accelerating voltage of 200 kV.X-ray diffraction experiments on the samples were conducted using a Bruker D8 Advance X-ray diffractometer (supplied by Bruker Co., Ltd., Billerica, MA, USA) with a tube voltage of 35 kV, a tube current of 40 mA, an incident wavelength of λ = 0.179 nm, and a scanning speed of 2 • /min.The nitrogen content in the printed specimens was measured using an NCS ONH-5500 analyzer (supplied by NCS Testing Technology Co., Ltd., Beijing, China). Results and Discussion Microstructure Figure 3a shows the microstructure of the built cubic specimen without heat treatment, which reveals a mosaic-like regular structure.The magnified metallographic image in Figure 3b further reveals that the mosaic-like grid structure is arranged in a regular pattern, with 100 µm as the minimum structural unit.The core of the mosaic unit consists of larger grains, while the edges consist of smaller grains similar to recrystallized grains.The track width formed by the units is 100 µm, arranged in two directions at a 90 • angle to each other.The aforementioned mosaic structure is mainly due to the special scanning method, where the laser spot radius is 100 µm, and the scan line spacing is also 100 µm.Due to the higher energy at the core of the laser, the powder in the core receives more energy compared to the powder at the edges, resulting in a difference in grain size between the core and the edges.At this point, the grain size distribution presents larger grains in the core and smaller grains on the sides.As the scanning progresses, after the completion of one layer, the scanning path of the next layer of powder is perpendicular to the previous layer at 90 • .The laser energy input of the next layer, to a certain extent, affects the already-formed matrix in the previous layer, causing secondary heating and recrystallization, ultimately leading to the grid-like structure.The microstructure metallography of the specimen when heat-treated at 1200 • C for 1 h is shown in Figure 3c, and it is worth noting that the grid-like mosaic structure in the microstructure remains intact and regular after heat treatment, which is significantly different from the destruction of the grid-like structure that has been observed in previous studies after heat treatment [29][30][31][32][33].The magnified microstructure shown in Figure 3d reveals that a certain amount of new phase has precipitated on the grain boundaries of the matrix phase and, even after 1 h of heat treatment at 1200 • C, the size remains small.The above microstructural characteristics are mainly attributed to the unique low-nitrogen (N)-composition design. Results and Discussion Microstructure Figure 3a shows the microstructure of the built cubic specimen without heat treatment, which reveals a mosaic-like regular structure.The magnified metallographic image in Figure 3b further reveals that the mosaic-like grid structure is arranged in a regular pattern, with 100 µm as the minimum structural unit.The core of the mosaic unit consists of larger grains, while the edges consist of smaller grains similar to recrystallized grains.The track width formed by the units is 100 µm, arranged in two directions at a 90° angle to each other.The aforementioned mosaic structure is mainly due to the special scanning method, where the laser spot radius is 100 µm, and the scan line spacing is also 100 µm.Due to the higher energy at the core of the laser, the powder in the core receives more energy compared to the powder at the edges, resulting in a difference in grain size between the core and the edges.At this point, the grain size distribution presents larger grains in the core and smaller grains on the sides.As the scanning progresses, after the completion of one layer, the scanning path of the next layer of powder is perpendicular to the previous layer at 90°.The laser energy input of the next layer, to a certain extent, affects the already-formed matrix in the previous layer, causing secondary heating and recrystallization, ultimately leading to the grid-like structure.The microstructure metallography of the specimen when heat-treated at 1200 °C for 1 h is shown in Figure 3c, and it is worth noting that the grid-like mosaic structure in the microstructure remains intact and regular after heat treatment, which is significantly different from the destruction of the grid-like structure that has been observed in previous studies after heat treatment [29][30][31][32][33].The magnified microstructure shown in Figure 3d reveals that a certain amount of new phase has precipitated on the grain boundaries of the matrix phase and, even after 1 h of heat treatment at 1200 °C, the size remains small.The above microstructural characteristics are mainly attributed to the unique low-nitrogen (N)-composition design.To further characterize the microstructural features before and after heat treatment, the phase distribution before and after heat treatment was statistically analyzed using EBSD.Figure 4a shows the EBSD phase map of the laser-irradiated surface before heat treatment, which predominantly consists of the ferrite phase.This is mainly due to the high temperature during the scanning process and the extremely fast cooling rate, which does not allow for the precipitation of austenite during the forming and cooling process.Figure 4b shows the EBSD phase map of the non-heat-treated scanning side, and it can be observed that each layer of ferrite has been melted by the laser into a solid structure with a width of approximately 100 µm.This solid structure will improve the impact toughness, which will be discussed in detail later.Figure 4c,e, respectively, show the phase distribution maps of the scanned front and side after 1 h of heat treatment at 1200 • C. A large amount of austenite precipitates at the grain boundaries of the ferrite, and some austenite precipitates at the low-angle grain boundaries within the ferrite.It can be observed that the size of the austenite precipitated on the high-angle grain boundaries is significantly larger than that precipitated on the low-angle grain boundaries within the grain.This is mainly because the high-angle grain boundaries have a higher energy, providing convenience for the rapid nucleation and growth of austenite.Figure 4d shows the phase distribution map of the scanned front after heat treatment at a lower solution temperature of 1100 • C for 1 h.It can be found that, compared to at 1200 • C, the amount of austenite increases significantly, and the austenite is distributed in a strip-like pattern connected to the grain boundaries of the ferrite.At a lower solution temperature, the morphological characteristics of the austenite phase have changed significantly compared to the discontinuous island-like distribution at a higher solution temperature. the phase distribution before and after heat treatment was statistically analyzed using EBSD.Figure 4a shows the EBSD phase map of the laser-irradiated surface before heat treatment, which predominantly consists of the ferrite phase.This is mainly due to the high temperature during the scanning process and the extremely fast cooling rate, which does not allow for the precipitation of austenite during the forming and cooling process.Figure 4b shows the EBSD phase map of the non-heat-treated scanning side, and it can be observed that each layer of ferrite has been melted by the laser into a solid structure with a width of approximately 100 µm.This solid structure will improve the impact toughness, which will be discussed in detail later.Figure 4c,e, respectively, show the phase distribution maps of the scanned front and side after 1 h of heat treatment at 1200 °C.A large amount of austenite precipitates at the grain boundaries of the ferrite, and some austenite precipitates at the low-angle grain boundaries within the ferrite.It can be observed that the size of the austenite precipitated on the high-angle grain boundaries is significantly larger than that precipitated on the low-angle grain boundaries within the grain.This is mainly because the high-angle grain boundaries have a higher energy, providing convenience for the rapid nucleation and growth of austenite.Figure 4d shows the phase distribution map of the scanned front after heat treatment at a lower solution temperature of 1100 °C for 1 h.It can be found that, compared to at 1200 °C, the amount of austenite increases significantly, and the austenite is distributed in a strip-like pattern connected to the grain boundaries of the ferrite.At a lower solution temperature, the morphological characteristics of the austenite phase have changed significantly compared to the discontinuous island-like distribution at a higher solution temperature. Mechanical Properties Tensile Properties Figure 5 shows the tensile properties of the sample before and after heat treatment.It can be observed that the sample has a very high yield strength (920 MPa) and tensile strength (922 MPa) in the state before heat treatment, but the elongation is only 2%. Mechanical Properties 3.2.1. Tensile Properties Figure 5 shows the tensile properties of the sample before and after heat treatment.It can be observed that the sample has a very high yield strength (920 MPa) and tensile strength (922 MPa) in the state before heat treatment, but the elongation is only 2%.Concerning the tensile properties of the sample after heat treatment, it can be observed that, as the solution temperature increases, the strength and plasticity of the sample simultaneously increase.After solution treatment at 1200 • C, even with only a weak solid solution strengthening due to a N content of only 0.098%, the yield strength is still as high as 712 MPa.In previous studies, the yield strength of standard-grade (higher-N-content, compared to this paper) 2205 and 2507 duplex stainless steel, whether through casting or additive manufacturing, typically ranged from 600 to 660 MPa [15,[34][35][36].The test results obtained in this paper showed that the yield strength of the samples after a 1 h solution treatment at 1200 • C was significantly higher than that shown in the experimental results of previous studies. as 712 MPa.In previous studies, the yield strength of standard-grade (higher-N-content, compared to this paper) 2205 and 2507 duplex stainless steel, whether through casting or additive manufacturing, typically ranged from 600 to 660 MPa [15,[34][35][36].The test results obtained in this paper showed that the yield strength of the samples after a 1 h solution treatment at 1200 °C was significantly higher than that shown in the experimental results of previous studies. Impact Properties Table 2 presents the impact toughness values of the samples before and after heat treatment, compared with the impact performance of the 2707 BPF samples with similar processes.It can be observed that the impact toughness of the experimental steel in this study is significantly higher than that of the reference, and that the impact toughness value of the untreated samples is significantly higher than that in the results for the reference.Although the samples without heat treatment only show a 2% elongation during the tensile process, they exhibit high toughness values.SEM images of the fractured surfaces of the untreated samples at different scales are shown in Figure 6a-c.The unique fracture morphology is an important reason behind the higher toughness value of the samples. Impact Properties Table 2 presents the impact toughness values of the samples before and after heat treatment, compared with the impact performance of the 2707 BPF samples with similar processes.It can be observed that the impact toughness of the experimental steel in this study is significantly higher than that of the reference, and that the impact toughness value of the untreated samples is significantly higher than that in the results for the reference.Although the samples without heat treatment only show a 2% elongation during the tensile process, they exhibit high toughness values.SEM images of the fractured surfaces of the untreated samples at different scales are shown in Figure 6a-c.The unique fracture morphology is an important reason behind the higher toughness value of the samples.From the macroscopic fracture morphology in Figure 6a, it can be observed that the fracture surface of the sample is divided into two sides.The side near the V-notch (the whiter side) shows irregular cleavage fracture, while the other side exhibits a mosaic-like structure similar to its microstructure.The magnified images in Figure 6b,c reveal more clearly the complex fracture forms.In each small structural unit, the central large grains exhibit smooth cleavage fracture planes.The small grain area at the edge of the structural unit consists of a certain number of smaller dimples.The smaller grain size and multiple interfaces in this region partly hinder the propagation of cracks during the fracturing process, thus improving the impact resistance.On the other hand, as mentioned earlier, the microstructure on the sample's side consists of ferrite arranged vertically as a whole.Cracks can only propagate through transgranular fracture within these ferrite regions, resulting in a fracture surface that is similar in appearance to the front surface's grid-like structure.The coexistence of mixed microstructures with different grain sizes, and the integrity of the ferrite in the side structure, contribute to the complex fracture form and the high toughness value of the samples.coexistence of mixed microstructures with different grain sizes, and the integrity of the ferrite in the side structure, contribute to the complex fracture form and the high toughness value of the samples.The SEM images of the fractured surface of the heat-treated impact samples are shown in Figure 6d-f.The macroscopic image in Figure 6d reveals a composite structure with smooth edges and an uneven core.The magnified image in Figure 6e of the core region shows a combination of dimples and cleavage planes, with some areas still exhibiting grid-like structures.The magnified image of the fracture edge in Figure 6f reveals cleavage planes.The structural differences between different regions of the fracture surface indicate a relatively poor continuity of crack propagation during the fracturing process, ultimately resulting in a more objective measure of the impact toughness. Discussion The extreme tensile behavior of the untreated sample is mainly due to the presence of a ferrite structure in the untreated state.Typically, at room temperature, the nitrogen The SEM images of the fractured surface of the heat-treated impact samples are shown in Figure 6d-f.The macroscopic image in Figure 6d reveals a composite structure with smooth edges and an uneven core.The magnified image in Figure 6e of the core region shows a combination of dimples and cleavage planes, with some areas still exhibiting grid-like structures.The magnified image of the fracture edge in Figure 6f reveals cleavage planes.The structural differences between different regions of the fracture surface indicate a relatively poor continuity of crack propagation during the fracturing process, ultimately resulting in a more objective measure of the impact toughness. Discussion The extreme tensile behavior of the untreated sample is mainly due to the presence of a ferrite structure in the untreated state.Typically, at room temperature, the nitrogen saturation solubility in ferrite is 0.007%, while most of the 0.098% nitrogen in the composition is dissolved in the ferrite, leading to a distorted ferrite lattice due to nitrogen oversaturation.Additionally, the repeated heating and rapid cooling during the printing process result in significant residual thermal stresses.The combination of these factors leads to a low plasticity and high strength in the untreated sample.The TEM characterization of the untreated sample confirms the above inference.Figure 7a shows the internal structure of the ferrite at the TEM scale.It can be observed that the interior of the undeformed ferrite is highly disordered.The magnified view in Figure 7b clearly shows a large number of randomly distributed and tangled dislocations within the ferrite grain.Even without deformation, the ferrite in this state is under a significant internal stress and, thus, fractures with only a small amount of deformation (2%). to a low plasticity and high strength in the untreated sample.The TEM characterization of the untreated sample confirms the above inference.Figure 7a shows the internal structure of the ferrite at the TEM scale.It can be observed that the interior of the undeformed ferrite is highly disordered.The magnified view in Figure 7b clearly shows a large number of randomly distributed and tangled dislocations within the ferrite grain.Even without deformation, the ferrite in this state is under a significant internal stress and, thus, fractures with only a small amount of deformation (2%).As mentioned earlier, the yield strength of the samples after a 1 h heat treatment at 1200 °C reached values as high as 712 MPa, while still maintaining a 27.5% elongation.This anomalous mechanical performance is mainly due to the combined effect of the unique phase morphology of austenite under special composition and the precipitation of high-temperature oxide phases.At lower solution temperatures, the austenite content is higher, and a large amount of austenite forms continuous bands along the ferrite grain boundaries [37,38].The softer and higher-content austenite leads to a decrease in yield strength, and the large distribution on the grain boundaries easily causes a deformation mismatch between the two phases, leading to fracture.Conversely, at higher solution temperatures, the austenite content is lower, and it is discretely distributed in island-like shapes along the ferrite grain boundaries.For this reason, there is less austenite with a smaller size and discrete distribution, which acts as a second-phase reinforcement by pinning on the ferrite grain boundaries, achieving an enhanced strength and plasticity. In addition, in previous studies, the introduction of high-temperature oxide Al2O3 into the system also leads to an improvement in strength [33,39].In traditional casting processes, the high-temperature-formed Al2O3 easily grows and producies inclusions during slow cooling, making it difficult to eliminate.However, in additive manufacturing processes, the extremely fast cooling rate prevents these high-temperature-formed Al2O3 from growing, and they ultimately become precipitates that are beneficial to mechanical properties.In this study, TEM observation also revealed fine dispersed circular Al2O3 precipitates, as shown in Figure 8a.The dark-field TEM image in Figure 8b provides a clearer view of the widely varying distribution sizes of Al2O3.To better illustrate the types of precipitates, Figure 8c shows the EDS mapping results of the region.It is evident that there is a clustering of Al and O elements in the precipitates.This type of precipitate strengthening is also one of the reasons behind the higher yield strength of the sample.We also characterized the uniform deformation zone of the samples after tensile fracture, using TEM. Figure 8d shows a TEM image of the deformed state, where a large number of dislocations are pinned by small Al2O3 inclusions in the ferrite.The interaction between the fine As mentioned earlier, the yield strength of the samples after a 1 h heat treatment at 1200 • C reached values as high as 712 MPa, while still maintaining a 27.5% elongation.This anomalous mechanical performance is mainly due to the combined effect of the unique phase morphology of austenite under special composition and the precipitation of high-temperature oxide phases.At lower solution temperatures, the austenite content is higher, and a large amount of austenite forms continuous bands along the ferrite grain boundaries [37,38].The softer and higher-content austenite leads to a decrease in yield strength, and the large distribution on the grain boundaries easily causes a deformation mismatch between the two phases, leading to fracture.Conversely, at higher solution temperatures, the austenite content is lower, and it is discretely distributed in island-like shapes along the ferrite grain boundaries.For this reason, there is less austenite with a smaller size and discrete distribution, which acts as a second-phase reinforcement by pinning on the ferrite grain boundaries, achieving an enhanced strength and plasticity. In addition, in previous studies, the introduction of high-temperature oxide Al 2 O 3 into the system also leads to an improvement in strength [33,39].In traditional casting processes, the high-temperature-formed Al 2 O 3 easily grows and producies inclusions during slow cooling, making it difficult to eliminate.However, in additive manufacturing processes, the extremely fast cooling rate prevents these high-temperature-formed Al 2 O 3 from growing, and they ultimately become precipitates that are beneficial to mechanical properties.In this study, TEM observation also revealed fine dispersed circular Al 2 O 3 precipitates, as shown in Figure 8a.The dark-field TEM image in Figure 8b provides a clearer view of the widely varying distribution sizes of Al 2 O 3 .To better illustrate the types of precipitates, Figure 8c shows the EDS mapping results of the region.It is evident that there is a clustering of Al and O elements in the precipitates.This type of precipitate strengthening is also one of the reasons behind the higher yield strength of the sample.We also characterized the uniform deformation zone of the samples after tensile fracture, using TEM. Figure 8d shows a TEM image of the deformed state, where a large number of dislocations are pinned by small Al 2 O 3 inclusions in the ferrite.The interaction between the fine dispersoids of Al 2 O 3 particles and dislocations hinders the initial movement of the ferrite during deformation, which contributes to the higher yield strength, compared to that under normal conditions. In summary, through a unique composition design that breaks away from the conventional 0.24-0.32%nitrogen (N) content in 2507, a unique austenite phase morphology has been induced, while a regular lattice structure is maintained.With the reinforcement of oxide precipitates, the sample exhibits a high yield strength of 712 MPa, while still maintaining a good elongation of 27.5%. dispersoids of Al2O3 particles and dislocations hinders the initial movement of the ferrite during deformation, which contributes to the higher yield strength, compared to that under normal conditions.In summary, through a unique composition design that breaks away from the conventional 0.24-0.32%nitrogen (N) content in 2507, a unique austenite phase morphology has been induced, while a regular lattice structure is maintained.With the reinforcement of oxide precipitates, the sample exhibits a high yield strength of 712 MPa, while still maintaining a good elongation of 27.5%. Conclusions In this study, a low-N 25Cr-type duplex stainless steel for additive manufacturing processes was fabricated through the L-PBF method.The product exhibits an excellent strength and ductility.The main conclusions are as follows: 1.When the strategy of alternating the layer scanning direction of 45° is used, the microstructure of the specimens shows a regular mosaic structure.The microstructure before heat treatment consists of a single ferrite phase.After heat treatment at 1200 °C, discrete and fine austenite precipitates at the ferrite grain boundaries, and the regular mosaic structure remains. 2. The unique morphology of the austenite phase, caused by the special low-N-composition design, along with the strengthening effect of oxide precipitation, leads to a high yield strength of 712 MPa after heat treatment in the experimental steel in this study, while a good elongation of 27.5% is maintained. Conclusions In this study, a low-N 25Cr-type duplex stainless steel for additive manufacturing processes was fabricated through the L-PBF method.The product exhibits an excellent strength and ductility.The main conclusions are as follows: 1.When the strategy of alternating the layer scanning direction of 45 • is used, the microstructure of the specimens shows a regular mosaic structure.The microstructure before heat treatment consists of a single ferrite phase.After heat treatment at 1200 • C, discrete and fine austenite precipitates at the ferrite grain boundaries, and the regular mosaic structure remains. 2. The unique morphology of the austenite phase, caused by the special low-Ncomposition design, along with the strengthening effect of oxide precipitation, leads to a high yield strength of 712 MPa after heat treatment in the experimental steel in this study, while a good elongation of 27.5% is maintained. 3. The specimen of additive-manufacturing low-N 25Cr-type DSS demonstrates an excellent V-notch impact performance, with a toughness value of 98.75 J/cm 2 before heat treatment and approximately 160 J/cm 2 after heat treatment. Figure 1 . 1 Figure 1.Powder morphology and phase information: (a) powder SEM image, (b) XRD diffraction pattern. Figure 1 . 1 Figure 1.Powder morphology and phase information: (a) powder SEM image, (b) XRD diffraction pattern. Figure 2 . 2 Figure 2. The dimensions of the specimens for testing mechanical properties: (a) the impact specimen dimensions; (b) the tensile specimen dimensions (unit: mm). Figure 2 . 2 Figure 2. The dimensions of the specimens for testing mechanical properties: (a) the impact specimen dimensions; (b) the tensile specimen dimensions (unit: mm). Figure 3 . 3 Figure 3.An optical microscope (OM) image of the specimen before and after heat treatment: (a) the specimen before heat treatment at 100× magnification, (b) the magnified microstructure of the selected area from (a), (c) the specimen heat-treated at 1200 °C for 1 h at 100× magnification, (d) the magnified microstructure of the selected area from (c). Figure 3 . 3 Figure 3.An optical microscope (OM) image of the specimen before and after heat treatment: (a) the specimen before heat treatment at 100× magnification, (b) the magnified microstructure of the selected area from (a), (c) the specimen heat-treated at 1200 • C for 1 h at 100× magnification, (d) the magnified microstructure of the selected area from (c). Figure 4 . 4 Figure 4. EBSD phase distribution maps: the front surface (a) and side surface (b) of the specimen before heat treatment, the front surface (e) and side surface (c) of the specimen heat-treated at 1200 °C for 1 h, the scan of the front surface (d) of the specimen heat-treated at 1100 °C for 1 h. Figure 4 . 4 Figure 4. EBSD phase distribution maps: the front surface (a) and side surface (b) of the specimen before heat treatment, the front surface (e) and side surface (c) of the specimen heat-treated at 1200 • C for 1 h, the scan of the front surface (d) of the specimen heat-treated at 1100 • C for 1 h. Figure 5 . 5 Figure 5.The tensile performance of the specimen: (a) the stress-strain curve, (b) the yield strength, tensile strength, and elongation at different treatment states. Figure 5 . 5 Figure 5.The tensile performance of the specimen: (a) the stress-strain curve, (b) the yield strength, tensile strength, and elongation at different treatment states. Figure 6 . 6 Figure 6.SEM images of the impact fracture surfaces: the fracture morphology of the specimen before heat treatment (a) and selected-area enlargement (b,c), the fracture morphology of the specimen heat-treated at 1200 ° for 1 h (d), and selected-area enlargement (e,f). Figure 6 . 6 Figure 6.SEM images of the impact fracture surfaces: the fracture morphology of the specimen before heat treatment (a) and selected-area enlargement (b,c), the fracture morphology of the specimen heat-treated at 1200 • for 1 h (d), and selected-area enlargement (e,f). Figure 7 . 7 Figure 7. TEM image of the specimen before heat treatment (a) and selected-area enlargement (b). Figure 7 . 7 Figure 7. TEM image of the specimen before heat treatment (a) and selected-area enlargement (b). Figure 8 . 8 Figure 8. TEM micrographs of oxide inclusions in the specimen after 1 h heat treatment at 1200 °C: (a) the undeformed state, (b) the dark-field image of (a,c) the EDS mapping of (a,d) the deformed state. Figure 8 . 8 Figure 8. TEM micrographs of oxide inclusions in the specimen after 1 h heat treatment at 1200 • C: (a) the undeformed state, (b) the dark-field image of (a,c) the EDS mapping of (a,d) the deformed state. Table 1 . 1 The chemical composition of the 25Cr-type DSS powder 1 . CrNiMoNMnOSiCPS24.706.523.740.0980.550.0280.350.00500.00580.0029 1 S, C, and O elements were determined via the infrared absorption method.Materials 2023, 16, x FOR PEER REVIEW 2 of 11 Table 1 . 1 The chemical composition of the 25Cr-type DSS powder 1 . CrNiMoNMnOSiCPS24.706.523.740.0980.550.0280.350.0050 0.0058 0.0029 1 S, C, and O elements were determined via the infrared absorption method. Table 2 . 2 V-notch impact toughness. 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World Journal of Orthopedics 755 October 18, 2023 Andrea Vescio MDGianluca Testa Alessia Caldaci Marco Sapienza Vito Pavone A O U Policlinico Rodolico-San Marco Jankowicz-Szymanska A VimalAKPoland, India Department of General Surgery and Medical Surgical Specialties Section of Orthopaedics and Traumatology University of Catania 95123CataniaItaly Depart-ment of General Surgery and Medical Surgical Specialties Section of Orthopaedics and Trau-matology A.O.U. Policlinico "Rodolico-San Marco" University of Catania Via Santa Sofia 7895123CataniaItaly Andrea Vescio 0000-0002-1677-927X, 0000-0001-5246-9714Gianluca Testa Alessia Caldaci Marco Sapienza 0000-0002-0191-087X, 0000-0002-8311-9132, 0000-0001-5664-8066 7041 Koll Center Parkway, Suite 16094566PleasantonCAUSA World Journal of Orthopedics 2218-5836 (online)755 October 18, 20230FE6BFB49BD2679ECB2496C4393BEE9010.5312/wjo.v14.i10.755Received: January 3, 2023 Peer-review started: January 3, 2023 First decision: April 13, 2023 Revised: April 26, 2023 Accepted: June 9, 2023Unsolicited articleExternally peer reviewed Pes planusScoreAssessmentClinicSurgeryOutcomeReparability BACKGROUNDFlexible flatfoot (FFF) is a very common condition in children, but no evidencebased guidelines or assessment tools exist.Yet, surgical indication is left to the surgeon's experience and preferences. AIM To develop a functional clinical score for FFF [Catania flatfoot (CTF) score] and a measure of internal consistency; to evaluate inter-observer and intra-observer reliability of the CTF Score; to provide a strong tool for proper FFF surgical indication. METHODS CTF is a medically compiled score of four main domains for a total of twelve items: Patient features, Pain, Clinical Parameters, and Functionality.Each item refers to a specific rate.Five experienced observers answered 10 case reports according to the CTF.To assess inter-and intra-observer reliability of the CTF score, the intra-class correlation coefficients' (ICCs) statistics test was performed, as well as to gauge the correlation between the CTF score and the surgical or conservative treatment indication.Values of 75% were chosen as the score cut-off for surgical indication.Sensitivity, specificity, positive likelihood ratio (PLHR), negative likelihood ratio (NLHR), positive predictive value (PPV), and negative predictive value (NPV). RESULTS Overall interobserver reliability ICC was 0.87 [95% confidence interval (CI): 0.846-0.892;P < 0.001].Overall intra-observer reliability ICC was 0.883 (95%CI: 0.854-0.909;P < 0.001).A direct correlation between the CTF score and surgical treatment indication [Pearson correlation coefficient = 0.94 (P < 0.001)] was found. INTRODUCTION The flexible flatfoot (FFF), known as pes planus, is a very common condition in children characterized by loss of the medial arch and an increase in the support base along with valgus of the hindfoot, yet 40 different definitions were formulated [1].FFF is associated with anatomical conditions, including valgus heel, subluxation of the subtalar joint with intra-rotation of the talus and flexion of plantar abduction of the mid-tarsal joint with naval dorsal subluxation [2].Generally, FFF is an age-related physiological variant, not a disease, and its incidence decreases significantly in terms of increased age: In children 3-years-old, it is 54%, whereas in children 6-years-old, it is 24% [3].A history should include pain, location, intensity, functional problems, while trauma or recurrent ankle sprains should be specifically questioned.FFF is typically an asymptomatic condition [4].Lower limb pain [5] and lower limb function [6] were found as the main manifestations in symptomatic FFF.Until 2022, more than 300 scientific articles were published, without evidence-based guidelines.The challenge for health professionals is to identify when a child's foot is consistent with developmental expectations, particularly in relation to foot posture, and/or function to reassure, monitor or intervene accordingly [7][8][9][10].Therefore, the measure to indicate where foot posture is outside of expected flatness in children (i.e., the diagnoses of flat foot) must be valid, reliable, and appropriate for developing foot posture typically observed.Recently, a systematic review [1] highlighted there was no consistency used to determine pediatric FFF in the literature or the choice of foot posture measures, in relation to validity and reliability, which was rarely justified.A surgical indication was in effect for the surgeon's experience [11,12].The purpose of the study was to develop new functional clinical scores for FFF to assess toddlers and adolescent patients' characteristic functionality [Catania flatfoot (CTF] Score) and measure of internal consistency; to evaluate inter-and intra-observer reliability of the CTF Score; and to provide a reliable tool for proper FFF surgical indication. MATERIALS AND METHODS CTF score development The CTF Score development was composed of two parts, the CTF Score Conception and CTF Score Composition and Scoring. CTF score conception: An orthopedic team was involved in developing the questionnaire.The CTF score was designed to be used in different clinical settings, including clinical research, survey studies, and clinical practice to assess FFF-affected patients and possibly assess changes with treatment.The development team was composed of two senior orthopedic and trauma surgeons (Vito Pavone and Gianluca Testa), and one pediatric orthopedic (fully-trained) resident (Andrea Vescio).At an early stage, an author (Andrea Vescio) search was done to analyze the functional foot and ankle score previously described and developed as the CTF score.Senior authors (Vito Pavone and Gianluca Testa) reviewed and validated the scores. CTF score composition and scoring: The questionnaire is a medically-compiled score of four main domains for a total of twelve items: Patient features (2 items), pain (1 item), clinical parameters (5 items), and functionality (4 items).Each item refers to a specific rate as reported in Supplementary Table 1.The lowest achievable value is -80, while the highest is 170.Calculation of the CTF score is based on the following formula: The value is expressed as a percentage: Higher percentages are associated with a lower clinical presentation. CTF score patient features domain: Patient features are composed of two items aimed to assess the principal general parameters of the evaluated subject.The first item is related to age; the second is linked to laxity.Hypermobility can be assessed according to the passive dorsiflexion of the fifth hand finger and thumbs, elbow, and knee hyperextension. CTF score pain domain: The pain domain was composed of one item to assess generalized pain of the foot or ankle, as well as in the plantar arch, heel, tibialis posterior tendon, and fascia. CTF score clinical parameters domain: The clinical parameters domain is composed of five items to assess the callous present, valgus of hindfoot, longitudinal arch, forefoot abduction, and triceps contracture.For each item, three answers are admissible: "none", "mild", and "severe".The first item "callous" allows for two answers: "yes" and "no". CTF score functionality domain: The functionality domain provides four items to evaluate the patient's capacities.Fatigue, inadequate physical and sport performance, and wear of orthosis is recorded.The first and last items of the section ("fatigue" and "orthosis") allow for two answers: "yes" and "no", while others provide "none", "mild", and "severe". Evaluation materials A review of all infants, toddlers, and adolescents admitted through the pediatric orthopedic ambulatory were carried out.For each patient the following demographic and clinical data captured: Gender, age, the involved side, and presence or absence of associated syndromes or deformities, past and recent medical history for foot and ankle discomfort or pain.Frontal, lateral, and posterior view photos were taken.The pictures were performed in the same positions to provide the more possible objectivity and recorded in an online database.The inclusion criteria were as follows: (1) Chronological age 17-years-old; (2) physical and podoscopic examination; (3) complete photographic history; and (4) positive Tip Toe and Jack test; all cases were examined by the same expected pediatric orthopedic team. Evaluation contributors Children in the study were independently examined and assessed by two orthopedic surgeons and three residents in pediatric orthopedics: All evaluators had previous experience of at least twenty-four months.Three assessors, two surgeons, and a resident completed a full program while treating over 50 FFF patients in the previous two years.All observers had 1 h of theoretical FFF clinical manifestation and score system training before patients' assessment.Each contributor was provided with a summary of the medical history and clinical examination of the frontal, lateral, and posterior view photos.As per the web-based score, observers were asked about conservative or surgical indication.Answers were submitted via a link hosted by https://www.google.com/forms and recorded by an Excel spreadsheet (Microsoft, Redmond, WA, United States).The CTF score was submitted at two different points. Primary outcome measurement To assess the inter-and intra-observer reliability of the CTF score, the intra-class correlation coefficients (ICCs) statistics test was performed.For scale development, it is generally accepted there should be at least five times the number of respondents as questions, for at least 60 in total [11]. Secondary outcome measurement To assess the correlation between the CTF score and surgical or conservative treatment, values of 75% were used as a score cut-off for surgery.Sensitivity, specificity, positive likelihood ratio (PLHR), negative likelihood ratio (NLHR), positive predictive value (PPV), and negative predictive value (NPV) were used. Statistical analysis Continuous data are presented as the mean and standard deviation when appropriate.The ICC (two-way random effects model, with single-measure reliability) was performed to evaluate intra-and interobservers' agreement.According to the Koo and Li guideline, agreement below 0.50 was considered "poor"; between 0.50 and 0.74 as "moderate"; between 0.75 and 0.89 as "good"; and above 0.90 as "excellent" [12].The Pearson correlation coefficient (PCC) was utilized to assess the correlation between conservative or surgical treatment and the CTF score.PCC vales between -1 and 1, where values close to -1 indicated high negative correlation, with values close to 1 indicating a high positive correlation, and values close to 0 indicating no or a very week correlation. A rule of thumb for interpreting the coefficient is provided by Colton et al [13]: (1) 0 to 0.25 (0 to -0.25) little or no relationship; (2) 0.25 to 0.50 (-0.25 to -0.50) fair degree of a relationship; (3) 0.50 to 0.75 (-0.50 to -0.75) moderate to good degree of a relationship; and (4) 0.75 to 1.00 (-0.75 to -1.00) very good to excellent relationship. The Bland and Altman plot was produced to analyze differences between cohort measurements.The limits of agreement were calculated as the mean difference ± 1.96 SD [14].A value of 75% was chosen as a score cut-off for surgical indication.Sensitivity, specificity, PLHR, NLHR, PPV, and NPV were recorded.P values of less than 0.05 were considered statistically significant.All statistical analyses were performed using IBM SPSS, Version 24.0 (IBM Corp., Armonk, NY, United States). RESULTS Five different experienced observers answered 10 case reports.For each patient, observers responded to 14 questions (12 items and 2 treatment indications) for a total of 140 responses.The web-based survey was submitted at two different times, while 280 observations were reported. Inter-and intra-observer reliability Overall interobserver reliability ICC was 0.87 [95% confidence interval (CI): 0.846-0.892;P < 0.001; "good"].The ICC value for specialists was 0.809 (95%CI: 0.761-0.849;P < 0.001; "good"), but was 0.852 (95%CI: 0.821-0879; P < 0.001; "good") for residents (Table 1). The overall intra-observer reliability ICC was 0.883 (95%CI: 0.854-0.909;P < 0.001) and considered "good" (Table 2 and Figure 1). The ICC value for specialists was 0.869 (95%CI: 0.832-0.901;P < 0.001; "good"), but was 0.878 (95%CI: 0.846-0.907;P < 0.001; "good") for residents (Table 1). CTF score treatment indication correlation A fair inverse correlation occurred between the CTF score and conservative treatment indication (PCC = -0.483;P < 0.001) (Figure 2A). The direct correlation between the CTF score and surgical treatment indication (PCC = 0.94; P < 0.001) was rated "from good to excellent" (Figure 2B). CTF score for linear regression According to the 75% cut-off, sensitivity was 100% (95%CI: 83.43%-100%), specificity was 85.71% (95%CI: 75.29%-92.93%),PLHR was 7 (95%CI: 3.94-12.43),NLHR was 0 (95%CI: 0-0), PPV was 75% (95%CI: 62.83%-84.19%),and NPV was 100% (95%CI: 100%-100%). DISCUSSION The CTF score was found to be a valid, effective tool in flatfoot assessment.The scale was seen as good or excellent for inter-and intra-observer reliability, done independently with experience levels.Higher score values were directly correlated with surgical treatment needs, while an increase in score reduced conservative management indication.In addition, the 75% CTF score values were discovered as reasonable cut-off points for surgical treatment, while high percentages of sensitivity and specificity guaranteed safe tool utilization. In recent surveys, European [9] and Italian [10] pediatric orthopedics underlined the absence of a specific and universally-recognized clinical evaluation score for juvenile FFF.The CTF Score fills the literature void and, considering the good results, can be proposed as a helpful tool for clinical research, survey studies, and clinical practice to assess FFFaffected patients as well as changes with treatment. Each domain scale was developed according to the weighted preferences of European and Italian pediatric orthopedics which ensure that each scale is internally consistent, i.e., measures a single trait and that each item has different levels of difficulty or severity. The final instrument comprises 12 questions divided into four domains which measure problems in domains titled Patient features (2 items), pain (1 item), clinical parameters (5 items), and functionality (4 items).Raw domain scores can be transformed into percentage scores to make them easier to interpret; higher scores indicate more severe disability.The item has strong face validity and is included as a categorical descriptive variable but not allied to any domain scale.The instrument is not suitable for those who are unable to walk, or who have a significant proximal component to their disability. In 2005, the American Orthopedic Foot and Ankle Society (AOFAS) members identified the Foot Function Index, and the American Academy of Orthopaedic Surgeons Foot and Ankle module scores as the most frequently used in the literature [15].Yet, AOFAS [16], Foot and Ankle Ability Measure [17], and the Rowan Foot Pain Assessment Questionnaire [18] were commonly utilized for foot and ankle disorder evaluation.On the other hand, previous scores were not specific for children or flatfoot, because they were developed for adult generalized foot and ankle disease or ankle osteoarthritis. The Oxford Ankle Foot Questionnaire for Children (OxAFQ-C) is the only validated tool in the pediatric population to measure the subjective well-being of children from 5-to 16-years-old with foot and ankle conditions [19].The major limit of the OxAFQ-C is its patient-reported nature, as several studies report a tendency in children to score themselves higher than their parents [20,21], while the physician CTF Score report an intra-observer reliability of 0.883, with the OxAFQ-C domain reliability rating at 0.6 and 0.83.In addition, the tool was useful for physicians with an intra-and interobserver reliability of 0.852 and 0.878, respectively.Since March 2020, the pandemic emergency raised questions about alternatives to normal clinical activity to avoid overcrowding in departments; for less risk of contagions, many checkups were procrastinated.This issue caused a possible loss of patient follow-up, which can reflect on the clinic and its outcomes.The necessity to develop management protocols highlights telemedicine as a valid alternative in particular conditions vs the face-to-face clinic, with safety margins and economic savings.The CTF score was administered with a web-based database, well-tolerated by observers; moreover, despite assessment of foot functionality, the CTF Score does not include a range of motion evaluation.The score was considered a good remote follow-up tool.The authors intend to promote the distribution of the score and faceto-face and remote validation. Surgical treatment is still debated, as Bouchard and Mosca [22] suggested that surgical management be used only in Achilles' tendon retraction, while several authors highlighted issues of fatigue, inadequate physical performance, and pain as the main parameters for the decision-making process [23].The 75% CTF score cut-off presented high sensitivity and specificity as reasonable cut-offs for surgical treatment.The tool does not replace the surgeon's experience, but represents a helpful orthopedic decision-making process.The CTF provides to general or pediatric physicians, podiatrist, physiotherapists, young or non-pediatric orthopedic trained orthopedic surgeons a common accepted and objective additional tool for the correct flatfoot grade and eventually surgical indication.The patient and family history, body posture assessment remain mandatory for the proper assessment.Future research into the development and validation of the questionnaire will assess whether the instrument is responsive to change.We will administer the questionnaire to general non-pediatric orthopedic surgeons, and reassess test-retest reliability while monitoring dimensionality and scaling of the instrument as more data become available. The limits of the score are related to the domains compilation, in fact, the valgus of the hindfoot, longitudinal arch, forefoot abduction, and triceps contracture assessment are related to the physician or surgeon experience, and the fatigue, inadequate physical and sport performance items are related to the patient consciousness.In the future, the development of new and more objective criteria could make the CTF more usable. CONCLUSION In conclusion, the CTF Score is useful for orthopedic surgeons in the juvenile FFF evaluation.The CTF score is derived from a high-quality questionnaire for clinical research, survey studies, or clinical practice.The 75% cut-off point is a good threshold for surgical indication and decision-making.Given widespread use of telemedicine, the CTF score is also seen as an objective remote clinical examination. Figure 1 Bland 1 Figure 1 Bland Altman plots according overall intra-observer reliability intra-class correlation coefficients.ICC: Intra-class correlation coefficients. Figure 2 2 Figure 2 Scatter plot.A: The correlation between the Catania flatfoot (CTF) score and the conservative treatment indication; B: The correlation between the CTF score and the surgical treatment indication. Table 1 Intra-observer reliability intra-class correlation coefficients values 195% confidence intervalSampleICCValueP valueLower limitUpper limitOverall0.8830.8540.9097.513< 0.0001Specialists0.8690.8320.90127.523< 0.0001Residents0.8780.8460.90744.351< 0.0001ICC: Intra-class correlation coefficients. Table 2 Interobserver reliability intra-class correlation coefficients values 295% confidence wintervalSampleICCValueP valueLower limitUpper limitOverall0.8700.8460.89234.479< 0.0001Specialists0.8090.7610.8499.475< 0.0001Residents0.8520.8210.87918.272< 0.0001ICC: Intra-class correlation coefficients. All study participants or their legal guardian provided informed written consent about personal and medical data collection prior to study enrolment.ARTICLE HIGHLIGHTSResearch backgroundFlexible flatfoot (FFF) is a very common condition in children, but no evidence-based guidelines or assessment tools exist.Yet, surgical indication is left to the surgeon's experience and preferences.Research motivationThe lack of common diagnostic criteria for FFF.Research objectivesTo develop a functional clinical score for FFF [Catania flatfoot (CTF) score] and a measure of internal consistency; to evaluate interobserver and intra-observer reliability of the CTF Score; to provide a strong tool for proper FFF surgical indication.Research methodsCTF is a medically compiled score of four main domains for a total of twelve items: Patient features, Pain, Clinical Parameters, and Functionality.Each item refers to a specific rate.Five experienced observers answered 10 case reports according to the CTF.To assess inter-and intra-observer reliability of the CTF score, the intra-class correlation coefficients' (ICCs) statistics test was performed, as well as to gauge the correlation between the CTF score and the surgical or conservative treatment indication.Values of 75% were chosen as the score cut-off for surgical indication.Sensitivity, specificity, positive likelihood ratio (PLHR), negative likelihood ratio (NLHR), positive predictive value (PPV), and Paediatric flexible flat foot: how are we measuring it and are we getting it right? 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The relationship between self-efficacy and treatment satisfaction among patients with anticoagulant therapy: a cross-sectional study from a developing country Samah W Al-Jabi Amal Abu Dalu Amer A Koni Maher R Khdour Adham Abu Taha Riad Amer Sa&apos;ed H Zyoud The relationship between self-efficacy and treatment satisfaction among patients with anticoagulant therapy: a cross-sectional study from a developing country 10.1186/s12959-022-00374-2R E S E A R C H Open Access Background: Thromboembolic events are a common complicated health problem. Although anticoagulants have several positive effects on these conditions, they also have several characteristics that strongly affect compliance and satisfaction. The purpose of this investigation is to explore the association between treatment satisfaction and selfefficacy in a sample of patients using anticoagulation therapy and determine the influence of sociodemographic and clinical factors on both aspects.Methods: This was a cross-sectional exploratory study carried out in Palestine. The Arabic version of the Anti-Coagulant Treatment Satisfaction Scale (ACTS) assessed treatment satisfaction. In addition, the Arabic version of the 6-Item Self-Efficacy for Managing Chronic Diseases (SES6C) was used to assess self-efficacy.Results: A total of 300 patients using anticoagulants (average age 51.95 and SD 17.98) were included. There is a modest correlation between treatment satisfaction and self-efficacy (r = 0.345; p < 0.001). The mean and median selfefficacy scores were 38.41 ± 9.88 and 39.00 (interquartile range: 33.00-46.00), respectively. Overall, patients reported a moderate burden and benefit score. The mean and median of the acting burden were 43.30 ± 10.45, and 43.30 (interquartile range: 36.00 to 51.00), respectively. The results showed that young age, higher education, employment, use of fewer medications, and having fewer diseases were significantly associated with higher self-efficacy behaviors. The results also showed that new oral anti-coagulants (NOACs) had a higher degree of self-efficacy and ACTS benefit scores (41.00 (33.75-47.00), p = 0.002; 13.00 (12.00-15.00), p < 0.001, respectively), than vitamin k antagonists (VKA). Conclusions: The results demonstrated a significant relationship between treatment satisfaction and self-efficacy, and certain sociodemographic and clinical characteristics influence both. We found that there is a higher degree of selfefficacy and treatment satisfaction among patients who use NOACs than those who use UFH / VKA. Therefore, patients should be motivated to increase their knowledge about anticoagulant therapy. Healthcare providers should play an active role in educating patients, increasing their self-esteem, and awareness about anticoagulant drugs. Importantly, this study was an explanatory one, and it includes a low proportion of patients with venous thromboembolism. This encourages future research on a large scale of patients, considering the indications of anticoagulant therapy. Keywords: Self-efficacy, SES6C, Treatment satisfaction, Burden, Benefit, ACTS, Anti-coagulant, NOAC Background Anticoagulant drugs with different mechanisms of action are indicated for many conditions, such as the prevention of systemic embolism in valvular heart disease, myocardial infarction [1], atrial fibrillation-related stroke [2], pulmonary embolism (PE), deep vein thrombosis (DVT) [3] and cancer-related thrombosis [4]. However, if non-anticoagulated, there is a significant increase in the mortality rate in patients with stroke risk factors [5], morbidity, and negative effects on quality of life [6]. Although anticoagulants have several positive effects on the disease, they also have several characteristics that can strongly affect compliance and satisfaction, inducing dissatisfaction and reducing the patient's quality of life (e.g., diet and activity restrictions, regular blood tests, bleeding, and/or bruises). These side effects and burdens can lead to poor compliance, and as a result, to failure of treatment failure [3]. In addition, long-term anticoagulant therapy, particularly with vitamin K antagonist (VKA), can be associated with low satisfaction and poor adherence [6]. Patient satisfaction with anticoagulant therapy and his quality of life in clinical practice may affect treatment outcomes [1], as satisfaction can increase compliance and treatment success. Individual-level patient characteristics and preferences for shared decision-making will improve the patient's treatment experience by achieving treatment satisfaction [7,8]. Treatment satisfaction was associated with better compliance and persistence, in addition to reducing treatment burden or regimen complexity [9]. This makes satisfaction for an individual patient an important factor in the selection or changing anticoagulant therapy in patients [10]. On the other hand, treatment dissatisfaction can negatively affect patients, such as the quality of the treatment regimen implemented [9]. Another aspect that can affect the success of treatment and its goal is the patient's self-efficacy. 'Self-efficacy' is a term that refers to the confidence one has in the ability to follow the behaviors needed to achieve a desired goal or effect. It is a psychological concept that is used in association with chronic diseases and medication management. There has been a growing interest in the role of self-efficacy as a predictor of the treatment outcome [11]. Self-efficacy helps participants have confidence and skills better to manage their chronic conditions [12,13]. Self-efficacy has been increasingly recognized as an essential prerequisite for effective self-management of all chronic diseases [14]. Low self-efficacy is one of the main problems that require physical and mental rehabilitation [15]. In this study, we hypothesize that if the patient has the required knowledge and skills, and is confident in managing his chronic condition and anticoagulant medication, he will be more adherent and satisfied with the therapy used. Thromboembolic events and many cardiovascular diseases are widely treated with anticoagulants, which have great benefit to patients [16] and have many characteristics that can affect patients' daily lives. Ample of studies have been established to study anticoagulant treatment satisfaction and self-efficacy; however, these studies did not correlate the two concepts with each other [8,10,[17][18][19][20][21][22][23][24][25][26][27][28]. Therefore, the purpose of this investigation is to explore the association between treatment satisfaction and self-efficacy in a sample of patients using anticoagulation therapy and determine the influence of sociodemographic and clinical factors on both aspects. To the best of our knowledge, this study is the first of its type in Palestine. Consequently, this study will provide baseline data and information on treatment satisfaction, selfefficacy, and the relationship between the two concepts, in addition to evaluating factors associated with patient self-efficacy and treatment satisfaction with anticoagulant therapy. Furthermore, it will help healthcare providers establish mechanisms that can achieve patient treatment satisfaction and train them to accept good self-efficacy. Methods Study design This study was a cross-sectional questionnaire-based exploratory study to measure patient self-efficacy and treatment satisfaction with anticoagulant therapy and to find the relationship between both aspects. This study was conducted in Jerusalem at Al Makassed Tertiary Hospital, from July 2019 to July 2020. Study population The population of this study was patients from hospitals or outpatient clinics who were using anticoagulants. Sample size calculation and sampling procedure The study was carried out in a group of patients who used anticoagulants attending Al Makassed Hospital. Based on the expected population during the research period (n = 1000 patients) and a 50% response distribution, the needed sample size was approximately 278 with a confidence level of 95% and a margin of error of 5%. An automated software program, the Raosoft sample size calculator (http://www.raosoft.com/samplesize.html) was used to calculate the required sample size for this study. The intended sample size was increased to 300 individuals to reduce erroneous results and increase the reliability of the study. Inclusion and exclusion criteria The inclusion criteria were patients over 18 years of age using any type of anticoagulant drug, Arab nationality, and who can read or understand Arabic. Exclusion criteria were patients who refused to participate in the study and those previously diagnosed with mental illnesses or severe cerebral vascular disease that affected cognitive ability. Data collection and management This quantitative study used a questionnaire as an instrument to collect data from respondents. This study's data collection forms were adopted from two different scales, the Anti-Coagulant Treatment Satisfaction Scale (ACTS) and the Self-Efficacy for Managing Chronic Disease 6-Item Scale (SES6C). The data collection form contains four sections. In the first section, we covered sociodemographic factors, such as age, gender (male, female), residency (city, village, or refugee camp), job, the primary healthcare centers they visit, marital status, body mass index (BMI) and educational level (illiterate, primary, secondary, or university). The second section of the questionnaire consists of questions related to clinical data, including co-morbid conditions, recent major surgery, history of major bleeding, risk of falls, laboratory findings, and type of anticoagulant drug type used, dose, and duration. The third section measures patients' satisfaction with anticoagulant therapy treatment using the ACTS scale. The ACTS scale is an anticoagulation-related quality of life measurement instrument. This scale is not generic, but it is condition-specific, focusing on the aspects of health-related quality of life specific to anticoagulant medications [1]. The ACTS questionnaire is an instrument used to measure treatment satisfaction with anticoagulant therapy through patient-reported outcomes (PRO). It consists of two parts; the first part includes 12 items that assess the perceived burden of anticoagulant treatment, and the second part consists of 3 items that assess the perceived benefits of anticoagulant treatment. It uses a 5-point intensity scale (from 1 'not at all' to 5 'extremely'). Its total burden scores are reversed (higher scores indicate less burden and vice versa) and range from 12 to 60. However, the total scores of the ACTS benefits are directly scored, ranging from 3 to 15 [23,29,30]. The questionnaire has been profoundly tested for content validity, question difficulty measures, readability, item/person reliability in many languages [30] (Cano et al., 2012) and specifically Arabic language [6,31]. We also registered our study with ePROVIDE -Mapi Research Trust and obtained permission for the use of ACTS use (ID: 42874). The fourth section is used to measure the self-efficacy in anticoagulant therapy using the SES6C. The SES6C is a valid and reliable instrument for assessing the self-efficacy in the management of chronic diseases. It is a self-reporting instrument that can be used to assess a degree of confidence of the patient who is suffering from chronic disease in trying to manage their disease [32]. It is a 6-item scale with a visual analogue, ranging from 1 (not at all confident) to 10 (totally confident). This scale covers several common domains in many chronic diseases, role function, emotional functions, symptom control, and communication with physicians. It is an important and reliable instrument for assessing selfefficacy in managing different chronic diseases. A higher score indicates greater confidence in the management of the patient's chronic disease. A qualified pharmacist collected the required data and administered the questionnaire. The pharmacist met the patient in a clinic or pharmacy. Patients were advised that the questionnaire would take approximately 20 min to complete. Ethical approval All aspects of the study protocol, including access to and use of patient clinical information, were approved by the Institutional Review Board (IRB) of An-Najah National University and local health authorities prior to the initiation of this study. In addition, a verbal consent form was obtained from each patient. Pilot study A pilot study of 30 participants has been conducted to test the tool, ensure the availability of the required data, estimate the time, and modify the data collection form, as appropriate. Patients who participated in the pilot study were not included in the final analysis. Statistical analysis Data were entered and analyzed using the Statistical Package for Social Sciences program version 15 (SPSS). Data were expressed as means ± SD for continuous variables and as frequencies (percentages) for categorical variables. Variables that are not normally distributed are expressed as medians (lower-upper quartiles). The normality of the variables was tested using the Kolmogorov-Smirnov test and found that the data were not normally distributed. Therefore, non-parametric tests of univariate analysis were used to test for differences in medians between categories. Indeed, the Mann-Whitney test was used to compare the two groups of patients' characteristics on the dependent variables (self-efficacy, burden, and benefit scores), while the Kruskal-Wallis was applied to compare three or more groups. The significance level was established at a p-value of < 0.05. Results Sociodemographic and clinical data Our study was carried out on 323 patients with a response rate of 92.88%. Table 1 indicates that most of our patients are women (61.7%), nearly half of them (54.3%) are middle-aged (30-60 years), and 35% are over 60 years old. Additionally, 55.7% of our patients had a secondary level of education or less (18% secondary, 17.0% primary, 14.0% elementary, and 6.7% were illiterate), and 41.3% had a high level of education. Of 300 patients, 64.7% were employed, 53.3% had a monthly income between 2000 and 5000 NIS, and 36.0% had a monthly income of more than 5000 NIS. Depending on the location, 57.3% of our patients were urban, 28.3% Rural, and 12.0% lived in camps. Almost half of the patients (48.7%) had three or more diseases. The reason for the anticoagulation therapy was AF 21%, DVT or PE 9.7%, as prophylaxis during hospital admission 24%, and other indications 45.3%. The mean number of medications used by our patients was 4.2. This number was used to categorize this variable. Therefore, 58.7% of the participants used four or less than four medications. Self-efficacy score according to sociodemographic variables The mean and median self-efficacy score was 38.41 ± 9.88, and 39.00 (interquartile range: 33.00-46.00), respectively. A significant difference in self-efficacy scores was found between participants and their age (p < 0.001), educational level (p < 0.001), employment status (p = 0.049), comorbidities (p = 0.034) and chronic medications (p = 0.029), and anticoagulant drug (p = 0.002). The median of No significant differences were observed between participants according to their gender, weight, income, location, and marital state ( Table 2). ACTS burden score according to sociodemographic variables The mean and median of the acting burden were 43.30 ± 10.45, and 43.30(interquartile range: 36.00 to 51.00), respectively. According to the gender of the participant, the ACTS burden score for the male participants was 46.00 (38.00-52.00), and the female 42.00 (35.00-49.00) (p = 0.006). The median ACTS burden scores significantly increase with increasing monthly income (p = 0.001), 40.00 (31.00-46.00) was for a monthly income of less than 2000 NIS, 42.00 (35.00-50.00) for 2000-5000 NIS, and 45.00 (40.00-53.00) for a monthly income of more than 5000 NIS. Married participants had a higher ACTS burden score 44.00 (37.00-51.00) than unmarried (single, divorced and widowed) 41.00 (31.00-49.00), (p < 0.001). According to the indications, no significant differences were found in the burden scores. Depending on the type of anticoagulant used, there were no significant differences in the median burden score of treatment satisfaction between UFH, VKA and NOACs (Table 3). ACTS benefit score according to sociodemographic variables The mean and median of the total ACTS benefit total score was 11.84 ± 2.17, and 12.00 (interquartile range 11.00-13.75), respectively. The median of the ACTS benefit scores in participants under 30 years old was 15.00 (14.00-15.00), middle age (30-60) was 12.00 (11.00-14.00), and older than 60 was 11.00 (9.00-12.00). Depending on the gender of the participants, the median ACTS benefit score for male was 12.00 (10.00-12.00), and female 12.00 (11.00-14.00). Illiterate participants had the lowest median ACTS benefit score of 9.50 (9.00-11.00), elementary school level was 11.00 (9.00-12.00), primary school was 12.00 (10.00-13.00), secondary school was 12.00 (11.00-14.00), and university 12.00 (11.00-15.00). Furthermore, participants with one or two diseases had a median score was 12.00 (10.00-14.00), while participants with three or more have a lower score of 11.00 (10.00-12.00). The ACTS benefit score was 12.00 (11.00-15.00) for participants using four or less and 11.00 (10.00-12.00) for participants using more than four medications. The median benefit score was significantly higher for those who received prophylactic doses of enoxaparin during hospital admission (p < 0.001). Finally, the median of the ACTS benefit score according to the anticoagulants used was 11.00 (10.00-12.00) for enoxaparin, 11.50 (10.00-13.00) for warfarin, and 13.00 (12.00-15.00) for NOAC users. Table 4 shows a significant acting benefit score based on the participant's age (p < 0.001), gender (p = 0.015), BMI (p = 0.003), educational level (p < 0.001), marital status (p = 0.003), comorbidities (p = 0.018), chronic medications (p < 0.001), indication (p < 0.001), and anticoagulant drug (p < 0.001). Correlations between treatment satisfaction and selfefficacy The values of the Spearman correlation coefficient between the total burden, benefits, and overall satisfaction score with the self-efficacy score were 0.325, 0.171 and 0.345, respectively. The results of this study indicated a significant modest positive correlation between all satisfaction domains (burdens (p < 0.001), benefits (p = 0.003) and overall satisfaction (p < 0.001)) and self-efficacy scores (Table 5). Discussion Our study was carried out at Al Makassed hospital, which is located in Jerusalem city in Palestine, in patients who use anticoagulant drugs prophylactically or as a treatment for different medical conditions. This study was one of the first conducted in Palestine to examine whether there is a significant relationship between patient treatment satisfaction using the ACTS scale and self-efficacy using the SES6C scale and to determine the factors that affected both scales. Our study demonstrated a mean self-efficacy score (SES6C score) of 38.41 and revealed that older patients, less educated, and unemployed patients had a significantly lower self-efficacy score. This necessitates appropriate education and counseling for these patients about their anticoagulant medication. The median score for those who were using Vit-K drugs was 41.00 out of 60. In addition, clinical factors, such as using Vit-K drugs, having a higher number of chronic diseases, and polypharmacy, significantly lower self-efficacy. Especially for Vit-K drugs, special attention and selfmanagement strategies are necessary because these medications have a greater risk of major bleeding [33] and therapeutic failure [34,35]. Since positive correlations were found between self-efficacy and treatment satisfaction, consequently affecting patient adherence to anticoagulant drugs and success of treatment. Health care providers (mainly physicians and pharmacists) should pay more attention to this issue and consider these factors in the treatment and monitoring plan. Several previous analyzes ensured that selfmanagement in patients using anticoagulants was applicable and positively affected treatment-related quality of life compared to routine care [36][37][38][39]. Selfmanaging patients may be more aware of any change in their INR due to their ability to measure it and take an adaptive action, having higher knowledge, and greater self-efficacy. This reinforces the evidence demonstrating that warfarin therapy self-management is an effective and safe alternative to routine management [40][41][42][43]. However, the impact of this strategy on the risk of bleeding and thrombosis should be tested. In recent years, greater interest has been shown in the study of treatment satisfaction. A Spanish study aimed to evaluate the satisfaction with treatment of nonvalvular atrial fibrillation in anticoagulant patients on anticoagulants in Spain in 2018 [23]. Regarding anticoagulation satisfaction, the ACTS burden scale was 49.69 ± 9.45 and the benefits scale was 11.35 ± 2.61. Higher scores denote a lower burden and higher perceived benefit for both scales, and consequently, greater satisfaction with treatment. Our patients reported a mean burden score of 43.30 out of 60 and a benefit score of 11.84 out of 15. However, our patients reported lower burden scores than other populations [6,23], which means a higher burden of this therapy. The burden mean score revealed that the male participants had a higher degree of satisfaction degree than females, which corresponds to a study carried out in Spain [23] and another study, which found that the more burdened and less satisfied participants who used warfarin were more likely to be women [44]. According to our results, young age and women were more perceived to benefit from anticoagulant therapy, but burden scores were not significantly different between age categories. A study conducted in California in patients with venous thromboembolism found that women of younger age were associated with a higher perceived anticoagulant burden [22]. Two Spanish studies, one that dealt with AF patient satisfaction with NOAC [28] and the second specified nonvalvular AF patients' satisfaction with VKA [21], showed that elderly patients were less burdened with anticoagulant treatment. Furthermore, the benefit score was reported to be significantly reduced in patients with low education level. A Sudanese cross-sectional study published in 2017 [20], evaluated patient satisfaction with oral anticoagulant therapies and their adherence to them and identified the predictors of the two study domains. Approximately half of the participants (50.5%) were satisfied with anticoagulant treatment. The study concludes that a multidisciplinary effort from healthcare providers is needed to ensure health education, in addition to continuously motivating patients continuously in order to increase treatment success, especially among patients with a low educational level. Patient satisfaction with each anticoagulant drug should be determined among different indications, in order to select a suitable therapy with a low treatment burden on patients that improves clinical outcomes and quality of life. An important finding was that patients who used NOAC had higher scores of both parts of treatment satisfaction; burden and benefit than patients on unfractionated heparin or vitamin k-dependent anticoagulants. However, the significant differences were only for benefit (p < 0.001) score only. A substudy of a multicenter research (SAKURA AF registry) aimed to compare the satisfaction of NOAC users and warfarin users in patients with AF using the ACTS scale [26]. The burden scores were significantly higher (54.5 ± 6.3 versus 52.7 ± 6.9, P < 0.0001) among NOAC users than among warfarin users, but the baseline benefit scores among NOAC users tended to be lower (9.8 ± 3.1 versus 10.1 ± 3.2, P = 0.0513), which is contrary to our results. The reduced burden of NOACs and constant patient education about the benefit of stroke prophylaxis will lead to greater adherence of patients to their treatment plan and therefore have a positive effect on clinical outcomes. SAFARI, a French observational study published in 2016 [24], aimed to investigate patient-reported satisfaction with treatment with rivaroxaban to prevent stroke in patients with AF. A total of 405 nonvalvular atrial fibrillation patients who had been previously treated with warfarin switched to rivaroxaban. Patient satisfaction improved after 3 months and persisted for 6 months. The same observation was found in a Japanese study AGAIN [10] and others [18,25]. The AGAIN study showed an improvement in treatment satisfaction after switching from warfarin to apixaban in patients with nonvalvular AF (NVAF) after switching from warfarin to apixaban from warfarin. Furthermore, the effect of NOAC therapy in improving treatment satisfaction compared to other anticoagulants was found in the treatment of patients with acute symptomatic DVT and PE [17,27]. According to the indications, patients who received prophylactic doses of enoxaparin during hospital admission had higher benefit scores, indicating higher treatment satisfaction. This could be explained by the ease of administration, as the prophylactic dose may have been administered by a nurse, this would be different from patients undergoing self-administration. Increasing patient satisfaction is a role that healthcare providers should be carried out through educating patients about the use of medications, their importance, side effects, and other drug-related factors. Educating the anticoagulated patient is often neglected because it is time-consuming, but pharmacists can play an essential role in this education strategy [45]; pharmacists (according to the National Patient Safety Agency) are in a prime position to provide good counseling about the anticoagulant and disease status [46], since patients forget 40-80% of the information that their doctors give them by their physicians [45]. Strengths and limitations To our knowledge, this study was one of the first to investigate the relationship between self-efficacy and treatment satisfaction among patients using anticoagulants in Palestine. On the other hand, this study has a number of limitations. First, this is a cross-sectional study; therefore, it is difficult to prove causal relationships between the scales that have been used and their associated factors. Second, this study did not explore other potential factors that may affect self-care/self-efficacy, such as the duration of the disease, smoking status, economic status (as NOACs were not covered by health insurance), INR monitoring practice, and duration of anticoagulation use. Third, the interviewer's bias in the results may have been introduced, since the data was collected via a faceto-face interview. Although the study included patients with different anticoagulant medications, bias related to the dosage form of the medication used (oral, intravenous, or subcutaneous) may be introduced as the nature of administration is different and its use is likely to be restricted to a distinct patient subset. Furthermore, there is a potential bias related to the indications since these relate to different patients, as for age, sex, and comorbidity. Finally, the sample size and the use of a single center to recruit patients are considered limiting factors in this study. Conclusions We found a relationship between patients' self-efficacy on anticoagulant drugs and treatment satisfaction. This correlation is expressed by the low correlation coefficient. A notable finding is that there is a higher degree of self-efficacy and treatment satisfaction among patients using NOACs than among those who use UFH/VKAs. Furthermore, certain factors were found to be significantly associated with self-efficacy and treatment satisfaction. These factors should be considered when creating a treatment plan. The study's findings have certain implications for future practice, including the following; we advocate policymakers and healthcare providers to make the necessary changes to improve healthcare services and facilities for all patients, especially outside the cities. In addition, we recommend conducting specialized therapy and training sessions on anticoagulant therapy. Patients should be motivated to increase their knowledge of anticoagulant therapy, which can be achieved through customized health promotions and counseling programs and tailored health promotions. Healthcare providers should play an active role in educating patients, increasing their self-esteem, and awareness about anticoagulant drugs. Importantly, this study was an explanatory one, and it includes a low proportion of patients with venous thromboembolism. This encourages future research on a large scale of patients, considering the indications of anticoagulant therapy. Authors' contributions AAD conducted the literature search, data collection, and analysis and drafted the manuscript. AAK, AA, RA, and MK discussed the findings and provided input and a critical review to all parts of the investigation. SWA and SHZ extensively reviewed the literature, developed the study idea and objectives, designed the methods, and took the lead in data analysis and interpretation. Then, all the authors read and agreed on the final version. Funding No funding was received for this study. Availability of data and materials All data used in this study are available from the corresponding author upon request. Declarations Ethics approval and consent to participate All aspects of the study protocol, including access to and use of patient clinical information, were approved by the Institutional Review Board (IRB) of An-Najah National University and local health authorities prior to the initiation of this study. In addition, a verbal consent form was obtained from each patient. The IRB of An-Najah National University approved only verbal consent. Because we did not collect any identifying information during the interview and our study did not pose a major risk to participants, our ethics committee waived the requirement for written consent. The authors confirmed that all methods were performed in accordance with the relevant guidelines and regulations. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Abbreviations ACTS: Anti-Clot Treatment Scale; AF: Atrial Fibrillation; BMI: Body Mass Index; CVD: Cardiovascular Disease; DVT: Deep Vein Thrombosis; HF: Heart Failure; IHD: Ischemic Heart Disease; IRB: Institutional Review Boards; NIS: New Israeli Shekel; NOACS: New Oral Anticoagulants; PE: Pulmonary Embolism; PRO: Patient Reported Outcome; SES6C: Self-Efficacy for Managing Chronic Disease 6-Item Scale; SPSS: Statistical package for Social sciences; UFH: Unfractionated Heparin; VKAs: Vitamin K Antagonists Acknowledgments Not applicable. Table 1 1Sociodemographic and clinical characteristics of the study sampleSociodemographic Variables Frequency (%) N = 300 Gender Male 115 (38.3) Female 185 (61.7) Age Less than 30 31 (10.3) 30-60 163 (54.3) More than 60 106 (35.3) BMI Normal weight (18.5-24.9) 65 (21.7) Overweight (25-29.9) 136 (45.3) Obese > 30 93 (31.0) Educational status a Illiterate 20 (6.7) Elementary 42 (14.0) Primary 51 (17.0) Secondary 54 (18.0) University 124 (41.3) Marital status a Married 229(76.3) Unmarried (single, divorced, widowed) 67(22.3) Income/month (NIS) a Less than 2000 23 (7.7) 2000-5000 160(53.3) More than 5000 108(36.0) Employment status a Employed 194(64.7) Unemployed 100(33.3) Locality a Camp 36(12.0) Rural 85(28.3) Urban 172(57.3) Chronic co-morbid disease a No co-morbid disease (pregnant) 42 (14.0) One Disease 44(14.7) Two diseases 63(21.0) Three diseases or more 146(48.7) Indications for anticoagulant Atrial fibrillation 63 (21.0) Deep vein thrombosis or Pulmonary embolism 29 (9.7) Prophylaxis b 72 (24.0) Other indications 136 (45.3) Chronic medications ≤ 4 176(58.7) Table 1 1Sociodemographic and clinical characteristics of the study sample (Continued) Abbreviations; NIS New Israeli Shekel (0.29 US Dollar), BMI Body mass index.Sociodemographic Variables Frequency (%) N = 300 > 4 124(41.3) a Some data were missing b Patients that received prophylactic doses of unfractionated heparin during hospital admission Table 2 2Total self-efficacy score by socio-demographic and clinical variablesVariable Frequency % N(300) Median (Q1-Q3) Mean ± SD Mean rank P-value Age category (years) < 0.001 b < 30 31 (10.3) 47.00(41.00-50.00) 44.32 ± 9.09 205.84 30-60 163 (54.3) 41.00(35.00-46.00) 39.36 ± 9.38 158.72 > 60 106 (35.3) 35.00(30.00-42.00) 35.23 ± 9.80 121.68 Gender 0.311 a Male 115 (38.3) 40.00(33.00-47.00) 39.09 ± 10.46 156.93 Female 185 (61.7) 39.00(32.00-45.00) 37.99 ± 9.52 146.50 BMI a 0.146 b Normal 65 (21.7) 41.00(39.00-49.00) 40.13 ± 9.51 163.11 Overweight 136 (45.3) 39.00(34.00-45.00) 38.35 ± 9.05 147.75 Obese 93 (31.0) 38.00(31.00-44.00) 37.09 ± 9.97 136.19 Education < 0.001 b Illiterate 20 (6.7) 29.50(21.75-41.50) 30.70 ± 11.37 90.18 Elementary 43 (14.3) 35.00(28.00-41.00) 36.14 ± 9.42 123.37 Primary 52 (17.3) 37.00(31.00-42.00) 36.45 ± 9.77 130.83 Secondary 54 (18.0) 41.00(36.75-48.00) 40.65 ± 9.91 167.40 University 124 (41.3) 41.50(34.00-47.00) 39.85 ± 9.18 159.59 Income (month) 0.432 b Less than 2000 NIS 23 (7.7) 41.00(31.00-45.00) 37.61 ± 11.70 145.93 2000-5000 NIS 160(53.3) 39.00(31.00-45.00) 38.00 ± 9.66 140.54 More than 5000 NIS 108(36.0) 41.00 (35.00-46.00) 39.31 ± 10.07 154.10 locality 0.675 b Camp 36 (12.0) 38.50(31.25-45.75) 37.53 ± 9.46 138.42 Rural 85(28.3) 39.00(31.00-44.00) 38.11 ± 9.51 143.67 Urban 172(57.3) 39.00(34.00-46.75) 38.76 ± 10.18 150.44 Marital status 0.103 a Married 229(76.3) 12.00(11.00-14.00) 39.00 ± 9.52 152.59 Unmarried (single, divorced, widowed) 67(22.3) 11.00(10.00-13.00) 36.25 ± 10.97 133.50 Employment status 0.049 a Employed 194(64.7) 42.00(34.00-48.00) 40.04 ± 10.26 161.07 Unemployed 100(33.3) 38.00(32.00-44.25) 37.82 ± 9.24 140.51 Chronic co-morbid disease 0.034 b No co-morbid disease 42 (14.0) 42.00(35.00-47.00) 40.29 ± 8.89 166.74 One disease 44(14.7) 42.00(34.00-49.00) 40.30 ± 10.56 164.71 Two diseases 63(21.0) 40.00 (33.00-47.00) 39.43 ± 8.89 156.02 Three diseases or more 146(48.7) 37.00(30.75-44.00) 36.76 ± 10.32 133.22 Indications for anticoagulant 0.001 b Atrial fibrillation 63 (21.0) 36.00 (27.00-41.00) 34.02 ± 10.51 115.01 Deep vein thrombosis or Pulmonary embolism 29 (9.7) 43.00 (34.51-49.50) 42.76 ± 7.79 187.07 Prophylaxis c 72 (24.0) 39.50 (35.00-46.75) 38.63 ± 8.83 152.99 Other indications 136 (45.3) 41.00 (33.00-47.00) 39.40 ± 9.90 157.82 Chronic medications 0.029 a ≤ 4 176(58.7) 41.00(33.25-47.00) 39.32 ± 9.95 159.76 > 4 124(41.3) 37.00(32.00-44.00) 37.11 ± 9.68 137.49 Table 2 2Total self-efficacy score by socio-demographic and clinical variables (Continued)Variable Frequency % N(300) Median (Q1-Q3) Mean ± SD Mean rank P-value Anticoagulant drug 0.002 b UHF 87(29) 40.00(35.00-46.00) 39.02 ± 8.45 154.05 Vit-K dependent 134(44.7) 35.00(30.25-42.00) 35.18 ± 9.74 119.38 NOACs 75(25) 41.00(33.75-47.00) 39.00 ± 8.20 161.20 a The statistical significance of the differences was calculated using the Mann-Whitney U test b The statistical significance of the differences was calculated using the Kruskal-Wallis test Bold P-value indicates a significant difference c Patients that received prophylactic doses of unfractionated heparin during hospital admission NIS: New Israeli Shekel (0.29 US Dollar) Table 3 3Total burden scores by socio-demographic and clinical variablesVariable Frequency % N(300) Median (Q1-Q3) Mean ± SD Mean rank P-value Age category (years) 0.651 b < 30 31 (10.3) 42.00(34.00-52.00) 41.68 ± 11.02 141.21 30-60 163 (54.3) 43.00(37.00-51.00) 43.99 ± 10.77 154.47 > 60 106 (35.3) 45.00(36.00-50.00) 42.73 ± 91.78 147.11 Gender 0.006 a Male 115 (38.3) 46.00(38.00-52.00) 45.01 ± 9.88 168.03 Female 185 (61.7) 42.00(35.00-49.00) 42.25 ± 10.68 139.60 BMI a 0.702 b Normal 65 (21.7) 42.00(35.00-50.00) 42.89 ± 9.89 140.45 Overweight 136 (45.3) 44.00(36.25-51.75) 43.44 ± 10.15 151.19 Obese 93 (31.0) 44.00(37.00-50.50) 43.55 ± 11.48 147.04 Education 0.160 b Illiterate 20 (6.7) 39.00(29.25-49.50) 39.60 ± 11.17 118.63 Elementary 43 (14.3) 46.00(36.75-50.25) 43.31 ± 9.19 149.86 Primary 52 (17.3) 42.00(35.00-49.00) 41.06 ± 10.13 131.38 Secondary 54 (18.0) 45.00(38.75-54.00) 45.67 ± 9.60 165.19 University 124 (41.3) 43.00(36.00-50.75) 43.80 ± 11.25 146.76 Income (month) 0.001 b Less than 2000 NIS 23 (7.7) 40.00(31.00-46.00) 38.60 ± 8.74 108.11 2000-5000 NIS 160(53.3) 42.00(35.00-50.00) 41.93 ± 9.44 136.66 More than 5000 NIS 108(36.0) 45.00(40.00-53.00) 46.06 ± 11.10 167.91 locality 0.123 b Camp 36(12.0) 41.00(35.00-48.50) 41.47 ± 8.55 131.01 Rural 85(28.3) 40.00(34.00-50.00) 41.83 ± 11.09 136.84 Urban 172(57.3) 44.00(37.25-51.00) 49.17 ± 10.13 155.37 Marital status 0.005 a Married 229(76.3) 44.00(37.00-51.00) 44.29 ± 10.26 Unmarried (single, divorced, widowed) 67(22.3) 41.00(31.00-49.00) 39.86 ± 10.32 Employment status 0.114 a Employed 194(64.7) 42.00(35.75-50.25) 44.76 ± 11.55 158.40 Unemployed 100(33.3) 45.00(36.25-52.00) 42.61 ± 9.88 141.88 Chronic co-morbid disease 0.696 b No co-morbid disease 42 (14.0) 44.00(38.00-52.00) 44.24 ± 8.25 156.31 One disease 44(14.7) 45.00(35.00-53.00) 44.67 ± 11.45 156.79 Two diseases 63(21.0) 42.00(36.00-51.00) 42.35 ± 9.26 141.24 Three diseases or more 146(48.7) 44.00(36.00-50.00) 43.41 ± 11.05 144.93 Indications for anticoagulant 0.366 b Atrial fibrillation 63 (21.0) 43.00 (37.00-50.00) 42.54 ± 8.60 145.65 Deep vein thrombosis or Pulmonary embolism 29 (9.7) 41.00 (34.00-46.50) 41.00 ± 8.41 126.03 Prophylaxis c 72 (24.0) 44.00 (37.00-52.00) 43.57 ± 10.16 154.79 Other indications 136 (45.3) 45.00 (36.00-51.00) 44.01 ± 11.71 155.69 Chronic medications 0.932 a ≤4 176(58.7) 43.00(36.00-51.00) 43.43 ± 11.05 150.86 Table 3 3Total burden scores by socio-demographic and clinical variables (Continued)Variable Frequency % N(300) Median (Q1-Q3) Mean ± SD Mean rank P-value > 4 124(41.3) 43.00 (36.00-50.00) 43.14 ± 9.57 149.99 Anticoagulant drug UHF 87(29) 42.00(35.00-51.00) 42.75 ± 9.63 143.83 0.596 b Vit-K dependent 134(44.7) 43.00 (36.00-51.00) 41.65 ± 11.06 143.90 NOACs 75(25) 44.00 (36.75-57.00) 44.12 ± 11.43 154.06 a The statistical significance of differences was calculated using the Mann-Whitney U test b The statistical significance of the differences was calculated using the Kruskal-Wallis test Bold P-value indicates a significant difference c Patients that received prophylactic doses of unfractionated heparin during hospital admission NIS: New Israeli Shekel (0.29 US Dollar) Table 4 4Benefits total by score sociodemographic and clinical variablesVariable Frequency % N(300) Median (Q1-Q3) Mean ± SD Mean rank P value Age category (years) < 0.001 b < 30 31 (10.3) 15.00(14.00-15.00) 14.55 ± 0.89 255.40 30-60 163 (54.3) 12.00(11.00-14.00) 12.08 ± 1.97 159.84 > 60 106 (35.3) 11.00(9.00-12.00) 10.69 ± 1.93 105.45 Gender 0.015 a Male 115 (38.3) 12.00(10.00-12.00) 11.47 ± 1.98 133.34 Female 185 (61.7) 12.00(11.00-14.00) 12.07 ± 2.26 159.92 BMI a 0.003 b Normal 65 (21.7) 12.00(11.00-15.00) 12.63 ± 2.08 178.75 Overweight 136 (45.3) 12.00(10.00-13.00) 11.57 ± 2.30 139.13 Obese 93 (31.0) 12.00(10.00-13.00) 11.58 ± 1.94 137.90 Education < 0.001 b Illiterate 20 (6.7) 9.50(9.00-11.00) 10.05 ± 1.57 75.45 Elementary 43 (14.3) 11.00(9.00-12.00) 10.76 ± 1.76 101.92 Primary 52 (17.3) 12.00(10.00-13.00) 11.73 ± 2.15 145.35 Secondary 54 (18.0) 12.00(11.00-14.00) 12.19 ± 2.13 159.62 University 124 (41.3) 12.00(11.00-15.00) 12.38 ± 2.15 166.65 Income (month) 0.641 b Less than 2000 NIS 23 (7.7) 12.00(11.00-15.00) 12.17 ± 2.61 160.98 2000-5000 NIS 160(53.3) 12.00(11.00-14.00) 11.82 ± 2.20 145.83 More than 5000 NIS 108(36.0) 12.00(10.00-13.00) 11.75 ± 2.09 143.06 locality 0.258 b Camp 36 (12.0) 12.00(11.00-14.75) 12.25 ± 2.08 161.86 Rural 85(28.3) 12.00(11.00-15.00) 12.02 ± 2.30 153.71 Urban 172(57.3) 12.00(10.00-13.00) 11.66 ± 2.14 140.58 Marital status 0.003 b Married 229(76.3) 12.00(11.00-14.00) 12.01 ± 2.14 127.51 Unmarried (single, divorced, widowed) 67(22.3) 11.00(10.00-13.00) 11.37 ± 2.14 154.64 Employment status 0.188 a Employed 194(64.7) 12.00(10.00-13.25) 12.10 ± 1.80 156.46 Unemployed 100(33.3) 12.00(11.00-14.00) 11.73 ± 2.32 142.88 Chronic co-morbid disease 0.018 b No co-morbid disease 42 (14.0) 14.00(12.00-15.00) 12.14 ± 1.96 216.07 One disease 44(14.7) 12.00(10.00-14.00) 11.99 ± 2.10 152.50 Two diseases 63(21.0) 12.00(10.00-14.00) 12.05 ± 2.06 154.00 Three diseases or more 146(48.7) 11.00(10.00-12.00) 11.20 ± 2.05 123.50 Indications for anticoagulant < 0.001 b Atrial fibrillation 63 (21.0) 11.00 (9.00-12.00) 10.84 ± 1.90 112.70 Deep vein thrombosis or Pulmonary embolism 29 (9.7) 12.00 (10.50-15.00) 12.31 ± 2.58 167.93 Prophylaxis c 72 (24.0) 13.00 (12.00-15.00) 13.11 ± 1.87 203.99 Other indications 136 (45.3) 11.00 (10.00-12.75) 11.53 ± 2.03 135.98 Chronic medications < 0.001 a ≤4 176(58.7) 12.00(11.00-15.00) 12.25 ± 2.23 167.30 > 4 124(41.3) 11.00(10.00-12.00) 11.26 ± 1.96 126.65 Table 4 4Benefits total by score sociodemographic and clinical variables (Continued) a The statistical significance of the differences was calculated using the Mann-Whitney U test b The statistical significance of the differences was calculated using the Kruskal-Wallis test Bold P-value indicates significant differences. c Patients that received prophylactic doses of unfractionated heparin during hospital admission NIS: New Israeli Shekel (0.29 US Dollar)Variable Frequency % N(300) Median (Q1-Q3) Mean ± SD Mean rank P value Anticoagulant drug < 0.001 a UHF 87(29) 11.00(10.00-12.00) 11.04 ± 1.74 113.63 Vit-K dependent 134(44.7) 11.50(10.00-13.00) 11.52 ± 2.22 135.54 NOACs 75(25) 13.00(12.00-15.00) 13.07 ± 1.89 198.53 Table 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Myelin oligodendrocyte glycoprotein antibody-associated optic neuritis with COVID-19 infection: A case report and literature review 2022 Buravej Assavapongpaiboon Department of Ophthalmology Faculty of Medicine Chulalongkorn University Rama 4 Road, Pathumwan Sub-district, Pathumwan District1873, 10330BangkokThailand Department of Ophthalmology King Chulalongkorn Memorial Hospital Thai Red Cross Society Rama 4 Road, Pathumwan Sub-district, Pathumwan District1873, 10330BangkokThailand Supanut Apinyawasisuk Department of Ophthalmology Faculty of Medicine Chulalongkorn University Rama 4 Road, Pathumwan Sub-district, Pathumwan District1873, 10330BangkokThailand Department of Ophthalmology King Chulalongkorn Memorial Hospital Thai Red Cross Society Rama 4 Road, Pathumwan Sub-district, Pathumwan District1873, 10330BangkokThailand Supharat Jariyakosol Department of Ophthalmology Faculty of Medicine Chulalongkorn University Rama 4 Road, Pathumwan Sub-district, Pathumwan District1873, 10330BangkokThailand Department of Ophthalmology King Chulalongkorn Memorial Hospital Thai Red Cross Society Rama 4 Road, Pathumwan Sub-district, Pathumwan District1873, 10330BangkokThailand Myelin oligodendrocyte glycoprotein antibody-associated optic neuritis with COVID-19 infection: A case report and literature review American Journal of Ophthalmology Case Reports 2610149120222451-9936/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). A R T I C L E I N F OKeywords:Optic neuritis Myelin oligodendrocyte glycoprotein Severe acute respiratory syndrome coronavirus 2 Coronavirus disease 2019A B S T R A C TPurpose: To demonstrate the association between SARS-CoV-2 infection and MOG antibody associated optic neuritis.Observations: A 35-year-old Thai woman presented with acute blurred vision of her left eye with pain on eye movement for six days and had dry cough for one week before the onset of visual loss. Her visual acuity was 20/ 32 in the right eye and counting fingers with a RAPD in the left eye. She had bilateral disc swelling, more prominent on the left eye. A CT scan of the brain and orbits showed swollen optic nerve sheath complex both eyes. Serology test was positive for serum anti-myelin oligodendrocyte glycoprotein (MOG) antibody. Her nasopharyngeal swab for SARS-CoV-2 PCR was also positive. The diagnosis of SARS-CoV-2 associated MOG antibody optic neuritis was made. Conclusions and importance: This case of MOG antibody associated optic neuritis after COVID-19 infection, along with several other cases reported in the literature, suggests that there may be an association between COVID-19 infection and MOG antibody-associated disease. However, larger case-controlled studies are required to confirm this association. Introduction Coronavirus disease 2019 (COVID-19) outbreak caused by novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a concern for all countries. Ocular manifestations of COVID-19 include conjunctivitis, keratoconjunctivitis, uveitis and retinitis. Various neuroophthalmology conditions are associated with COVID-19, including optic neuritis. Case report Here we report a case of a 35-year-old Thai woman without a significant past medical history, presented with acute blurred vision of her left eye with pain on eye movement for six days. She denied a history of weakness, numbness, or other neurologic symptoms. Reviews of systems revealed dry cough for one week without fever or anosmia, before the onset of visual loss with no known COVID-19 contact. Her visual acuity was 20/32 in the right eye and counting fingers in the left eye. There was a RAPD in the left eye. Anterior and posterior segment examinations were unremarkable except for bilateral optic disc edema (more prominent in left eye). Her nasopharyngeal swab for SARS-CoV-2 PCR was positive. Due to the hospital's COVID-19 precaution guideline, an MRI scan was not permitted. However, a CT scan of the brain and orbits showed swollen optic nerve sheath complex of both eyes, more prominent in the left eye without definite enhancement. The brain parenchyma and other parts were unremarkable. Serum myelin oligodendrocyte glycoprotein (MOG) antibody (fix cell-based assay method) was sent based on the typical characteristic of bilateral optic disc swelling and optic nerve sheath involvement, which later returned positive. The antibody titer was not quantified. Serum aquaporin-4 antibody, anti-nuclear antibody, rheumatoid factor, and syphilis serology were all negative. Routine CSF analysis was negative for other infectious and inflammatory disorders, including SARS-CoV-2 PCR and MOG antibody. Chest X-ray revealed no active pulmonary disease. She was diagnosed with SARS-CoV-2 associated MOG antibody optic neuritis (MOG-ON). The treatment included 1 g intravenous methylprednisolone for five days, followed by oral prednisolone with slow tapering, and oral favipiravir for five days. At eight days after treatment her visual acuity improved to 20/30 in both eyes. At four weeks after the onset, her visual acuity was 20/25 in the right eye and 20/20 in the left eye with residual subjective dyschromatopsia in the left eye. Discussion To our best knowledge, there have been nine reported cases of SARS-CoV-2 associated MOG-ON (Table 1). [1][2][3][4][5][6][7][8][9] Eight of nine were newly diagnosed MOG-ON. Only one report was a case with relapsing MOG-ON after COVID-19 infection. 6 We hypothesize that there might be an association with the first-episode MOG-ON and COVID-19 infection, in which the pathophysiology could be explained by the following two hypotheses. First, a molecular mimicry, in which the viral antigen triggers human antibodies directed toward endogenous central nervous system (CNS) myelin proteins, might explain the association. The process usually takes 5-10 days or 1-3 days for primary and secondary immune response, respectively. The supporting evidence is that, in most cases, the onset of optic neuritis followed the COVID-19 for at least a week. Second, SARS-CoV-2 may disrupt and increase permeability of blood-brain barrier by increased expression of pro-inflammatory cytokines, happened early after infection as seen in an animal model. 10 This allows entry of pre-existing circulating anti-MOG antibodies into CNS causing pathology. This hypothesis could explain the rapid onset of optic neuritis after the COVID-19 reported by Zhou et al. 1 However, MOG antibody-associated disease (MOGAD) has been believed to be mediated by an immune response to a non-specific post-viral infection since before the COVID-19 pandemic. Myelitis associated with MOG antibody is also related to post-infection and presents with prodromal symptoms up to 61%. 11 Therefore, it might be too early to conclude that MOG-ON is specifically associated with SARS-CoV-2 infection and requires a case-controlled study to evaluate the association. However, a small number of cases is probably a potential limitation of the study. Although an MRI study was not performed in concern of SARs-CoV-2 contamination, we believe the CT scan showing optic nerve sheath complex abnormalities was sufficient to support the clinical diagnosis of optic neuritis and exclude other causes of optic neuropathy. Patients Table 1 Details of nine case reports compared to our case of SARS-CoV-2 associated MOG antibody optic neuritis. with MOG-ON tended to respond to intravenous methylprednisolone treatment rapidly and dramatically as seen in this case as well as other reports. Most previous reports including ours did not show that corticosteroid treatment worsen COVID-19 symptoms. Conclusion In summary, MOG-ON may be associated with SARS-CoV-2 infection. Ophthalmologist should ask the recent history of COVID-19 and should be aware for the possible concurrent SARs-CoV-2 infection in patients presenting with MOG-ON. The treatment of MOG-ON should not be delayed in COVID-19 patients. Prompt corticosteroid treatment provides excellent outcomes with minimal complications in all patients. Abbreviation: M male, F female, CXR chest X-ray, D day, W week, OD right eye, OS left eye, OU both eye, PJ light projection, HM hand motion, FC finger count, Ft feet, CSF cerebrospinal fluid, SARS-CoV-2 severe acute respiratory syndrome coronavirus 2, COVID-19 Coronavirus disease 2019, PCR polymerase chain reaction, IVMP intravenous methylprednisolone, NA not available.Author Age Sex Underlying illness Onset after COVID-19 symptoms COVID-19 symptoms Onset of optic neuritis Laterality Presenting visual acuity CSF SARS- CoV-2 PCR Treatment Final visual acuity after admission Zhou 1 26 M none a few D dry cough 3 D OD > OS OD HM OS 20/250 Negative IVMP 5D followed by oral steroid OU 20/50 at D 7 OU 20/30 at W 3 Sawalha 2 44 M none 2 W shortness of breath and cough 1 W OD > OS OD 20/200 OS 20/30 NA IVMP 5D followed by oral steroid NA (significant improvement) Khan 3 11 M none 2 W a brief febrile illness and redness of both eyes 1-2 D OD > OS OD PJ OS 20/30 NA IVMP 5D followed by oral steroid OD 20/30 at D 10 OS 20/20 at D 10 Kogure 4 47 M post adrenal resection due to hyperaldosteronism, recurrent paranasal sinusitis detected during admission asymptomatic NA OS OD NA OS 20/400 Negative IVMP 3D followed by oral steroid OS 20/200 at D 10 OS 20/16 at W 2 Zoric 5 63 M hypertension, diabetes mellitus 4 W pneumonia CXR resolved before onset of optic neuritis 1 W OD OD 20/630 OS 20/20 NA IVMP 5D followed by oral steroid OD 20/63 at D 5 OD 20/25 at W 3 Woodhall 6 39 F MOG antibody optic neuritis (remission) 6 D malaise, coryzal symptoms, sweating. NA OD OD HM OS 20/20 NA IVMP 5D followed by plasma exchange OD 20/125 at W 2 Rojas- Correa 7 69 M diabetes mellitus 45 D fever, rhinorrhoea, cough NA (subacute) OD > OS OD 20/60 OS 20/30 Negative IVMP 5D OD 20/30 at D 5 OS 20/25 at D 5 de Ruijter 8 15 M none 2-3 W fever, nausea, and a cough 7 D OD > OS OD 1/300 OS 1/70 NA IVMP 3D NA (almost fully improvement) Jossy 9 38 M none 2 W and recurrent at 6 W NA (mild symptom, home isolation) 5 D (2nd episode) OS OD 20/20 OS HM (2nd episode) NA IVMP followed by oral steroid for 1st episode; IVMP 3D followed by oral prednisolone as per ONTT protocol for 2nd episode OS 20/20 at D 7 (2nd episode) Our case 35 F none 1 W dry cough 6 D OS > OD OD 20/32 OS FC 2 ft Negative IVMP 5D followed by oral steroid OU 20/30 at D 8 OD 20/25 at W 4 OS 20/20 at W 4 AuthorshipAll authors attest that they meet the current ICMJE criteria for Authorship. Buravej Assavapongpaiboon: Conceptualization, Data Curation, Writing -Original draft, Supanut Apinyawasisuk: Conceptualization, Writing -Review & Editing, Validation, Supharat Jariyakosol: Conceptualization, Writing -Review & Editing, Supervision.AcknowledgementsFunding: No funding or grant support.Patient consentThe patient consented to the publication of the case verbally. This report does not contain any personal information that could lead to the identification of the patient.Declaration of competing interestAll authors have no financial disclosures. Myelin oligodendrocyte glycoprotein antibody-associated optic neuritis and myelitis in COVID-19. S Zhou, E C Jones-Lopez, D J Soneji, C J Azevedo, V R Patel, 10.1097/WNO.0000000000001049J Neuro Ophthalmol. 403Zhou S, Jones-Lopez EC, Soneji DJ, Azevedo CJ, Patel VR. Myelin oligodendrocyte glycoprotein antibody-associated optic neuritis and myelitis in COVID-19. J Neuro Ophthalmol. 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Comment Assessing the effectiveness of COVID-19 vaccines in older people in Latin America-NC-ND 4.0 license Published Online March 21, 2022 Alfonso J Rodriguez-Morales Oscar H Franco Faculty of Medicine Faculty of Health Sciences Universitaria Autónoma de las Américas Fundación, Risaralda, Colombia (AJR-M) School of Medicine Universidad Científica del Sur Lima, Perú (AJR-M) Institute of Social and Preventive Medicine Universidad Privada Franz Tamayo CochabambaBolivia (AJR-M) University of Bern BernSwitzerland (OHF Comment Assessing the effectiveness of COVID-19 vaccines in older people in Latin America-NC-ND 4.0 license Published Online March 21, 2022Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre -including this research content -immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. www.thelancet.com/healthy-longevity Vol 3 April 2022 e219 See Articles page e242 We declare no competing interests. Following their introduction towards the end of 2020, COVID-19 vaccines have been evaluated in terms of prevention of complications, hospitalisation rates, and mortality levels among those who are vaccinated compared with those who are not. 1 Assessing the efficacy of vaccines is crucial for their use, but studies that assess real-world effectiveness of vaccines are also highly relevant, especially those comparing the efficacy of available vaccines in a specific country and those assessing efficacy in specific population groups that differ on certain factors (eg, age and gender). 2 Nevertheless, even as late as March, 2022, there is still a low number of such studies in low-income and middleincome countries, particularly in Latin America and Africa. 3 A study analysing the effectiveness of COVID-19 vaccines in Latin America was conducted in Chile, 4 with a cohort of approximately 10·2 million people. This study assessed the CoronaVac vaccine in people aged 60 years or older, and showed effectiveness of 86·3% (95% CI 84·5-87·9) for the prevention of COVID-19related hospitalisation and 86·5% (84·6-88·1) for the prevention of COVID-19-related deaths among people who were fully vaccinated (ie, (≥14 days after receipt of the second dose), after adjusting for covariates. . In The Lancet Healthy Longevity, Leonardo Arregocés-Castillo and colleagues 5 explored the effectiveness of Ad26.COV2-S, BNT162b2, ChAdOx1 nCoV-19, and CoronaVac, which were the first four available COVID-19 vaccines in Colombia, using a populationbased matched-pair cohort study (called the ESPERANZA cohort) that included over 2 million participants (n=2 828 294) aged 60 years and older. This older age group is considered a population with a higher risk of severe disease, complications, and death than younger age groups due to age and associated risk factors (eg, the higher presence of comorbidities, such as obesity, diabetes, hypertension, and cardiovascular disease). 6 In their study, Arregocés-Castillo and colleagues found that the overall effectiveness of the available vaccines for preventing hospitalisation without subsequent death was 61·6% (95% CI 58·0-65·0), 79·8% (78·5-81·1) for preventing death after hospitalisation for COVID-19, and 72·8% (70·1-75·3) for preventing death without previous COVID-19 hospitalisation. The overall effectiveness did not differ significantly by specific vaccine, though BNT162b2 and ChAdOx1 nCoV-19 were shown to be most effective. These highly relevant findings reinforce the importance of vaccination among older people to prevent not only the risk of infection and severe disease, but also hospitalisation and death. This study also highlights the importance of these assessments amid the pandemic, accounting for dynamic changes that include the circulation of multiple variants of concern. 7 These new variants pose novel challenges for the effectiveness of vaccines, especially in high-risk populations. 8 Arregocés-Castillo and colleagues' findings in Colombia were consistent with the benefit of vaccination in older people identified by the aforementioned study done in Chile. 4 Nevertheless, a limitation of the Colombian study is that it only assessed the effectiveness of vaccines against the circulating variants of concern during the study period. Future studies should consider this limitation. As Arregocés-Castillo and colleagues indicated, although expected, the effectiveness of vaccines decreased by age. An additional or booster dose can effectively contribute to improving the immune response, therefore becoming a possible solution to the decreased effectiveness of vaccines in older people. On the basis of these findings, in early October, 2021, Colombia started offering booster doses to people older than 50 years and later extended this to younger groups. "Esperanza" means hope in Spanish. Indeed, the findings of the study by Arregocés-Castillo and colleagues are part of the hope that health authorities continue to use effective COVID-19 prevention measures, including vaccination, jointly with other public health measures, while maintaining an evidencebased approach to managing COVID-19. Only through a thorough and collective approach can there be hope to achieve appropriate control of the pandemic and, hopefully, shortly bring an end to the ongoing pandemic phase of COVID-19 and the deleterious consequences on populations worldwide. Therefore, Colombia, as well as other low-income and middle-income countries in Latin America, Africa, and Asia, need to assess the effectiveness of COVID-19 vaccine boosters in older people throughout 2022 and broaden the access and availability of vaccines to all individuals who could benefit within and beyond the limits of specific counties. International, national, and regional health authorities must keep working on population access to and education on basic hygiene and sanitation, as well as the safety, efficacy, and effectiveness of COVID-19 vaccines. Despite the overwhelming evidence for the effectiveness of vaccines, vaccination coverage is far from 100%, even in high-income countries. There is large room for improvement, in which public health scientists and practitioners could have a key role. 9 Evidence-based information is crucial for disseminating correct health messages to enhance public trust and interest to adhere to vaccine schedules and boosters, 10 especially in highrisk populations such as older people. Only a global approach that secures access to vaccines for all who need them presents a meaningful pathway to an effective resolution of this catastrophe. The solutions have been developed in record time, with not only one, but multiple effective vaccines. These vaccines now need to be distributed to all that need them. Adequate distribution, education, and information are key factors and represent some of the essential learnings that have been extracted from the current crisis. These learnings need to be implemented to avert and better respond to future pandemics. Effectiveness and safety of SARS-CoV-2 vaccine in real-world studies: a systematic review and meta-analysis. Q Liu, C Qin, M Liu, J Liu, Infect Dis Poverty. 10132Liu Q, Qin C, Liu M, Liu J. Effectiveness and safety of SARS-CoV-2 vaccine in real-world studies: a systematic review and meta-analysis. Infect Dis Poverty 2021; 10: 132. Estimated effectiveness of COVID-19 messenger RNA vaccination against SARS-CoV-2 infection among older male veterans health administration enrollees. Y Young-Xu, G M Zwain, E I Powell, J Smith, JAMA Netw Open. 42138975Young-Xu Y, Zwain GM, Powell EI, Smith J. Estimated effectiveness of COVID-19 messenger RNA vaccination against SARS-CoV-2 infection among older male veterans health administration enrollees, January to September 2021. JAMA Netw Open 2021; 4: e2138975. Effectiveness of ChAdOx1 vaccine in older adults during SARS-CoV-2 Gamma variant circulation in São Paulo. Mdt Hitchings, O T Ranzani, M Dorion, Nat Commun. 126220Hitchings MDT, Ranzani OT, Dorion M, et al. Effectiveness of ChAdOx1 vaccine in older adults during SARS-CoV-2 Gamma variant circulation in São Paulo. Nat Commun 2021; 12: 6220. Effectiveness of an inactivated SARS-CoV-2 vaccine in Chile. A Jara, E A Undurraga, C González, N Engl J Med. 385Jara A, Undurraga EA, González C, et al. Effectiveness of an inactivated SARS-CoV-2 vaccine in Chile. N Engl J Med 2021; 385: 875-84. Effectiveness of COVID-19 vaccines in older adults in Colombia: a retrospective, population-based study of the ESPERANZA cohort. L Arregoces-Castillo, J Fernández-Niño, M Rojas-Botero, 10.1016/S2666-7568Lancet Healthy Longev. 22published onlineArregoces-Castillo L, Fernández-Niño J, Rojas-Botero M, et al. Effectiveness of COVID-19 vaccines in older adults in Colombia: a retrospective, population-based study of the ESPERANZA cohort. Lancet Healthy Longev 2022; published online March 21. https://doi.org/10.1016/ S2666-7568(22)00035-6. Clinical, laboratory and imaging features of COVID-19: a systematic review and meta-analysis. A J Rodriguez-Morales, J A Cardona-Ospina, E Gutiérrez-Ocampo, Travel Med Infect Dis. 34101623Rodriguez-Morales AJ, Cardona-Ospina JA, Gutiérrez-Ocampo E, et al. Clinical, laboratory and imaging features of COVID-19: a systematic review and meta-analysis. Travel Med Infect Dis 2020; 34: 101623. Variants, vaccines and vaccination passports: challenges and chances for travel medicine in 2021. P Schlagenhauf, D Patel, A J Rodriguez-Morales, P Gautret, M P Grobusch, K Leder, Travel Med Infect Dis. 40101996Schlagenhauf P, Patel D, Rodriguez-Morales AJ, Gautret P, Grobusch MP, Leder K. Variants, vaccines and vaccination passports: challenges and chances for travel medicine in 2021. Travel Med Infect Dis 2021; 40: 101996. The biological and clinical significance of emerging SARS-CoV-2 variants. K Tao, P L Tzou, J Nouhin, Nat Rev Genet. 22Tao K, Tzou PL, Nouhin J, et al. The biological and clinical significance of emerging SARS-CoV-2 variants. Nat Rev Genet 2021; 22: 757-73. Cross-sectional analysis of COVID-19 vaccine intention, perceptions and hesitancy across Latin America and the Caribbean. D Urrunaga-Pastor, G Bendezu-Quispe, P Herrera-Añazco, Travel Med Infect Dis. 41102059Urrunaga-Pastor D, Bendezu-Quispe G, Herrera-Añazco P, et al. Cross-sectional analysis of COVID-19 vaccine intention, perceptions and hesitancy across Latin America and the Caribbean. Travel Med Infect Dis 2021; 41: 102059. Public trust, misinformation and COVID-19 vaccination willingness in Latin America and the Caribbean: today's key challenges. A J Rodriguez-Morales, O H Franco, Lancet Reg Health Am. 3100073Rodriguez-Morales AJ, Franco OH. Public trust, misinformation and COVID-19 vaccination willingness in Latin America and the Caribbean: today's key challenges. Lancet Reg Health Am 2021; 3: 100073.

Effect of diet on pathogen performance in the microbiome 26 Mar 2022 Ronan Strain APC Microbiome Ireland Biosciences Institute University College Cork T12 YT20CorkIreland Teagasc Food Research Centre P61 C996Moorepark, FermoyCo. CorkIreland Catherine Stanton APC Microbiome Ireland Biosciences Institute University College Cork T12 YT20CorkIreland Teagasc Food Research Centre P61 C996Moorepark, FermoyCo. CorkIreland ProfRoss R Paul APC Microbiome Ireland Biosciences Institute University College Cork T12 YT20CorkIreland School of Microbiology University College Cork College RoadT12 K8AFCorkIreland APC Microbiome Ireland Biosciences Institute University College Cork College RoadT12 YT20CorkIreland Effect of diet on pathogen performance in the microbiome 26 Mar 202256E033BDF2580463CDF9AF1A69D1A6FB10.20517/mrr.2021.10Received: 2 Dec 2021 First Decision: 29 Dec 2021 Revised: 17 Feb 2022 Accepted: 23 Feb 2022Colonisation resistancegut microbiotapathogendietinfection Intricate interactions among commensal bacteria, dietary substrates and immune responses are central to defining microbiome community composition, which plays a key role in preventing enteric pathogen infection, a dynamic phenomenon referred to as colonisation resistance.However, the impact of diet on sculpting microbiota membership, and ultimately colonisation resistance has been overlooked.Furthermore, pathogens have evolved strategies to evade colonisation resistance and outcompete commensal microbiota by using unique nutrient utilisation pathways, by exploiting microbial metabolites as nutrient sources or by environmental cues to induce virulence gene expression.In this review, we will discuss the interplay between diet, microbiota and their associated metabolites, and how these can contribute to or preclude pathogen survival. INTRODUCTION The gut microbiota has coevolved with its host over millions of years and augments the coding potential of the human genome (~22,000 genes) by upwards of 500-fold [1] .Indeed, the human genome itself, encodes at most only 17 enzymes involved in food digestion, mainly the digestion of starch, sucrose and lactose [2] .On the other hand, our gut microbiota encodes upwards of 60,000 Carbohydrate-Active Enzymes, with diverse specificities, facilitating the depolymerisation and fermentation of complex dietary polysaccharides into host utilisable short-chain fatty acids (SCFAs) [3] .This gut microbiota is heterogeneous and highly personalised, and while bacterial enterotypes cluster independently of nationality, ethnicity, sex, age, or body mass index [4] , diet is the dominant selective force that defines microbiota membership and functionality [5] .Diet is also the simplest to customise and therefore presents the most straightforward route for therapeutic intervention. The distribution of bacteria throughout the gastrointestinal (GI) tract varies, from 10 3 -10 4 cells/mL in the stomach and upper small intestine, to 10 11 cells/mL in the colon [6] .Furthermore, the taxonomic composition of these communities is niche-specific, and largely defined by the nutritional requirements of the residing bacteria.These contentions are supported by observations in gnotobiotic mouse models whereby concentrations of individual dietary components correlate with the relative abundance of specific microbiota members [7] .For example, Bacteroides cellulosilyticus has the ability to be controlled by administration of different concentrations of the prebiotic fibre arabinoxylan [8] .This phenomenon opens up the potential for therapeutic probiotic colonisation, which has been demonstrated by Kearney et al. [9] ; administration of the seaweed polysaccharide polyphyran and a polyphyran-degrading commensal Bacteroides plebius enabled successful engraftment of this species. The majority of human enteric pathogens are part of a small group of bacterial families that belong to the phylum Proteobacteria; the Enterobacteriaceae, i.e., Escherichia coli (E.coli), Yersinia, Salmonella, Shigella; the Vibrionaceae (Vibrio cholera) and the Camplyobacteriaceae (Camplyobacter) [10] .Bacterial pathogens must overcome an array of obstacles such as oesophageal peristalsis, stomach pH and locating a permissible niche in the intestine in order to access nutrients to begin replication and to achieve successful colonisation in the GI tract.The final hurdle is to overcome resident commensal bacteria of the large intestine.Continuous competition for nutrients, and compartments, the production of antimicrobial substances by commensals, and the barrage of immune responses evoked by the commensals themselves collectively give way to the phenomenon of colonisation resistance.For example, commensal symbionts and their related pathogens often compete with each other for metabolic resources compared with distant unrelated species.These metabolites include diverse carbon sources, bile acids, trace metals and vitamins.An overview of some metabolites influencing pathogen virulence and fitness can be found in Table 1.Those bacteria which possess high-affinity transporters for available nutrients will ultimately define the microbial community structure, but also serve as a barrier for GI pathogens.However, many pathogens confer the ability to subvert competition with commensal bacteria, often by generating their own specific niche to suit their metabolic needs.As an example, invasion of epithelial cells provides an environment suited to intracellular pathogens.Additionally, there is accumulating evidence to suggest that intestinal pathogens/pathobionts may subvert and exploit the host immune response to induce microbial dysbiosis and improve conditions for their subsequent colonisation [11] .Furthermore, intestinal pathogens have evolved unique nutrient utilisation pathways in relation to their symbiotic counterparts; Escherichia can utilise alternative sugar sources to that of their commensal rivals [12] , and gain a competitive advantage.In this regard, what we consume may have the potential to alter bacterial networks and shift the balance in favour of or against pathogen survival. Accumulating evidence over the past decade has linked the high-fat/high-sugar/low-fibre "Western diet" with a myriad of the ever-increasing glycemic index and metabolic disorders.It is widely acknowledged that the microbial dysbiosis resulting from this lifestyle is a major contributing factor to the epidemic of glycemic index and metabolic disorders [13] .One could speculate whether long-term dietary regimes could increase or decrease the host's colonisation resistance to enteric pathogens or opportunistic pathobionts.In this review, the interactions among diet, the microbiota, colonisation resistance and pathogen performance will be examined by focusing on the keystone taxa and metabolites involved. BREAST MILK AND SOLID FOODS IN EARLY LIFE PROTECT AGAINST ENTERIC INFECTION BY MODULATING THE GUT MICROBIOTA Neonates face an increased susceptibility to GI infections.This susceptibility in newborns has been generally attributed to the immaturity of the adaptive and innate immune systems; premature newborns often display a heightened risk of suffering from excessive inflammation, which decreases as they age [14] .Given that the gut microbiota is involved in the development of the immune system [15] and the neonatal microbiota is less diverse than the adult microbiota, research is shifting toward the potential role that specific gut taxa play in the maturation of immune function and colonisation resistance. Naturally delivered infants are generally dominated by bacterial groups associated with maternal vaginal microbiota (e.g., Atopobium, Bacteroides, Clostridium, E. coli, Streptococcus spp.and Prevotella), whereas Csection-born infants are dominated by the taxa associated with the skin microbiota such as Staphylococcus spp. [16].Breastfeeding aids in the initial colonisation of key taxa: Bifidobacterium and Lactobacillus [17] , with the former involved in the digestion of human milk oligosaccharides (HMOs), which are resistant to human enzymatic digestion [18] .The resulting fermentation of these compounds produces lactate [19] and SCFAs, specifically acetate, which accounts for 80% of the total SCFA production in the infant gut [20] compared with 50% in the adult gut [21] .The acidic end products of HMO fermentation are linked to a lower intestinal pH and may be crucial to maintaining colonisation resistance in infants [Figure 1].The interactions between pathogens and SCFAs will be discussed later. The distinction between microbiota community composition of human breast milk-fed and formula-fed infants is clear, with the general consensus that human breast milk directs the propagation of beneficial bacteria and their related metabolites [22] .Lactation drives the colonisation of Bifidobacterium in infants, and has been shown to play a key role in the maturation of the immune system (for a review, see Ref. [23] ).Moreover, in mice, IgG in breast milk derived from mothers previously infected with Citrobacter rodentium, is passed to the offspring, enhancing colonisation resistance when challenged by this pathogen [24] .Probiotics derived from breast milk have shown great promise in mitigating the risk of necrotising enterocolitis (NEC), the most common GI disease in preterm infants and the leading cause of death in extremely preterm infants from 2 weeks to 2 months of age [25] .Perhaps the most promising "probiotic" is Bifidobacterium longum subsp.infantis, an extremely proficient coloniser of the infant gut, and exhibits decreasing NEC incidence in neonates [26] .More recently, a human breast milk-derived commensal Propionibacterium strain UF1, belonging to the same phylum as Bifidobacterium, the Actinobacteria, has been shown to mitigate NEC-like injury in mice [27] and conferred protection against Listeria monocytogenes infection [28] . Along with Bifidobacterium, the other key taxa involved in the fermentation of HMOs are Bacteroides [29] . Vaginal birth and breastfeeding significantly improve Bacteroides colonisation [30] , suggesting a long coevolved symbiotic relationship between the host and taxa, and this symbiosis is reflected in studies demonstrating their role in immune system development [31,32] .For example, the immunosuppressive effect of the capsular exopolysaccharide, polysaccharide A from B. fragilis is achieved by promoting differentiation of regulatory T cells (T reg ) [32] , which may be beneficial to the host given that many pathogens favour a gutinflamed environment.However, the supposed reliance on Bacteroides spp.for the development of the infant immune system is not clear, as an observational study in Northern Europe observed reduced prevalence of Bacteroides and Type I diabetes (T1D) in Russian children, relative to their counterparts from Finland and Estonia, where Bacteroides and T1D were more common [33] .The Bacteroidetes phylum primarily produces acetate and propionate [34] .Acetate has been observed to decrease the frequency of autoreactive T cells, and a diet designed to release large amounts of acetate protected against the development of T1D in mice [35] .However, Bacteroides spp. that may be a risk factor for T1D, might be unrelated to the production of acetate and its associated metabolites, but more likely related to the overexposure to Bacteroides -associated LPS derived from HMO-utilising Bacteroides [33] . As the diet changes from maternal milk to fibre-rich foods as the infant matures, the infant microbiome acquires members of Bacteroidales and the butyrate-producing Clostridiales.Clostridium species have been associated with the increasing abundance and activity of T reg cells in the colon [36] by providing bacterial antigens.Administration of members of the Clostridiales, but not Bacteroidales, provided protection to germ-free adult mice, colonised with neonatal microbiota, against infection from Salmonella and Citrobacter [37] .These researchers hypothesise that in the first days of life, oxygen consumption by aerobic bacteria or facultative anaerobes enhances the ability of the strict anaerobes, Clostridiales, to colonise the gut, which, in turn, provides protection against pathogen infection.Furthermore, when succinate was administered in the drinking water to mice, it reduced oxygen intestinal content, which in turn, enhanced Clostridales colonisation.Interestingly, the authors demonstrated that it was these bacterial groups that abrogated infection and that host immunity did not contribute to the Clostridia-mediated effect.Similarly, a longitudinal study examining faecal samples from an asymptomatic infant carrier of Clostridioides difficile (an infant female born by C-section), from pre-weaning to weaning, revealed a dramatic change in microbiota composition within the first five days of transition from breast milk to cow's milk and solid foods [38] .A rapid decline and eventual disappearance of C. difficle, accompanied by an increase in the relative abundance of Bacteriodales/Clostridiales observed during weaning, were likely responsible for the expulsion of C. difficile. THE ROLE OF DIETARY FIBRE IN PROMOTING MICROBIAL DIVERSITY, MAINTAINING THE MUCUS BARRIER AND THE LINK WITH COLONISATION RESISTANCE There is an appreciation that the diet of westernised nations has declined in the quantity of fermentable fibre intake, which has been associated with the coincidental rise in diseases such as heart disease, diabetes and colorectal cancer [39] .Human populations with a diet rich in dietary fibre exhibit increased diversity of the microbiota, such as experienced by the Hadza tribal people from Tanzania [40] .Conversely, a "westernised" low fibre diet can result in a progressive loss of microbial diversity [41] .While there is a greater prevalence of GI pathogen infections in developing countries compared with their western counterparts [42] , this may be attributed to poverty-related risk factors and sanitation practices.Interestingly, in a study comparing the microbiota of children from Burkina Faso and their European counterparts, it was observed that Enterobacteriaceae (Shigella/Escherichia) were underrepresented in the Burkina Faso cohort relative to European children [43] .The authors hypothesised that the high fibre diet consumed by the Burkina Faso children selects for a bacterial community capable of maximising energy intake from the fibres, while at the same time protecting them from potential enteropathogens.However, whether diet-induced increases in microbial diversity or specific taxa and their related metabolites can improve colonisation resistance in humans is an intriguing but essential question to answer.The majority of studies to date supporting the inverse link between diversity and infection stem from murine models.Both germ-free and antibiotictreated mice display increased susceptibility to enteric infections, which are associated with reduced microbiota diversity [44,45] .While the bulk of studies supports microbial-induced benefits derived from fermentable fibre, recent evidence suggests that cellulose, an insoluble fibre, also exerts enrichment in protective microbial species and provides colonisation resistance [46] .A comparison of the impact of soluble dietary fibre (oat β-glucan) versus insoluble dietary fibre (microcellulose) in mice gut microbiota reported reduced alpha-diversity (distribution of species abundances) and higher relative abundance of fibredegrading Bacteroides and pathogenic Proteobacteria in the former [47] , indicating potential cross-feeding between commensals and pathogens.Higher alpha-diversity was observed when the two fibres were mixed, and may be explained by an increase in carbon sources providing substrates for a larger range of bacterial taxa.Such mixtures would reflect better a human diet, which contains a mixture of soluble and insoluble fibres. SCFAs are the end products of the fermentation of dietary fibres and have a broad range of effects on mammalian host physiology, can attenuate inflammation, and alter the microbial composition and pathogen virulence.SCFAs are saturated aliphatic organic acids comprised of one to six carbons, of which acetate (C2), propionate (C3) and butyrate (C4) are the most abundant (> 95%) [48] .The success of the invading pathogen depends on the biotic interactions within the community, including exchanging and competing for metabolites [49] .The effect of SCFAs can be a double-edged sword for invading pathogens, with beneficial or inhibitory effects depending on concentration and environmental pH. The glycoprotein-rich layer that covers the gut epithelium provides the first line of defence against both commensal and pathogenic bacteria.Evidence has suggested that reduced dietary fibre intake is associated with a thinner colonic mucus [50] .Indeed, a recent study demonstrated that during chronic or intermittent fibre deficiency in mice, the gut microbiota degraded the host-secreted glycoproteins as an energy source, and in turn, resulted in an unstable mucus barrier function, increasing susceptibility to infection by C. rodentium [51] .One of the key players in the mucosal-microbiota environment is Akkermansia muciniphila, comprising 1%-4% of colonic microbes, which preferentially degrades mucin as its primary nutrient source [52] and is inversely correlated with a myriad of GI diseases.The distribution of Akkermansia in the GI tract of vertebrates is vast [53] , suggesting a long-term co-evolutionary relationship with their hosts and underlining their symbiotic importance.Mucus consists primarily of the heavily O-glycosylated protein, mucin 2, which Akkermansia can degrade with an arsenal of enzymes [54] .Continuous production of mucin by the goblet cells contributes to both mucin presence in the mucus layer and in the colonic lumen [55] .Akkermansia turnover of mucin contributes to the maintenance of intestinal integrity and microbial community homeostasis.Other species, including Bacteroides thetaiotaomicron and Bifidobacterium bifidum, that possess the capacity to break down mucus O-glycans have been identified in some studies [56] .Acetate producers like B. thetaiotaomicron may require a commensal adjuvant, e.g., Faecalibacterium prausnitzii, an acetate consumer and butyrate producer, in order to maintain colonic epithelial homeostasis [57] .F. prausnitzii is capable of immunosuppression through blocking of NF-κB activation and anti-inflammatory cytokine production, and reduced abundance of F. prausnitzii has been observed in inflammatory bowel disease (IBD) subjects [58] , indicating a harmonious relationship between mucosal commensals and the host in a healthy gut environment.Conversely, the glycans released from the mucin may actually provide a food source for GI pathogens.Ng et al. [59] demonstrated that Salmonella and C. difficile thrived on B. thetaiotaomicron-liberated glycans following antibiotic-induced disruption of mono-colonised mice compared to germ-free mice. The western-style diet (high-fat/low-fibre) has been associated with a decrease in Bacteroides, Bifidobacterium and Akkermansia [60] , subsequently affecting intestinal mucosal homeostasis and permeability; the effects of which can be ameliorated by the addition of dietary fibres [60] , and thereby potentially protecting against infection [51] .Moreover, the Western diet is characterised by an increase in the Firmicutes/Bacteroidetes ratio and weight gain in humans.A link between obesity and risk of infection with the enteric pathogen C. difficile has been identified [61] , and C. difficile colonisation has been attenuated in mouse models by the addition of dietary fibre [62] , suggesting a fibre deficient lifestyle may be a risk factor for C. difficile infection (CDI) and persistence.Collectively, these data suggest that individual fibres select for bacteria that are best at metabolising the specific fibre, leading to reduced diversity and hence a higher risk for colonisation of pathogens.Mixtures of dietary fibres that better represent a human diet promote higher gut microbiota diversity, and thus improve colonisation resistance and mucosal integrity. THE DOUBLE-EDGED SWORD OF SHORT-CHAIN FATTY ACIDS Of the three major SCFAs, acetate is the most abundant, constituting approximately 60% in the colon and stool [63] .It can be produced from pyruvate via acetyl-CoA by most of the enteric bacteria (Akkermansia muciniphila, Bacteroides spp., Bifidobacterium spp., Prevotella spp., Ruminococcus spp.) or from pyruvate via the Wood-Ljungdahl pathway (Blautia hydrogenotrophica, Clostridium spp., Streptococcus spp.) [21] .Acetate may have anti-inflammatory effects in vivo, by decreasing the LPS-stimulated TNFα response from neutrophils [64] , albeit to a lesser extent than butyrate and propionate. Many pathogens use SCFAs as environmental cues to determine their biogeographical location within the gut and switch on genes accordingly, e.g., virulence factors to colonise the preferential location.For example, Salmonella typhimurium preferably colonises the ileum [65] , where the typical concentration of acetate is 30 mM.This concentration enhances the expression of SPI-1 (Salmonella Pathogenicity Island 1)encoded T3SS (Type three secretion system), which is involved in the invasion of the host.Similarly, SPI-1 gene expression is promoted in the presence of minute concentrations of formate (~8 mM), like those encountered in the ileum [66] , suggesting S. typhimurium has multiple mechanisms to determine its biogeographical location.Furthermore, streptomycin-treated mice were more susceptible to S. typhimurium infection in the ileum compared with untreated mice, where the SCFA concentrations remained unchanged, suggesting that ileal commensal bacteria can also affect S. typhimurium virulence, likely by physically blocking colonisation or contributing to the immune response [67] .On the contrary, higher concentrations of propionate and butyrate, or the absence of formate, i.e., similar to conditions found in the colon, suppress the expression of T3SS [68] , and invasion is inhibited.Colonic environmental cues likely initiate adaptation of S. typhimurium gene expression to endure environmental insults and/or preparation for transmission to a new host.Interestingly, a recent study observed reduced ileal colonisation of Salmonella in mice which were pre-treated with a consortium of Bacteroides spp. with a high capacity for production of propionate [69] , through disruption of intracellular pH homeostasis. Similarly, Enterohaemorrhagic E. coli (EHEC) utilises SCFAs for virulence gene regulation; its preferred site of colonisation and infection is the colon [70] , where the ratio of acetate/butyrate tends to be lower.However, some studies have demonstrated that acetate can be refractory to the virulence of EHEC [71] by lowering intestinal pH [72] .Mixtures of SCFAs that represent the small intestine significantly upregulate EHEC flagellar genes and motility, whereas colonic SCFA concentrations have a down-regulatory effect [73] .Expression of the iha gene that encodes an adherence-conferring outer membrane protein, however, is upregulated by EHEC in the small intestine [74] , and is crucial for colonisation and infection.Consequently, ileal SCFA concentrations activate EHEC flagellar production and motility, followed by expression of genes involved in type III secretion and adherence when approaching colonic SCFA concentrations [75] , thereby permitting efficient adherence in EHEC's preferred niche.Production of acetate by Bifidobacterium has been demonstrated to inhibit the translocation of Shiga toxin of the EHEC 0157:H7 from the gut lumen [76] and prevent 0157:H7-induced colonic epithelial cell death via Bifidobacterium acetate-upregulated carbohydrate transporters [77] .Campylobacter jejuni has similar mechanisms to sense metabolites and hence spatial distribution.In avian hosts, where C. jejuni behave as symbionts in the lower GI tract, concentrations of acetate are high and allow for the expression of genes that permit commensal colonisation [78] .Conversely, high concentrations of lactate, similar to those observed in the upper GI tract, where C. jejuni colonises less efficiently, repress the genes involved in colonisation.The authors speculate whether C. jejuni utilises similar environmental cues in order to colonise humans and, thereby, cause diarrhoeal disease. SCFAs have also been associated with concentration-dependent negative effects on C. difficile growth [79] , and as SCFA concentrations are reduced following antibiotic treatment, this could be a contributing factor to its subsequent colonisation.Other studies have demonstrated that SCFAs increase the expression of Toxin B (TcdB), an essential virulence factor [80] .SCFAs may serve as a signal to C. difficile of an inhospitable and competitive environment; therefore, upregulation of TcdB may provide a survival mechanism.The success of faecal microbiota transplants (FMT) in the treatment of CDI reinforces the role of commensal microbiota (Bacteroides, Clostridium clusters IX and XIVa) in the treatment of CDI [81] .Early in vitro studies have demonstrated that dietary fibre polysaccharides induce a bifidogenic effect and hence increase SCFA production, which may result in enhanced colonisation resistance against C. difficile [82] .Likewise, an in vivo study found that C. difficile-infected mice fed a diet rich in dietary fibre had stimulated the growth of fibreutilising taxa (Bacteroides spp.) and their associated metabolites, i.e., SCFAs [62] , with decreased C. difficile fitness and numbers, while toxin expression was increased [62] . Among the major SCFAs, butyrate is the most extensively studied, largely due to its beneficial effects on both colonocyte energy metabolism and intestinal homeostasis [83] .Butyrate is the least abundant of the three SCFAs produced, comprising 15% of the total SCFA pool in humans [84] .Butyrate can upregulate mucin 2, reinforcing the mucus layer of the intestinal mucosa, and leading to enhanced protection against luminal pathogens [85] .Butyrate is formed in the so-called "classical pathway", by the condensation of two molecules of acetyl-CoA, and the subsequent reduction to butyryl-CoA, which can be converted to butyrate by members of the Clostridia family (Anaerostipes spp., Coprococcus catus, Eubacterium rectale, Eubacterium hallii, F. prausnitzii, Roseburia spp.) [21,86] .Anaerostipes spp.and E. hallii are also capable of utilising lactate as the substrate for the production of butyrate.Alternatively, butyrate can be synthesised from butyryl-CoA by the phosphotransbutyrylase/butyrate kinase route (Coprococcus comes, Coprococcus eutactus) [86] . Butyrate is at its highest concentration in the colon, and specific pathogens use the high concentrations of butyrate as an environmental cue in order to express virulence factors.For example, EHEC exhibited high adherence to Caco-2 cells in the presence of butyrate, whereas acetate and propionate had little effect [87] . Similarly, Shiga toxin-producing E. coli (STEC) exhibit increased adherence in the presence of SCFA concentrations that reflect those that are found in the colon [74] .In a study by Zumbrun et al. [88] , the authors demonstrated that a high fibre diet contributed to elevated butyrate and reduced commensal Escherichia compared to a low fibre diet; these resulting changes led to higher STEC colonisation, more weight loss and higher mortality in high fibre diet-fed mice.In IBD patients, faecal butyrate concentrations are higher than healthy controls, despite the lower abundance of butyrate-producing taxa [89] , and this may be explained by its impaired uptake and oxidation by inflamed colonocytes.While colonic SCFA concentrations seem to exacerbate certain Escherichia pathologies, colonic concentrations of butyrate [90] and propionate [91] have an antagonistic effect towards invasion gene expression in Salmonella, by down-regulating expression of SPI-1. The butyrate-producing Clostridia are obligate anaerobes capable of maintaining healthy gut homeostasis.Under eubiosis, the Clostridia-derived butyrate is the major energy source for the colonocytes and activates epithelial signalling through the intracellular butyrate sensor PPAR-y, driving mitochondrial β-oxidation of this substrate.Salmonella virulence factors induce inflammation during the early stages of infection, and these virulence factors have been shown to deplete the butyrate-producing Clostridia from the gutassociated community, leading to an epithelial aerobic environment which ultimately favours the aerobic expansion of the pathogen [92] .Moreover, antibiotic treatment resulting in a reduction of PPAR-y signalling, i.e., increased bioavailability of oxygen, has been shown to exacerbate this effect [93] .The reduced butyrate concentrations observed during Salmonella infection stimulate the colonocytes to switch from β-oxidation of butyrate to lactate fermentation and increase luminal lactate.Salmonella exploits this increase in lactate and utilises this carbon source for subsequent expansion [94] .Recently, genes involved in the direct βoxidation of butyrate have been identified in Salmonella, and excision of the operon drove the transition from a GI to an extraintestinal pathogen, i.e., non-typhoidal to typhoidal [95] , suggesting utilisation of butyrate plays a crucial role in Salmonella GI disease.Moreover, specific members of Clostridia are some of the few bacterial species capable of utilising fructose-asparagine, a known food source for Salmonella, which improves its fitness [96] , and could explain an evolutionary competition between these species. In silico analysis of butyrate production pathways in GI pathogens has identified members of the Fusobacterium genus and a few pathogenic strains of Clostridium (C.tetani and C. tetanomorphum) with the ability to produce butyrate [97] .However, their capacity to synthesise butyrate involves amino acids, primarily glutamate and lysine, as substrates, and are different to those observed in commensals, which primarily ferment pyruvate for butyrogenesis.The end product of this amino acid fermentation yields ammonia, higher concentrations of which are associated with colorectal cancer (CRC) [98] .Additionally, increased Fusobacterium nucleatum abundances have been observed in CRC patients when compared with healthy controls, and has been suggested as a possible microbial biomarker in CRC development [99] .It remains to be seen whether CRC tumorigenesis is a cause or consequence of microbiota alterations; however, there are associations of an "inflammatory diet", i.e., high consumption of red meat, processed meat and refined grains, with the prevalence of F. nucleatum-positive colorectal carcinomas [100] . On the whole, the effects of SCFAs on pathogen colonisation are concentration-dependent, with higher or lower concentrations having the capacity to be either antagonistic or hospitable, respectively, depending on the species and its preferred niche.Some examples of these differential effects of SCFAs can be found in Figure 2. The studies mentioned here only skim the surface on the complexity of the microbial and chemical interactions in the microbiota that influence health and disease. PREBIOTICS Prebiotics are selectively fermented ingredients that beneficially affect the host by stimulating the growth and/or functional activity of one or a limited number of bacteria in the colon, and thus improve host health [101] .The premise that these can potentially inhibit or obstruct the growth or virulence of pathogens is nothing new, and has been suggested as an alternative to antibiotic growth promoters in animals.The bestdocumented benefits stem from the use of indigestible oligosaccharides, such as fructans and galactans [102] ; however, consideration into the ability of pathogens to ferment or utilise the prebiotics or the associated metabolites should be taken into account [103] .Cereals, fruits and legumes are natural sources of prebiotics, whilst the active components are often synthesised using industrial chemical and enzymatic methods.The majority of prebiotic studies on pathogen inhibition to date involve livestock and animal models, which vary in terms of physiology and microbiota composition; therefore, care should be taken in translating these results to humans.Prebiotics such as fructo-oligosaccharides (FOS) and galacto-oligosaccharides (GOS) are preferentially fermented into SCFAs by Bifidobacterium and Lactobacillus which have historically been viewed as beneficial bacteria, resulting in lowered luminal pH.The addition of oligosaccharides to poultry feed has been shown to increase Bifidobacterium and Lactobacillus populations, while also being refractory to E. coli [104] and Salmonella colonisation [105,106] .This outgrowth of prebiotic-utilising taxa has also been demonstrated to eliminate C. difficile in mice [62] .The prebiotic inulin is primarily composed of FOS and has been shown to ameliorate low-grade inflammation through microbiota-dependent induction of IL-22 expression [107] and was able to prevent increased bacterial mucus penetration in high-fat diet-fed mice [108] .Furthermore, supplementation of high-fat diet-fed mice with B. longum restored mucus growth [108] , suggesting that prebiotic and probiotic treatments have the potential to prevent intestinal mucus abnormalities, which is a consequence of a high-fat diet.However, a study by Miles et al. [109] demonstrated that inulin may actually have the potential to exacerbate disease severity in response to inducers of colitis such as dextran sodium sulphate (DSS) in both low-fat and high-fat diets.Moreover, inflammation is crucial for the successful colonisation of Salmonella, through the inflammatory-mediated expression of virulence factors; studies in rats have shown increased translocation of Salmonella when FOS is added to the diet [110] . Although the overwhelming data suggest the beneficial effects of inulin supplementation, more care is needed to define the specific mechanisms by which inulin impact the gut microbiota to protect against the effects of inflammation and improve mucosal integrity. The other extensively studied prebiotic is GOS, which is commercially produced from lactose using glycoside hydrolases that catalyse transgalactosylation reactions [111] .In vitro studies have demonstrated the protective effect of GOS against EHEC and Cronobacter sakazakii, through an anti-adherence mechanism [112] .Interestingly, a recent study on the in vivo protective effects of GOS on the murine EHEC model pathogen, C. rodentium, showed that GOS treatment prevented pathogen-induced intestinal tissue damage, independent of anti-adherence activity and C. rodentium abundance [113] .In terms of the effect of GOS on microbiota composition, studies have identified a clear bifidogenic effect of GOS, while simultaneously lowering E. coli, Helicobacter and Clostridium spp.abundances [114] .The combined effects of the prebiotic on SCFA production, microbiota composition, pathogen virulence and fitness make it difficult to pinpoint the exact mechanism involved in providing a protective effect.This lack of a known mechanism underscores the importance of thorough analysis in animal studies before extrapolating results to humans. DIETARY LIPIDS AND BILE ACIDS Bile acids are amphipathic biological detergents produced by the liver with the primary function of metabolising lipids in the GI tract [115] , and their production is linked to the ingestion of fatty foods.Those bile acids, which are not reabsorbed into the liver (~5%), can serve as substrates for colonic microbial metabolism, i.e., hydrolysis of conjugated bile acids by bile salt hydrolases or biotransformed into secondary bile acids by 7α-dehydroxylation, where they are either excreted in faeces or recirculated back into the liver through the enterohepatic circulation [116] .Thus, changes in microbiota composition culminate in changes in the bile acid pool, and this homeostatic imbalance is associated with a range of disease states, including CRC, IBD and recurrent CDI [117] .Recent advances have identified specific operational taxonomic units, i.e., closely related bacteria, involved in bile acid biotransformations and correlate to a loss of specific taxa with the development of disease.For example, the previous infection of mice with Yersinia pseudotuberculosis remodels the microbiota to enrich for Deltaproteobacteria, a taurine metabolising class of bacteria which provide colonisation resistance to the pathogen Klebsiella pneumoniae [118] .Thus, commensal bile acid interactions are intrinsically linked in both mitigation and amplification of colonisation resistance. Identifying the biochemical mechanisms which underpin the effect on colonisation resistance will improve our knowledge going forward and open up new avenues for therapeutic manipulation of the microbiota. Antibiotic-induced destruction of the microbiota is associated with recurrent CDI.The protective role of the microbiota against C. difficile can be consolidated by the success of FMT [81] .C. difficile spores must germinate in vivo to develop into actively growing bacteria to produce enough toxins to initiate infection.In vitro, primary bile acids stimulate germination, and secondary bile acids inhibit this process [119] .Indeed, these interactions [Figure 2] have been shown in vivo, whereby depletion of secondary bile acids in the ileum resulted in C. difficile germination and growth [120] .Moreover, inflammation induced by C. difficile toxins subsequently changes this pathogen's nutrient metabolism pathways and enables it to thrive in the inflamed gut, particularly on products of collagen degradation, outcompeting commensal bacteria aside from members of the Bacteroides genus, which can also utilise collagen degradation products [121] .In a study by Buffie et al. [122] , the authors identified a single commensal that conferred resistance to C. difficile; Clostridium scindens, a bile acid 7α-dehydroxylating intestinal bacterium, which enhanced resistance in a secondary bile acid-dependent fashion.C. scindens-mediated restoration of secondary bile acids from hostderived bile salts were sufficient in inhibiting C. difficile germination, underpinning the pivotal role commensal bile acid-metabolising bacteria play in preventing recurrent CDI.Patients successfully treated for recurrent CDI have an enrichment of bile salt hydrolase-producing bacteria, the abundance of which negatively correlates with faecal concentrations of taurocholic acid, a primary bile acid [123] .While obesity has been identified as a risk factor for CDI [61] , and the microbiota composition of obese subjects is characterised by a decrease in the Bacteroidetes/Firmicutes ratio, there is no evidence to suggest that bile acid synthesis or enterohepatic circulation is altered by obesity.However, an obesity-driven altered and less diverse microbiota coupled with antibiotic elimination of bile-acid metabolising bacteria may very well contribute to an increased risk of CDI. High-fat diets promote the biosynthesis of bile, which can impact commensal microbiota that are sensitive to bile acid concentrations.Additionally, pathogens such as S. typhimurium are quite resilient to high bile acid concentrations, and colonisation resistance to this pathogen is alleviated upon oleic acid or high-fat diet supplementation in mice [124] , and colonisation resistance is improved when switched back to a plantbased diet.Commensal E. coli that can compete with S. typhimurium through bile acid [124] or oxygen [125] competition could provide a means of protection against this pathogen. PROTEIN AND AMINO ACIDS Aberrations in microbiota community structure driven by antibiotics, infection and/or diet will affect protein homeostasis and can increase free amino acids in the gut, providing a nutritional niche upon which some pathogens can capitalise.Indeed, many gut pathogens such as EHEC [126] , Vibrio cholerae [127] , C. jejuni [128] , and C. rodentium [129] have genes involved in amino acid biosynthesis upregulated upon gut colonisation.Moreover, the host relies on amino acid metabolism to support its immune responses against invading pathogens, with diets deficient in protein having a counterproductive effect on immune function, independent of the microbiota [130] .On the other hand, dietary administration of high protein and amino acid by-products stimulates the growth of pathogens [129] and protein-fermenting bacteria contribute to disease susceptibility [131] .Therefore, identifying commensal competitors and pathogen metabolic pathways involved in protein fermentation and amino acid biosynthesis may help in developing new strategies to encourage colonisation resistance through diet. D-amino acids are biosynthesised by gut bacteria as opposed to L-amino acids which humans biosynthesise or obtain from the diet.Tryptophan is an essential amino acid from the diet in mammals and is primarily catabolised by commensal bacteria into various indole-containing metabolites.While tryptophan is required for optimal immune responses, such as T-cell proliferation, the commensal-mediated tryptophan metabolites can have differential effects on gut pathogens.In S. typhimurium, the tryptophan metabolite indole induces expression of genes related to efflux-mediated multidrug resistance [132] , while concomitantly decreasing the expression of genes involved in invasion located on the SPI-1 pathogenicity island [133] .On the other hand, indole upregulates EHEC secretion of EspA and EspB via the type III secretion system, enhancing this pathogen's ability to form attaching and effacing (A/E) lesions [134] , while other indolederivatives can inhibit biofilm formation, motility and formation of A/E lesions [135] .The enzyme indoleamine 2,3-dioxygenase (IDO) catalyses the conversion of tryptophan into kynurenine, reducing the tryptophan pool in the gut, and thereby directly impacting various immune responses.C. difficile infection upregulates the expression of IDO, increasing the production of kynurenine, subsequently depleting the tryptophan pool, and thereby diminishing the immune responses of the host toward this pathogen [136] .All in all, tryptophan and its associated metabolites have direct effects on immune function and pleiotropic effects on gut pathogens, suggesting that this molecule will be of interest in colonisation resistance studies moving forward. Many gut pathogens switch metabolic pathways depending on the environment, e.g., in inflammation.The pathobiont Adherent Invasive E. coli (AIEC) shifts its metabolism to catabolise L-serine in the inflamed gut to maximise growth potential [137] , with L-serine having little effect on AIEC fitness in a healthy gut environment.Interestingly, AIEC bloom in the inflamed gut, and are significantly reduced when amino acids are decreased in the diet.C. difficile exploits the niche created following particular antibiotic treatments, and this dysbiotic environment has increased the availability of amino acids.Specifically, a recent study demonstrated that C. difficile is dependent on L-proline metabolism, as L-proline knockout C. difficile strains were unable to colonise the gut of germ-free mice transplanted with either a dysbiotic or healthy microbiota [138] .Furthermore, low-protein or low-proline diets given to mice substantially decreased wild-type C. difficile expansion suggesting that C. difficile is dependent on proline for adequate colonisation, which can potentially be mediated through dietary intervention.Furthermore, commensals such as members of the Clostridia class, decreased the fitness advantage of C. difficile's ability to ferment proline, through competition for this amino acid [139] .Likewise, EHEC was found to be reliant on proline for colonisation, with commensal E. coli that compete for proline attenuating the expansion of EHEC [140] . As discussed above, the microbiota can limit the colonisation of invading pathogens by depleting the concentration of amino acids in the gut.Some pathogens can overcome this problem by inducing amino acid biosynthesis to subvert such a deficiency.Transposon sequencing is a powerful tool and allows for the generation of a library of random pathogen mutants, for example, those defective in amino acid biosynthesis pathways [141] .This technique provides a means to estimate the fitness contribution or essentiality of each genetic component in a bacterial genome.Caballero-Flores et al. [129] applied this to C. rodentium and found that specific mutants deficient in the production of arginine, threonine, histidine, tryptophan, or isoleucine lost their competitive advantage in mice, compared to wild-type C. rodentium.Moreover, these genes were significantly upregulated in conventional mice as opposed to germ-free mice, suggesting that C. rodentium specifically use these pathways to outcompete the microbiota.Feeding of a high-protein diet to mice produced markedly better colonisation of C. rodentium compared to normal chow.While mouse studies like these inform a mechanistic understanding of pathogen colonisation, the importance of these findings in relation to human disease warrant further investigation. TRACE ELEMENTS As mentioned earlier, the majority of human enteric pathogens belong to the phylum Proteobacteria.In the normal intestine, which is largely inhabited by commensals, mainly Bacteroidetes and Firmicutes, Proteobacteria only constitute < 1% of microbiota populations.The outgrowth of Proteobacteria "blooms" are a hallmark of gut "dysbiosis" resulting from microbial perturbations caused by antibiotic therapy, dietary changes or inflammation. The availability of micronutrient trace elements is essential to the successful colonisation of pathogens during infection.Nearly 60% of known enzymes contain at least one metal cofactor, with zinc being the most common, followed by iron and manganese [142] .In the inflamed gut, these dietary trace elements are heavily sequestered by high affinity binding proteins or kept in organelles that are not accessible to bacteria, in a process known as "nutritional immunity" [143] .Many proteobacterial pathogens are equipped with an array of high affinity siderophores, to help them overcome the restriction of available metals and ultimately drive key cellular processes, which in turn sustains and propagates infection.Deficiency or increased supplementation of dietary trace elements may disrupt the commensal microbial populations and predispose individuals to infection. Zinc deficiency is associated with increased Enterobacteriaceae and Enterococcus, with concomitant decreases in abundance of Clostridiales and Verrucomicrobia (A. muciniphilia) [144] .Moreover, in a mouse model of enteroaggregative E. coli (EAEC), a cause of traveller's diarrhoea, zinc-deficient mice exhibited altered immune responses and an increase in EAEC virulence factors [145] .Furthermore, dietary zinc supplementation abrogated disease progression, reduced EAEC colonisation and expression of virulence factors [146] .In another study, zinc supplementation protected from uropathogenic E. coli haemolysininduced gut barrier dysfunction [147] .These observations indicate a beneficial impact of zinc supplementation on zinc-deficient subjects; conversely, excessive zinc supplementation can have a detrimental impact on microbial homeostasis and host immune responses.In a study by Zackular et al. [148] zinc supplementation stimulated the growth of Enterococcus and Clostridium XI cluster while concomitant reductions in Turicibacter and Clostridium (unclassified) were observed.Ultimately, excess zinc selected for a microbiota that was much more prone to destruction by antibiotics, thus exacerbating C. difficile colonisation and associated disease [148] . For many bacterial pathogens, the availability of iron is often the limiting factor for colonisation and infection.During inflammation, nutritional immunity limits the bioavailability of iron in the gut, and thus bacterial species equipped with an array of iron acquisition systems are often the most successful and pathogenic.Given the ability of bacterial siderophores to hijack host iron homeostasis, it is not surprising that the innate immune system has evolved mechanisms to counteract bacterial iron acquisition, such as the production of Lipocalin-2 (LCN2).In the acute phase response to infection, LCN2 is expressed to bind bacterial siderophores and neutralises bacterial capacity to sequester iron.However, some bacteria have evolved resistance mechanisms to counteract this immune response, such as the stealth siderophore salmochelin produced by Salmonella, thereby gaining a competitive advantage in the inflammatory milieu [149] .Interestingly, the probiotic E. coli strain Nissle shares many fitness properties to uropathogenic E. coli, including iron uptake systems.In the presence of LCN2, Nissle is capable of outcompeting Salmonella in a mouse model [150] , underscoring the evolved synergy between commensal and host immune response in thwarting pathogen colonisation.Some pathogens, such as V. cholerae, have the ability to obtain iron from haem only when Cholera toxin (CTX) is produced [151] .The production of CTX induces inflammation and thus decreases gut iron concentrations but enables the bioavailability of host haem, while concurrently changing the transcriptomic gene signature of V. cholerae to one that is capable of utilising iron from haem.This change allows the expansion of V. cholerae by providing an iron-limited metabolic niche and a competitive advantage over commensals by this pathogen's unique ability to acquire iron from haem. In both developing and developed nations, iron deficiency remains the most common form of nutritional deficiency, in many cases prompting iron supplementation to alleviate symptoms of malnutrition.Given the importance of iron to GI pathogens, the effect of iron in bolstering colonisation resistance should perhaps be considered as a detrimental effect by inducing microbial dysbiosis.Indeed, an outgrowth of Enterobacteriaceae and increased risk of infection has been observed in both mice [152] and humans [153] , following iron supplementation.Intriguingly, the adverse effects can be mitigated simply by the addition of prebiotics to the diet [154,155] , inducing growth of beneficial Bifidobacterium and Lactobacillus.Bifidobacterium have also been demonstrated to efficiently sequester iron [156] .The reliance of the host immune system on sequestration of iron, coupled with commensal sequestration capacity and subsequent SCFA production, play multifactorial roles in reducing pathogen colonisation. In addition to the production of LCN2, the host can produce another antimicrobial molecule, Calprotectin, whose primary function is to bind to free zinc and manganese in the gut lumen.Bacteria utilise manganese as a cofactor for a number of proteins; perhaps the best studied is the role of manganese as a detoxifier of reactive oxygen species, of which numerous are encountered following an immune response.Salmonella has evolved high-affinity cation transporters to bypass the action of calprotectin and therefore promote growth in an inflamed intestine [157] .It is unclear how excess or deficient dietary manganese influences gut microbiota populations.It is possible many pathogens behave similarly to Salmonella in subverting calprotectin when manganese is in excess, or perhaps behave similarly to Staphylococcus aureus, which can switch to iron in manganese-deplete conditions; thereby bypassing nutritional immunity and causing infection [158] . Pinpointing the delicate balance between trace element toxicity and deficiency while simultaneously understanding the mechanisms involved in both nutritional immunity and colonisation resistance remains complicated.Understanding the complex pathways dietary trace elements play in microbial respiration in infection and inflammation will undoubtedly uncover novel treatments.For example, recently, dysbiotic Enterobacteriaceae blooms were ameliorated by tungstate treatment, which inhibited molybdenumcofactor-dependent respiratory pathways and reduced the severity of inflammation in mouse models [159] . CONCLUSION Disentangling the direct and indirect impact of dietary ingredients and nutrients on commensal bacteria in the gut, their associated metabolites, immune function and pathogen virulence will no doubt be challenging.This knowledge will require multidisciplinary collaborations between experts in nutrition, immunology, and microbiology, to name a few.Given that gnotobiotic and antibiotic-treated mice are more susceptible to infection, and that this phenotype can be reversed upon supplementation with even a simplified consortium of commensal bacteria [160] strongly supports the paradigm of colonisation resistance.Reductionist or modular approaches like these can help identify potential probiotic or synbiotic candidates and generate insights into diet-microbe/host-microbe/microbe-microbe interactions. Studies in neonates and infants support the importance of breast milk-feeding, in providing antibodies and colonisation of human breast milk-metabolising taxa, both of which have been shown to enhance colonisation resistance in the offspring.Moreover, C-section delivery disrupts the mother to infant transmission of specific commensals, resulting in a reduced immunostimulatory potential passed to the offspring [161] .Perhaps a critical, yet, unexplored area of research would be the impact of infant formulametabolising taxa in reducing/providing protection against pathogens.Identifying specific species and their associated functions, which improve neonatal colonisation resistance, has the potential to optimise the production of infant formula through the selection of desired carbon sources. The mucus layer provides the first line of defence against exogenous microorganisms, the integrity of which is greatly determined by microbial composition, which in turn, is influenced by dietary components, in particular fibre.Patients with IBD should be cautious when consuming specific dietary fibre or prebiotics; inulin has been demonstrated to exacerbate DSS-induced colitis in mice, whereas others such as psyllium have been successful in ameliorating gut inflammation [162] .In the context of human GI disease, it is equally ambiguous, as a study observed avoidance of dietary fibre associated with flares in Crohn's disease patients but not in ulcerative colitis patients [163] .Research focusing on individual fibres or prebiotics must be interpreted cautiously as they may inadvertently select a small subset of taxa, while care must be taken when comparing mouse chow diet controls, which contain a mixture of fibres, and thus selecting for a larger subset of taxa.More research is required in humans, in terms of the impact of dietary fibre and prebiotics on the function, stability and characterisation of specific taxa and their associated metabolites.These data could complement in vitro, ex vivo and "humanised" mouse studies to identify mechanisms when the host is challenged with a GI pathogen. Microbiota alterations, driven either by pathogen-induced inflammation or other diseases such as IBD, will ultimately impair normal SCFA homeostasis through changes in commensal SCFA producers and/or pathogen/pathobiont utilisation of SCFAs.Given that SCFAs provide an energy source for colonocytes and the majority of human studies measure SCFA concentrations from excreted faeces, faecal SCFA concentrations may not be representative of those found in other parts of the GI tract.Thus, extrapolating findings from human dietary intervention studies in providing SCFA-mediated effects should be approached with caution. Mechanisms of protein or trace element homeostasis in the gut, with respect to interactions with complex and diverse commensal and pathogenic bacteria, are largely uncharacterised, especially in the context of human disease.This uncertainty is further complicated by interindividual variations underpinned by yet-tobe-determined genetic, environmental or epigenetic factors.While simplified mouse models may fail in recapturing the complexities observed in humans, they can be valuable assets in identifying commensal bacteria with, for example, a high degree of affinity for trace elements, e.g., iron siderophores.Uncovering dietary ingredients that promote their growth could, in theory, boost colonisation resistance through nutrient competition.Future research into the role of branched-chain fatty acids derived from the catabolic products of branched-chain amino acids and how they impact colonisation resistance could be an attractive area of research, given that they may influence metabolic homeostasis in the gut. Seasonal variations in the mouse chow diet, industrial variations in the processing of prebiotics and feed supplements, choice of laboratory pathogen/commensal strains and breed of the mouse all contribute to disparities among research groups.Thus, tightly controlled models are a necessity, before translating to novel therapeutics and functional foods.Moreover, the fine line between commensal and pathogen in genetically predisposed individuals only adds to the uncertainty and personalised dietary interventions [164] in these individuals for the prevention of infection is an interesting prospect, but further research is warranted. DECLARATIONS Authors' contributions Conceived the idea for the review: Ross RP, Stanton C Researched the literature and wrote the review: Strain R Corrected and edited the review: Ross RP, Stanton C Figure 1 . 1 Figure 1.Metabolism of human milk oligosaccharides (HMOs) lowers gut pH boosting colonisation resistance; HMOs are metabolised by initial colonisers (Bifidobacterium longum ssp.infantis and Bacteroides) in the infant gut, producing SCFAs and subsequently lowering pH & increasing colonisation resistance against gastrointestinal pathogens.SCFAs: Short-chain fatty acids. Figure 2 . 2 Figure 2. Differential effects of dietary metabolites on pathogens; metabolites from fibre degradation by commensals have differential effects on the success of an invading pathogen.Acetate promotes the growth of Enterohemorrhagic E. coli (EHEC), whereas butyrate and propionate repress growth.Conversely, acetate represses Salmonella growth, whereas butyrate and propionate promote growth.Colonisation of Clostridioides difficile is increased by the cholesterol metabolites, primary bile acids.The commensal Clostridium scindens can limit the availability of primary bile acids by converting these to secondary bile acids, thus increasing colonisation resistance to C. difficile.SCFAs: Short-chain fatty acids. Table 1 . Metabolites influencing pathogen virulence and fitness 1PathogenGene expressionGene functionMetaboliteRef.EHECLEE-encoded T3SSAdherence↑Butyrate[87]EHECLEE-encoded T3SSAdherence↑Ethanolamine[165]EHECLEE-encoded T3SSAdherence↑Succinate[166]EHECLEE-encoded T3SSAdherence↑Oxygen[167]EHECLEE-encoded T3SSAdherence↓D-serine[168]EHECAdhesionsAdherence↑Ethanolamine[169]EHECAdhesionsAdherence↑Choline[169]EHECihaAdherence↑Butyrate[74]EHECFliCMotility↑Acetate[73]EHECGvrA/LEE-encoded T3SSAcid resistance↑Bicarbonate[170]EHECQseQuorum sensing↑Ethanolamine[169]EHECQseQuorum sensing↑Fucose[171]EHECStxToxin↑Ethanolamine[169]EHECSdhARespiration↑Fumarate[172]EHECEspA/EspBA/E lesions↑Indole[134]AIECtdc/sdaInflammed gut colonisation↑L-serine[137]SalmonellaSPI-1Invasion↑Acetate[68]SalmonellaSPI-1Invasion↑Formate[66]SalmonellaHybInvasion↑Hydrogen[173]SalmonellaSPI-2Intracellular replication↑Ethanolamine[174]SalmonellattrSR ttrBCAInflammed gut respiration↑Tetrathionate[175]SalmonellafraBDAECarbon/nitrogen utilisation↑Fructose-asparagine[176]SalmonellapduA-XInflammed gut respiration↑1,2-propandiol[177]SalmonellaSPI-1Invasion↓Indole[133]C. difficileTcdABToxin↑Butyrate[178]C. difficileTcdABToxin↓Pyruvate[179]C. difficileSlecGermination↑Taurocholate[180]C. difficileCD2344Respiration↑Succinate[181]C. difficilePrdBNutrient utilisation↑Proline[139]C. difficiletcdB/Spo0AToxin/sporulation↓Secondary bile acids DCA LCA[182]V. choleraetcpAPilus/colonisation↑Autoinducer-2 (AI-2) synthase[183]V. choleraertxA/hylAToxin↑Autoinducer-2 (AI-2) synthase[183]V. choleraeflrAMotility↑Cholesterol[184]V. choleraectxAB/tcpAToxin/motility↓Sodium[185]↑Upregulates. ↓Downregulates. 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Computational design and experimental characterisation of a stable human heparanase variant † RSCCopyright RSC2022. 2022 Cassidy Whitefield Nansook Hong Joshua A Mitchell Colin J Jackson Computational design and experimental characterisation of a stable human heparanase variant † RSC Chem. Biol RSC33412022. 202210.1039/d1cb00239b Heparanase is the only human enzyme known to hydrolyse heparin sulfate and is involved in many important physiological processes. However, it is also unregulated in many disease states, such as cancer, diabetes and Covid-19. It is thus an important drug target, yet the heterologous production of heparanase is challenging and only possible in mammalian or insect expression systems, which limits the ability of many laboratories to study it. Here we describe the computational redesign of heparanase to allow high yield expression in Escherchia coli. This mutated form of heparanase exhibits essentially identical kinetics, inhibition, structure and protein dynamics to the wild type protein, despite the presence of 26 mutations. This variant will facilitate wider study of this important enzyme and contributes to a growing body of literature that shows evolutionarily conserved and functionally neutral mutations can have significant effects on protein folding and expression. Introduction Heparan sulfate (HS) consists of 1-4 linked disaccharide units that are negatively charged and structurally heterogeneous due to variable sulfation, deacetylation and epimerization during biosynthesis. 1 HS is often covalently linked to proteins and peptides to form heparan sulfate proteoglycans (HSPGs). 2 HSPGs are themselves a major component of the extracellular matrix (ECM) and basement membranes, forming a protective barrier by interacting with other major components of the ECM such as collagen, fibronectin and laminin. Their structural diversity and negative charge attract various cationic proteins and water, forming porous hydrogels that are able to store bioactive molecules including growth factors, 3,4 chemokines 5 and enzymes. 6 Heparanase (HPSE) is the only mammalian enzyme that is known to hydrolyse HS. [7][8][9][10] In adults, HPSE is normally expressed at low levels, found only in platelets, immune cells and the placenta, [11][12][13] but increased expression of HPSE has been observed in many disease states, including cancer and Covid-19. [14][15][16] When overexpressed, HPSE catalyses the hydrolysis of HS, resulting in weakening of the ECM barrier, which can promote inflammation, 17,18 cancer cell invasion, growth and migration, 19,20 as well as angiogenesis. 21,22 HPSE is also associated with tumour initiation by up-regulating proinflammatory mediators. 23,24 Moreover, animal studies have shown HPSE genetic knock-outs improve cancer prognosis and increased survival without significant side effects. 25,26 Owing to its roles in many diseases, HPSE has been a drug target for many years. For instance, HPSE expression promotes resistance to chemotherapy, whereas targeting HPSE with inhibitors can overcome chemoresistance and tumour relapse. 27 Indeed, many groups have attempted to produce drug-like HPSE inhibitors over recent decades. 1,26,28,29 However, HPSE production currently relies on complex and expensive eukaryotic expression systems such as mammalian 7,8,30 and insect cells. 31,32 While some prokaryotic HPSE expression methods have been reported, 33,34 they have not been sufficiently robust for widespread adoption. HPSE has many features that are known to reduce soluble expression prokaryotic systems, such as Escherichia coli, including multiple disulfide bonds and large positive regions on the surface, [35][36][37] as well as N-glycosylation. 22,38 Moreover, HPSE is natively expressed as a pre-proheparanase which undergoes proteolytic cleavage of a signal peptide then a linker segment, resulting in an active heterodimer composed of 8 kDa and 50 kDa subunits ( Fig. 1) 39 Thus, in prokaryotic expression systems the 8 kDa and 50 kDa subunits have to be expressed separately and assemble into a heterodimeric complex. 33,34 There are many experimental and computational methods that have been developed to improve enzyme function and stability, such as bioinformatics-based approaches like consensus design 40,41 or ancestral sequence reconstruction, 42,43 or forcefieldbased approaches like Rosetta 44 or FoldX. 45 However, both approaches have limitations. The Protein Repair One Stop Shop (PROSS) algorithm combines forcefield-based Rosetta modelling and phylogenetic sequence information to create variants with improved stability. 46 Here, we describe the use of PROSS to generate the first stable human HPSE variant to be expressed in E. coli. We demonstrate that it has significantly increased solubility, very similar catalytic activity and identical inhibition by competitive inhibitors, when compared to wild type human HPSE produced from mammalian cells. Our results are supported by an X-ray crystal structure and molecular dynamics simulations, which demonstrate that the introduced mutations stabilise HPSE with almost no effect on the three-dimensional structure or dynamics. This mutant HPSE should significantly reduce the costs and technical barriers to the development of HPSE inhibitors and its widespread study. Results Computational design of a soluble HPSE variant We first tested bacterial expression of human HPSE (wild type) by cloning two subunits (8 kDa and 50 kDa, Tables S1 and S2, ESI †) into the dual expression vector (pETDuet-1). To optimize the chances of obtaining soluble, properly folded protein, we co-expressed the protein with chaperones (trigger factor and GroEL/GroES), [47][48][49] and used E.coli Shuffle T7 Express 50 cells, which allow disulfide bonds to form in the cytosol. Under these conditions, the 50 kDa subunit was totally insoluble, while the 8 kDa subunit was partially soluble (Fig S1, ESI †). Given that the molecular structure of HPSE has recently been solved, 31,32 it is now an appropriate candidate to be engineered to allow expression in simple and inexpensive expression systems, such as E. coli. Recently the PROSS algorithm 46 has demonstrated its utility in designing stable variants of challenging proteins for soluble and functional expression in bacteria. [51][52][53] Unlike conventional consensus mutagenesis approaches, in which poorly conserved residues are mutated back to their consensus identity (from a multiple sequence alignment), 54 PROSS combines this approach with computational modelling with Rosetta, 55 generating a set variants, each containing multiple mutations that ideally act together to increase stability. 46 We therefore used PROSS to redesign the insoluble 50 kDa subunit based on the crystal structure of the insect cell expressed human HPSE (PDB ID: 5E9C). The substrate binding site and the heterodimer interface residues were restrained to maintain function and preserve the interaction with the 8 kDa subunit. Seven variants with accumulated mutations were generated (Fig. S2, ESI †), which were subsequently synthesized and sub-cloned into multiple cloning site 2 of pETDuet-1 vector. The 8 kDa subunit with a N-terminal poly histidine tag was sub-cloned into multiple cloning site 1. All variants were tested (Fig. S3, ESI †) and the most soluble design, containing 26 amino acid substitutions was identified and purified (HPSE P6), using Ni 2+ -NTA, heparin and size exclusion chromatography. This resulted in pure, homogeneous, heterodimeric HPSE with a final yield of 4 mg from 1 L E. coli culture (Fig. 2). Notably, PROSS is not infallible; many of the designs did not produce soluble protein, which emphasises the need to test multiple different variants. In mammalian cells, pre-proheparanase (Met1-Ile543) undergoes successive cleavage events of the N-terminal signal peptide (Met1-Ala35) and linker (Ser110-Gln157, red cartoon) segments to produce mature HPSE. The resulting heterodimer assembly of two subunits (8 kDa subunit (Gln36-Glu109, yellow cartoon) and 50 kDa subunit (Lys158-Ile543, blue cartoon)) consists of a TIM barrel (b/a) 8 and b-sandwich fold. The sequence of HPSE is shown on top as a bar representation in which glycosylation sites are shown as green sticks and cysteine residues are indicated as black arrows. In non-mammalian systems active protein must instead be produced via co-expression of the two subunits. 31 (B) Crystal structure of human HPSE expressed in an insect expression system (PDB ID: 5E8M) is shown as grey cartoons (bottom-left). Six N-glycosylation sites (Asn162, 178, 200, 217, 238, 459) are shown as green sticks and four cysteines (Cys179, 211, 437, 542) are shown as yellow sticks whereby two of them form a disulfide bond (Cys437-542) at the b-sandwich domain. Catalytic residues (Glu343, Glu225) at the TIM face are shown as red sticks. (C) Electrostatic potential surface was calculated using amino acid residues in the crystal structure by APBS (glycans were not included in the calculation). This shows two large positively-charged patches at the TIM domain and at the b-sandwich domain, which may promote aggregation in the nucleic acid rich micro environment. 37 HPSE P6 exhibits wild-type like activity and response to inhibitors Given the large number of mutations and loss of glycosylation sites, it was important to test whether these changes had any effect on the activity of the protein. The catalytic activity of purified HPSE P6 was tested by colorimetric assay using fondaparinux 56 (Arixtra), a synthetic analogue of HS. Although, the catalytic rate (k cat ) of HPSE P6 was slightly (less than 2-fold) higher (k cat = 2.94 AE 0.13 s À1 ) compared to HPSE WT (k cat = 1.72 AE 0.07 s À1 ), the binding affinity (K M = 11.6 AE 2.8 mM and 11.83 AE 2.7 mM respectively) was the same, demonstrating that the introduced mutations have no effect on the interaction between enzyme and substrate ( Fig. 2C, Table 1). In fact, the slight increase in k cat is most likely due to the higher purity of the HPSE P6, compared to the commercially available HPSE WT. The loss of the six glycosylation sites no effect on the activity of the enzyme, suggesting these sites may be important for protein solubility in mammalian systems. Having established the enzyme kinetic parameters are comparable to HPSE WT, we then tested whether the HPSE P6 variant would interact identically with heparan sulfate mimetic inhibitors; in this case pentosan polysulfate 57 ( Fig. 2C and D). As with the enzyme kinetics, the inhibitory response to the model inhibitor pentosan polysulfate was near identical between HPSE WT and HPSE P6, with an IC 50 of 12.46 AE 1.26 nM and 12.43 AE 2.47 nM, respectively (Fig. 2D). HPSE P6 is thermostable and structurally isomorphous to HPSE WT The thermal stability HPSE P6 was measured using circular dichroism (CD) by observing the loss of helicity at 222 nm over 20-90 1C. This revealed that HPSE P6 is somewhat thermostable, undergoing a transition to an unfolded state with a T m value of 63.6 AE 0.19 1C, (Fig. 3A). This Tm value is similar to other engineered variants of human proteins produced through the use of PROSS, 46,53 and significantly exceeds the normal temperature range that human proteins are exposed to (B37 1C). To understand how the 26 mutations in HPSE P6 result in enhanced protein folding and stability, we solved the crystal and HPSE WT, where catalytic rate (k cat ) of HPSE P6 was 70% higher (k cat 2.94 AE 0.13) compared to (k cat 1.72 AE 0.07) for WT. The binding affinity (K M 11.6 AE 2.8 mM and 11.83 AE 2.7 mM respectively) was the same. (D) Pentosan polysulfate was used to compare the design with the human HPSE expressed in mammalian cell. This was measured using colorimetric method with fondaparinux. Error bars represent standard error from a minimum of three measurements. structure of HPSE P6 at 1.30 Å resolution (Table S3, ESI †). The protein crystallised in the P2 1 2 1 2 1 space group within 1 day, forming rod shaped crystals. This compares to WT HPSE crystallising in the P2 1 space groups, after 1-3 days. Despite 26 mutations, the crystal structure of HPSE P6 shows almost identical overall backbone and active site structures to HPSE WT expressed in an insect system (with the exception of the absence of any glycosylation). The Ca RMSD was 0.645 Å, with an alignment score of 0.017 (Fig. 3C). Many subtle changes were observed due to the 26 mutations. Firstly, surface polarity, which is known to positively contribute to folding and stability, 59 was increased by substitutions to surface leucine and alanine residues to more polar or smaller amino acids (e.g. Leu197Gly, Leu354Gly, Leu498Gln, Leu230Arg, Ala195Ser) (Fig. 3C). Secondly, additional stabilising interactions, including increased hydrogen bonding networks and hydrophobic packing were introduced, which stabilise the folded state. For example, new hydrogen bonding interactions were introduced by Leu483-His, lle318Thr, Lys477Gln and Ser322Gln and new hydrophobic interactions were introduced by Ser530Ala, Ser292Ala and Arg307Leu in the partially solvent exposed areas. Interestingly, we observed indirect conformational change of Phe258 by Ser212-Ala (Fig. 3C.iv). Finally, the disulfide bond (Cys437-Cys542) was possibly stabilized by introduction of proline at the position 540 (Ala540Pro) on the loop (Fig. S4, ESI †). In previous applications of PROSS, it was noted that large positively charged patches, which could promote aggregation in the nucleic acid rich micro environment, 37 were eliminated or reduced. 60,61 Here, in the case of HPSE, the electrostatic potential surface of HPSE WT shows two large positive patches around the active site and the b-sandwich domain (Fig. 3B). For HPSE P6, the theoretical isoelectric point was the same as the wild type (pI 9.4), and the electrostatic surface potential shows that while one of the large positive patches around the b-sandwich domain was slightly diminished by two lysine mutations (Lys427Asp and Lys477Gln), the electrostatic potential around the active site at the TIM face was maintained as the area was constrained during the design process (Fig. 3B). Molecular dynamic simulations to account stabilization It has previously been shown that the dynamics and function of similar proteins can be very different despite ground state structures appearing very similar in terms of Ca RMSD. 62 Crystallographic B-factors are commonly used to probe differences in the conformational flexibility of proteins within a crystal lattice, although this approach can be limited by the existence of crystallographic artifacts, whereby flexible regions on the protein surface could be stabilized by interactions with the lattice symmetry mates. Comparison between the B-factors of HPSE WT and HPSE P6 reveal the overall trend in terms of regions with high or low B-factors is conserved, although a decrease in the overall B factors of the P6 variant in the TIM (b/a) 8 domain fluctuations, mostly around the surface loops of the active site (Fig. S4A, ESI †). However, this analysis is confounded by the higher resolution, lower Wilson B-factor and different crystal packing of the HPSE P6 variant. To complement the structural analysis, we also performed molecular dynamics simulations to examine the effects of these mutations on the conformational sampling and motions of the protein. To identify whether the dynamic range of HPSE P6 is the same as the HPSE WT, a total simulation time of 1 ms per protein was completed. Principal component analysis was conducted to visualize motions that represent the major fluctuations of the system. Principal components 1 and 2 of the HPSE WT and HPSE P6 (10.4% and 9.0%) overlap, demonstrating that the breathing motion of the active site is conserved (Fig. 4A). The third major component, which only contributes 6.5% of the total movement of the protein, shows slight differences, being comprised predominately of the movement of surface-exposed loops. No other principal component showed any difference between the two proteins (up to 20 components). Root mean square fluctuations were also analysed to identify the displacement of amino acids throughout the course of the simulation (Fig. 4B). The average RMSF and their 95% confidence intervals, (where 95% of the residue's displacement occurs in that region) are overlaid for both proteins. This demonstrates that HPSE WT and P6 fluctuations overlap closely. There were very few differences overall, where the most consistent change is a decrease in magnitude of surface loops for HPSE P6 in comparison to WT simulations. Even though these residues have a very slightly decreased magnitude, the RMSF still has the same overall shape. The only residues with RMSF values outside of the 95% CI are residues 488-495. This is a surface loop on the b-sandwich domain with two introduced mutations; Leu483His and His486Asp. These mutations allow an increase of hydrogen bonding causing a slight rigidification of this loop (Fig. 4C). Overall, the conservation of the protein dynamics despite 26 mutations is striking and unexpected. Indeed, the great majority of these mutations (identified as grey spheres in Fig. 4C) do not cause any significant difference on the dynamics of the protein. This analysis is also fully consistent with the functional data, which revealed almost to effect of the mutations on activity or inhibition. Discussion Despite the widespread interest in HPSE as an important enzyme in human physiology and a drug target, the difficulty related to obtaining large quantities of pure recombinant protein has limited the ability of many groups to study the protein. Bacterial expression systems, such as E. coli, are widely accessible, allow protein to be expressed in high yields and at low cost. While prokaryotic HPSE expression methods have been reported, 33,34 none have been repeated in the literature or have been widely adopted. Here, a stable version of human HPSE has been computationally designed, which allowed the mature heterodimeric enzyme to be expressed at reasonable yield in soluble form in E.coli. The subsequent characterization of this designed version (HPSE P6) showed the enzyme to behave essentially identically to HPSE WT in terms of its interactions with substrates (Table 1) and inhibitors (Fig. 1). Thus, this computationally designed HPSE P6 variant should be a useful surrogate for the wild-type enzyme in structural biology, inhibitor screening and kinetic analyses. It is notable that despite 26 mutations, the enzyme is essentially structurally isomorphous to the wild-type, with no significant changes to the C-a backbone or side chain rotamer sampling, especially in the vicinity of the active site. Additionally, the dynamics of the enzyme were also largely identical to the wild-type enzyme, suggesting that there were no significant changes to the relative stabilities of different conformational substates. This reinforces the functional neutrality of many mutations and the power of bioinformatics inspired approaches such as consensus design and PROSS; these mutations were acquired through phylogenetic analysis i.e., they are known to be tolerated in related enzymes. Indeed, while their individual effects might be small, the summation of the effects can become considerable. However, the route to HPSE P6 was not simple or trivial; P6 was the only design of the seven that we tested that was effective. Thus, while the combinatorial effects of the mutations can be powerful, the unpredictable effects of the mutations, and their epistatic interactions, make it imperative that a range of designs are trialled. Our structural analysis of HPSE P6 shows that many of the mutations appear to have effects that can be rationalised in terms of our understanding of how proteins fold: increasing surface polarity, forming additional non-covalent interactions such as hydrogen bonds, increased packing within the hydrophobic core, etc. The lack of major structural changes, such as strong salt bridges or significant changes to internal cavities, which are characteristic of rational or computationally designed stabilising mutations, meant that the structural dynamics of the protein, and thus its catalytic function, was largely unperturbed. This study is thus an interesting example of protein stabilisation: on the one hand, 26 mutations could be considered to be a large number of mutations, but the counter argument is that 26 functionally neutral mutations that have almost no effect on the structure and dynamics is in fact a very conservative method for stabilizing a protein, in comparison to a smaller number of mutations that might have a larger effect on the structure, dynamics and function of the enzyme. Experimental Stability design Chain A of the crystal structure of the ligand bound human HPSE (PDB ID: 5E9C) was submitted to the PROSS stability design algorithm 46 on the web server (http://pross.weizmann. ac.il), with constrained residues, which have contacts with the ligand (Dp4) and with the chain B. This generated 7 mutants. Cloning The linear 8 kDa (Gln36-Glu109) and 50 kDa (Lys158-Ile543) subunits of the human HPSE were E. coli codon optimized and synthesized by IDT (Australia). The seven PROSS designs were E. coli codon optimized and synthesized by Twist bioscience. The 8 kDa subunit was amplified and sub-cloned into the multiple cloning site 1 of the linearized pETDuet-1 vector (Novagen) through the BamHI and NotI restriction sites (Fast Digest,Thermo) by Gibson one-step isothermal assembly. 63 The resulting plasmid DNA was linearized using NdeI and XhoI restriction enzymes (Fast Digest, Thermo) and designs were inserted into the multiple cloning site 2 by Gibson assembly. 63 The ligated DNA was transformed to E. coli TOP10 cells and the plasmid DNA was extracted and sent to Garvan Institute (Australia) for Sanger sequencing to confirm the sequences. Protein expression and purification The wild type and the 7 designs were transformed in E. coli Shuffle T7 Express cells (NEB), together with different combinations of chaperones in a pACYC vector and spread on an Agar plate with ampicillin and chloramphenicol. 1% overnight seed culture from a single colony was inoculated into 1 L of LB medium supplemented with ampicillin (100 mg L À1 ) and chloramphenicol (34 mg L À1 ), then incubated at 37 1C for 5 hours. Overexpression was induced by adding IPTG to a final concentration of 0.05 mM and the culture was further incubated for 3 hours at 37 1C. The cell pellets were resuspended in buffer A (20 mM HEPES pH 8, 300 mM NaCl, 5 mM b-mercaptoethanol, 10% glycerol, 0.05% Tween, 20 mM Imidazole) with Turbonuclease RSC Chemical Biology Paper (Sigma) and lysed by sonication (Omni Sonic Ruptor 400 Ultrasonic homogenizer). The lysate was filtered (0.45 mm) and loaded onto Ni-NTA column (GE healthcare) and eluted with 100% buffer B (buffer A + 500 mM Imidazole). The peak eluent was diluted 5 times with buffer C (20 mM HEPES pH 7.4, 200 mM NaCl, 1 mM DTT, 10% glycerol, 0.05% Tween20) and loaded to heparin affinity column (GE healthcare) and eluted with 100% buffer D (buffer C + 1.5 M NaCl). The peak eluent was loaded onto a size exclusion column (HiLoad 26/600 Superdex 200 pg, GE healthcare) and eluted in a buffer E (20 mM HEPES pH 7.4, 200 mM NaCl, 10% glycerol, 1 mM DTT, 0.05% Tween20). The final concentration of the monomeric heparinase from the gel filtration was estimated by absorbance at 280 nm using NanoDrop One (Thermo) and the yield was more than 2 mg per litre of LB culture. Colorimetric assay using fondaparinux Assays were conducted using the colorimetric assay designed by Hammond et al. 56 The plates were resealed and developed at 60 1C for 60 minutes, and the absorbance was measured at 584 nm. Kinetics were carried out with a standard curve constructed with D-galactose as the reducing sugar standard, prepared in the same buffer and volume over the range of 0-2 mM. All curve fitting to calculate IC 50 values and Michaelis-Menten constants, was done using GraphPad Prism software (version 8.1). Circular dichroism analysis The size exclusion fraction was directly used to measure the CD using the Chirascan CD spectrometer (Applied Photophysics). The thermal stability of the protein (0.15 mg mL À1 ) was measured in a range of temperatures 20-90 1C by monitoring the ellipticity at 222 nm using a cuvette with 1 mm path length. Data analysis was performed using GraphPad Prism, within which the mid-point of the melting curve was calculated using Boltzmann sigmoid equation. Structure determination and refinement Well diffracted single crystals were obtained by the hangingdrop vapor-diffusion method at 18 1C by combining the protein (6-8 mg mL À1 ) and the well solution (1.9 M (NH 4 ) 2 SO 4 ) with a ratio of 1.5 : 1.5 mL. Crystals appeared within a week and continued to grow for 1-2 months. The crystal was cryoprotected with additional 30% glycerol to the mother liquor before flash freezing in liquid nitrogen. Crystallographic data were collected at 100 K at the Australian Synchrotron (MX2, 64 0.9537 Å). The obtained diffraction data were indexed and integrated with XDS. 65 Resolution estimation and data truncation were performed using aimless program in CCP4 66 on the basis of the datasets overall half-dataset correlation, a CC 1/2 value of 0.3. 67 All structures were solved by molecular replacement using the Molrep program in CCP4 66 using the structure deposited under PDB accession code 5E9M as a starting model. The models were refined using phenix.refine, 68 and the model was subsequently optimized by iterative model building with the program COOT v0.8. 69 The alternative conformations were modelled based on mF o -DF c density and the occupancies and B-factors were determined using phenix.refine. 68 The structures were then evaluated using MolProbity 70 in Phenix. Details of the refinement statistics were produced by Phenix v1.17 71 and summarized in Table S3 (ESI †). The structures were visualized and analysed using PyMol v2.3 72 or Maestro, 73 whereby APBS 58 program in PyMol was used to calculated the electrostatic potential and protein alignment program in Maestro was used to calculate the Ca RMSD. Molecular dynamics Molecular dynamic simulations were performed using the GROMACS 2018.8 engine with parameters from the Charm22* force field. 74,75 All chain termini were capped with neutral acetyl or methylamide groups. Protonation states were assigned with the PDB2PQR server for pH 5.0. 58 Completed structures were solvated with a TIP3P water model 76 using a rhombic dodecahedron simulation box with a minimum distance of 12 Å between the protein and simulation box, followed by the addition of 200 mM NaCl to the aqueous phase and sufficient ions to neutralise the system charge. Simulation systems of WT and PROSS 6 were relaxed using the standard steepest descent minimization using at least 10 000 steps before being equilibrated for 1 ns in the isothermal-isobaric (NPT) ensemble to stabilize the system. Ten replicates of each system were simulated for 100 ns under NPT. Periodic boundary conditions were used, and long-range electrostatics were calculated using the particle-mesh Ewald method with a cutoff of 1.2 nm. 77 Non-bonded interactions were evaluated using a Verlet cut-off scheme. The temperature in all simulations was set to 300 K and controlled via the Bussi-Donadio-Parrinello stochastic velocity rescaling thermostat; 78 the initial velocities of all particles were pseudo-randomly generated. Pressure coupling was handled with the Berendsen barostat during equilibration and the Parrinello-Rahman barostat for production. 79,80 The LINCS (Linear Constraint Solver) algorithm was used to constrain bonds involving hydrogen in conjunction with an integration time step of 2 fs. 81 Constraints were applied to the starting configuration of the production run. Analyses of simulations were preformed using the tools provided in the GROMACS package. Data was collected from the last 90 ns of each production simulation, as RMSF had stabilised by this time. Principal component analysis Principal component analysis was performed using the MDTraj python library and the scikit-learn machine learning tool. 82,83 Paper RSC Chemical Biology Using the aligned and concatenated trajectory, a merged dataset was created, from which the WT and P6 systems were projected. Data was plotted in Graphpad prism. Data availability Coordinates and structure factors have been deposited in the Protein Data Bank under accession code PDB 7RG8. Conclusions This study describes the production of a new variant of HPSE, which is functionally identical to the wild-type protein in terms of activity, inhibition, structure and dynamics that is easily expressed in E. coli and crystallises within a day, yielding high resolution crystals. This protein should make the study of HPSE function and the development of inhibitors significantly easier and less expensive. It is notable that the large number of mutations in HPSE P6 were functionally neutral. This contributes to a growing understanding of the relationship between protein sequence and folding, where evolutionarily conserved and functionally neutral consensus-like mutations can be understood to significantly affect the efficiency of protein folding and expression and protein thermostability. Conflicts of interest CJ has received funding from Beta Therapeutics to work on heparanase inhibitors. Fig. 1 1Native maturation and folding of HPSE in mammalian cells compared with heterologous production in insect or bacterial systems. (A) Fig. 2 2Expression, purification and activity of the successful HPSE P6 (A) Ni-NTA elution fractions (lanes a), heparin column flow in and flow through fractions (lanes b) and size exclusion elution fractions (lanes c). LMW protein marker (GE healthcare) is on the first lane. The sizes corresponding to the two subunits of the HPSE sit at 44 kDa, and 8 kDa, noting that the size of the large subunit is smaller than the previously reported value of 50 kDa due to the lack of glycosylation. (B) Size exclusion chromatography (HiLoad 26/600 Superdex 200 pg, GE Healthcare) shows one major peak corresponding to monomeric HPSE. (C) Kinetics HPSE P6 Fig. 3 3Thermal stability and structural insight of the designed HPSE. (A) The ellipticity at 222 nm was measured using circular dichroism over 20-90 1C, resulting in the melting temperature of 63.6 1C, similar to the values of other engineered proteins by the PROSS algorithm. 46,51,53 (B) The front views of wild type (WT) and designed HPSE (HPSE P6) are calculated using APBS 58 and visualized using PyMol. (C) The superimposed structures of wild type (grey) and HPSE P6 (orange, mutated residues are shown as sticks) are shown as overall (top-left) and detailed views (I-IV). Overall (top-left) and active site view (I) show closely aligned Ca backbones (RMSD of 0.645 Å) and the side chain conformations in the active site. Overall, the mutations reduce the hydrophobicity and increase polarity (II and III), to introduce new hydrogen bonds (black dotted lines, II) and to increase the hydrophobic packing (IV). The Phe258 side chain folds into the hydrophobic packing area (shown as black arrow) with a nearby mutation Ser212Ala causes a loss of an interaction with Thr257. PDB ID: 5E8M (WT), 7RG8 (HPSE P6). Fig. 4 4Molecular dynamic simulations of the wild type the pross design (A) PCA of the two proteins comparing PC1, PC2 and PC3, showing PC3 to have slight differences between the two proteins. (B) RMSF plot of the 95% CI of the wild type, and the mutant average RMSF showing that the mutant stays within the 95% CI, suggesting similar fluctuations. (C) HPSE P6 with average RMSF overlaid. Large RMSF is represented in orange putty, mostly seen around the active site. Mutations are represented as grey spheres. Bovine serum albumin-coated 96 well microplates were used for all assays and were prepared by incubation of the plates with 1% BSA dissolved in phosphate-buffered saline (PBS) with 0.05% Tween-20 (PBST) at 37 1C for 75 minutes. The plates were then washed three times with PBST, dried and stored at 4 1C. Assay mixtures contain 40 mM sodium acetate buffer (pH 5.0), 0.8 nM HPSE in 0.01% Tween 20 sodium acetate buffer and 100 mM fondaparinux (GlaxoSmithKline) with or without increasing concentrations of inhibitor. Plates were incubated at 37 1C for 2-20 hours before the reaction was stopped with 100 mL of 1.69 mM 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3d-benzene disulfonate (WST-1) in 0.1 M NaOH. C . W., N. H. and C. J. J. conceived the study and analysed data. C. W designed variants. N. H. conducted cloning and expression trials. C. W. expressed, purified and crystallised HPSE P6. C. W and N. H. processed crystallography data. N. H. analysed crystal structures. C. W. and J. A. M. performed and analysed molecular dynamics simulations and principal component analysis. C. W., N. H. and C. J. J. wrote the manuscript. All authors provided intellectual input and edited and approved the final manuscript. Table 1 1Kinetic and Inhibition parameters for HPSE WT and P6 proteinsParameter HPSE WT HPSE P6 K M (mM) 11.8 AE 2.7 11.6 AE 2.8 k cat 1.72 AE 0.07 2.94 AE 0.13 IC 50 (nM) 12.5 AE 1.3 12.4 AE 2.5 © 2022 The Author(s). Published by the Royal Society of Chemistry © 2022 The Author(s). Published by the Royal Society of Chemistry RSC Chem. Biol., 2022, 3, 341-349 | 349 AcknowledgementsWe acknowledge the ARC Centre of Excellence for Innovations in Peptide and Protein Science (CE200100012), the ARC Centre of Excellence in Synthetic Biology (CE200100029) and an ARC Linkage Grant (LP160101552). We thank the staff of the MX2 beamline at the Australian Synchrotron, part of ANSTO, which made use of the Australian Cancer Research Foundation (ACRF) detector. The table of contents entry was created with BioRender.com. . S Rivara, F M Milazzo, G Giannini, Future Med. Chem. 8S. Rivara, F. M. Milazzo and G. 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Apoptosis Quantification in Tissue: Development of a Semi-Automatic Protocol and Assessment of Critical Steps of Image Processing Published: 15 October 2021 Juliette De Noiron Laboratoire de Génétique et de Biologie Cellulaire (LGBC) UVSQ Université Paris-Saclay 78000VersaillesFrance Ecole Pratique des Hautes Etudes, PSL Research University 75014ParisFrance Marion Hoareau Laboratoire de Génétique et de Biologie Cellulaire (LGBC) UVSQ Université Paris-Saclay 78000VersaillesFrance Jessie Colin Laboratoire de Génétique et de Biologie Cellulaire (LGBC) UVSQ Université Paris-Saclay 78000VersaillesFrance Ecole Pratique des Hautes Etudes, PSL Research University 75014ParisFrance Present adress: Unité Biologie des ARN des Pathogènes Fongiques, Institut Pasteur F-75015ParisFrance Isabelle Guénal Laboratoire de Génétique et de Biologie Cellulaire (LGBC) UVSQ Université Paris-Saclay 78000VersaillesFrance Apoptosis Quantification in Tissue: Development of a Semi-Automatic Protocol and Assessment of Critical Steps of Image Processing Biomolecules 2021 11Published: 15 October 202110.3390/biom11101523Received: 22 July 2021 Accepted: 12 October 2021biomolecules Article Academic Editor: Stéphen T. Manon Citation: de Noiron, J.; Hoareau, M.; Colin, J.; Guénal, I. Apoptosis Quantification in Tissue: Development of a Semi-Automatic Protocol and Assessment of CriticalSteps of Image Processing. Introduction Apoptosis is a programmed cell death characterized by caspase activation, subsequent dismantling of cell components, including DNA fragmentation, and final phagocytosis of so called "apoptotic bodies" by surrounding cells or macrophages [1]. Importantly, apoptosis is not only critical for correct development of metazoan organisms, but also for their survival. Indeed, apoptosis failure is observed in many diseases including cancers. Therefore, it is widely studied, and new actors are regularly identified. Apoptosis detection can be performed by multiple methods based on various features of apoptotic steps or regulators. Imaging of apoptosis in whole tissues can rely on a more limited number of methods. The first developed and best known among them is TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) which relies on labeling of DNA 3 ends whose numbers increase during the DNA fragmentation step of apoptosis. However, TUNEL is costly, time consuming, and also detects necrotic cells [2]. Alternatively, the use of antibodies raised against cleaved-and thus activated-executioner caspases has proved to be more specific and convenient since immunodetection protocols are less time consuming as they include fewer steps than TUNEL. In mammals, the cleaved form of executioner caspase 3 is often targeted [2]. In Drosophila melanogaster, the antibody used was raised against the executioner caspase Dcp-1 cleaved at Asp 216. This antibody was recently shown to actually detect the cleaved forms of both Dcp-1 and DrICE executioner caspases [3]. Results Choosing a Readout According to the Biological Question The readout is the data used to translate the intensity of the biological effect into numbers. Therefore, the chosen readout should be consistent with the biological question and the tool used to study it. For instance, intensity can be measured to assess the amount of a stained component. In the study of apoptosis rate, regardless of the assay, a cell is apoptotic or not. Thus, quantifying the staining intensity, even if it can somehow make sense, does not seem to be the best option for accurate quantification of apoptosis. Conversely, as long as apoptotic cells can be separated from each other (low apoptosis rate, scattered pattern, or intracellular discrete staining), counting the number of objects equates counting the number of apoptotic cells, which constitutes a valid readout. If this readout cannot be used, another valuable readout is the stained area. These count or area measurements can be used as a readout per se or can be used as primary data for calculation of an apoptotic index or score. For instance, such apoptotic index can correspond to a percentage of apoptotic cells obtained by dividing the number of apoptotic cells (count readout) by the total number of cells in the tissue section. As well, another apoptotic index can be obtained from the stained area by dividing it by the cells number (obtained by plasma membrane or nuclear co-staining), by the area of interest (surface of a cellular clone or of the tissue section). These apoptotic indexes do not indicate the actual number of apoptotic cells, which can be estimated using the count readout, but this number of apoptotic cells is rarely necessary, and those readouts provide estimates that are sufficient to compare the apoptosis rate between different samples. Moreover, in a heterogenous tissue, indicating the percentage of apoptotic cells among a specific cell type usually makes more sense. Here, we used TUNEL and anti-cleaved Dcp-1 to detect apoptosis. These highlight different features of apoptosis as TUNEL labels fragmented DNA in the nuclei while anticleaved Dcp-1 staining is cytosolic. As rbf1 overexpression is a potent apoptosis inducer in the wing imaginal discs, the probability to have clusters of adjacent apoptotic cells is rather high. This can possibly become problematic for accurate quantification. Indeed, when adjacent cells are apoptotic, TUNEL labeling is expected to remain punctiform as nuclei remain spaced by cytoplasms (Figure 1a,c). On the contrary, with anti-cleaved Dcp-1 staining, it is expected that such adjacent apoptotic cells become indistinguishable from each other and thus appear as a single object (Figure 1b,d). Biomolecules 2021, 11, x FOR PEER REVIEW 4 of 19 apoptotic cells is rarely necessary, and those readouts provide estimates that are sufficient to compare the apoptosis rate between different samples. Moreover, in a heterogenous tissue, indicating the percentage of apoptotic cells among a specific cell type usually makes more sense. Here, we used TUNEL and anti-cleaved Dcp-1 to detect apoptosis. These highlight different features of apoptosis as TUNEL labels fragmented DNA in the nuclei while anti-cleaved Dcp-1 staining is cytosolic. As rbf1 overexpression is a potent apoptosis inducer in the wing imaginal discs, the probability to have clusters of adjacent apoptotic cells is rather high. This can possibly become problematic for accurate quantification. Indeed, when adjacent cells are apoptotic, TUNEL labeling is expected to remain punctiform as nuclei remain spaced by cytoplasms (Figure 1a,c). On the contrary, with anti-cleaved Dcp-1 staining, it is expected that such adjacent apoptotic cells become indistinguishable from each other and thus appear as a single object (Figure 1b,d). Therefore, when the readout is the count of apoptotic cells, these clusters of labeled cells are not expected to alter the quantification for TUNEL while they may cause an underestimation of the number of apoptotic cells with anti-cleaved Dcp-1 staining. The extent of this underestimation is difficult to anticipate as it depends on many parameters. Still, this underestimation surely increases with the apoptotic rate-as the probability to have clusters of apoptotic cells increases-which could lead to an artificial flattening of the difference of apoptosis rate that may exist between two conditions. As for the area readout, the size of the wing imaginal disc cells (and their nucleus) being homogenous, the stained area indirectly reflects the number of apoptotic cells without being impacted Therefore, when the readout is the count of apoptotic cells, these clusters of labeled cells are not expected to alter the quantification for TUNEL while they may cause an underestimation of the number of apoptotic cells with anti-cleaved Dcp-1 staining. The extent of this underestimation is difficult to anticipate as it depends on many parameters. Still, this underestimation surely increases with the apoptotic rate-as the probability to have clusters of apoptotic cells increases-which could lead to an artificial flattening of the difference of apoptosis rate that may exist between two conditions. As for the area readout, the size of the wing imaginal disc cells (and their nucleus) being homogenous, the stained area indirectly reflects the number of apoptotic cells without being impacted by their relative localization. In the end, counting cells seems, at least at first sight, a more precise, more direct, readout of apoptosis than area. However, this readout might be altered by apoptotic cell clusters. As it is not possible to anticipate how these clusters will affect the quantification in our experimental set up, we chose to use both count and area readouts. With ImageJ/Fiji, these two readouts can be obtained using the "Analyze Particles" function, which only works on binary 2D images. This means that our image processing protocol should include both z-projection and binarization using a threshold, these two treatments being compatible with our set up. Indeed, as our tissue of interest is a monolayer, z-projection should not affect quantification. Moreover, whether a cell is apoptotic or not, our readouts do not depend on signal intensity and binarization by itself should not affect the quantification. Designing an Image Processing Protocol Steps of image processing directly depend on the chosen readout. In order to get both the number of objects and the stained area, our image processing protocol is based on three major steps: 1. Background noise reduction, 2. Compression of our 3D images into 2D by a z-axis projection, 3. Thresholding. Those steps allow appropriate segmentation required for relevant quantification by the "Analyze Particles" function. Importantly, on ImageJ/Fiji, there are many ways to minimize background signal, several ways of compressing a 3D image in 2D and multiple ways to determine a threshold, resulting in countless combinations of possible image processing. In this study, we investigated the weight of these parameters on signal segmentation to end up with an optimized and unbiased protocol for apoptosis quantification. Median Filter and Size Limitation Efficiently Reduce Artefacts When quantification is automatized, definition of the signal of interest by segmentation is even more critical. Indeed, wrong segmentation can lead to quantification of unreal objects and thus give useless results. Thus, the signal of interest boundaries has to be better defined while background noise has to be decreased. General background noise can be minimized in many ways depending on the kind of images, the desired readout, and the defaults faced. In our case, mandatory use of a threshold would blacken every low intensity pixel constitutive of the general background noise. However, if binarization of the image efficiently removes diffuse low intensity background noise, it is not sufficient to erase artifactual pixels with aberrant high intensity, i.e., whose intensity is higher than the threshold value. Fortunately, isolated aberrant high pixels can be dealt with filters. Filters are matrix operations that re-calculate a pixel intensity value based on itself and its neighbors. The two mainly used filters are "Mean Filter" and "Median Filter" (Figure 2A). A "Mean Filter" with a radius of 1 gives a pixel an intensity value that corresponds to the mean of its value and those of its direct neighbors (Figure 2A(b)). Hence, the value of an isolated pixel with aberrant high intensity is attenuated by the intensity value of its neighbors. However, as extreme values impact mean calculation, every neighboring pixel is affected by the isolated aberrant high pixel and their intensity is artificially increased (Figure 2A(b)). By contrast, a "Median Filter" with a radius of 1 gives to a pixel an intensity value corresponding to the median of its value and those of its direct neighbors (Figure 2A(c)), which is expected to be much closer to local intensity value. Besides, as extreme values effect on median calculation is low, high intensity isolated pixel impact on its neighbors is negligible. Overall, the "Mean Filter" tends to spread an aberrant high value whereas a "Median Filter" tends to confine it. This effect is illustrated in Figure 2B, where the "1" arrows of the "No filter" panel show typical isolated aberrant high pixels that are efficiently erased by a "Median Filter" with a radius of 1 ( Figure 2B compare (b) and (c)). Aside from this benefit, the area pointed by the "2" arrow exemplifies the median filter ability to preserve edges of an object. Indeed, on the illustration, human eyes easily detect that the "2" arrow targets a marked cell ( Figure 2B(a)). However, this object is heterogeneous: in a restricted space, it contains few pixels of high intensity and many pixels of low intensity (i.e., below the chosen threshold). Without any filter, only high intensity pixels are kept after the thresholding step thereby fragmenting this object in several small groups of pixels ( Figure 2B(b)). Thus, with no further treatment, multiple objects will be counted in this area, which does not reflect reality. However, as these high intensity pixels are close to each other, the "Median Filter" with a radius of 1 homogenizes intensity values within this object. This allows its reconstruction and gives a segmentation consistent with reality ( Figure 2B(c)). Thus, when the "Median Filter" with a radius of 1 is applied, the "number of objects" decreases only to be closer to what a human eye would count. A "Mean Filter" with a radius of 1 gives a pixel an intensity value that corresponds to the mean of its value and those of its direct neighbors (Figure 2A(b)). Hence, the value of an isolated pixel with aberrant high intensity is attenuated by the intensity value of its neighbors. However, as extreme values impact mean calculation, every neighboring pixel is affected by the isolated aberrant high pixel and their intensity is artificially increased (Figure 2A(b)). By contrast, a "Median Filter" with a radius of 1 gives to a pixel an intensity value corresponding to the median of its value and those of its direct neighbors (Figure (c) same as (b) but a "Median Filter" with a radius of 1 was applied to the image before Max Intensity z-projection. White bar corresponds to 10 µm. Arrows with circled numbers 1, 2, and 3 target areas of interest. The number of visible objects in each panel was manually counted. Although the "Median Filter" with a radius of 1 efficiently reduces the number of artifactual objects by erasing isolated high pixels, the issue of groups of pixels with an intensity higher than the threshold value remains. A way to eliminate most of those artifacts is to limit our analysis to objects with a size consistent with the smallest biological object of interest. In our case, this smallest biological object is a TUNEL-labeled nucleus, we assessed their size on a few random images and thus set a size limit at 2 µm. Importantly, this ">2 µm size limitation" fits our data but should not be taken as a default value and must be adapted for other kinds of signals or cell types. Moreover, as the "Analyze Particles" function records the size of every object, this filtering can be carried out after quantification. In Figure 2B, the ">2 µm limitation" eliminates artifactual objects pointed out by the "3" arrow as well as individual pixels such as those pointed by the "1" arrows. It thus appears very powerful in order to "clean" the image. However, as efficient as the size limitation may be, it cannot replace "Median filter". Indeed, as already explained, in the absence of a "Median Filter", the cell indicated by the "2" arrow in Figure 2B becomes fragmented in several small groups of pixels, each one being smaller than 2 µm ( Figure 2B(b)). Thus, without the "Median Filter", these pixels are eliminated by the ">2 µm limitation" and the actually labeled cell indicated by the "2" arrow is not included in the quantification of the apoptotic signal. Here, reconstruction of the object by the "Median Filter" prevents its elimination by the ">2 µm limitation" ( Figure 2B(c)). In the end, combination of a "Median Filter" with a radius of 1 and ">2 µm limitation" allows a better segmentation and a more accurate quantification. Max Intensity Z-Projection Improves Contrast Confocal microscopy gives the possibility to capture objects in 3D. However, image processing often requires transforming volumes into 2D images by compressing the z-axis. In our case, the "Analyze Particles" function used to quantify the signal of interest requires 2D images. Flattening a 3D volume can seem counterproductive, as separate objects on the same z-axis will be reduced to a single one on the final 2D image. In our case, this is unlikely to happen since imaginal disc cells are organized in a monolayer with only limited folding and this can also be true for tissue sections as long as they are thin enough. In the case of a multilayer tissue, z-projection can still be used if the apoptosis rate is low enough to prevent the occurrence of having multiple apoptotic cells in the same z-axis. Projection consists of compressing the signal contained in every pixel of a z-axis into a single one. With ImageJ/Fiji, projection can be performed in several ways but two of them are mainly used. The first method, Average Intensity (AI), calculates the average intensity of all the pixels of a z-axis. The second, Max Intensity (MI), only retains the maximal intensity value along the z-axis. Figure 3 presents examples of these projection methods on a virtual object without any other treatment (i.e., median filter). The z-axis presented in "A" shows only one illuminated pixel on slice #2 and this pixel is included in the object. The "B" z-axis shows numerous pixels highly illuminated, comprised in the object. The "C" z-axis presents an artifactually illuminated pixel on slice #2 that is not comprised in the object. Comparison of the projection methods shows that AI projection decreases the importance of the artifactual pixel of the "C" z-axis, while the MI projection increases its weight. However, in the processing protocol, the preceding use of a "Median Filter" with a radius of 1 eliminates most of those artefacts that are thus not present anymore at the projection step. Conversely, the AI projection of the "A" z-axis leads to loss of signal even if it is part of the object. Furthermore, with the AI projection, the contrast between object and background is weak, therefore the range for the appropriate threshold value is limited (Figure 3). After a MI projection, contrast is enhanced and thus threshold determination is easier for the experimenter, which helps limiting the experimenter bias. This is particularly important for signals with low contrast such as TUNEL. In our case, these two projection methods do not end up in drastically different results but, overall, MI projection presents more benefits than AI projection. The z-axis presented in "A" shows only one illuminated pixel on slice #2 and this pixel is included in the object. The "B" z-axis shows numerous pixels highly illuminated, comprised in the object. The "C" z-axis presents an artifactually illuminated pixel on slice #2 that is not comprised in the object. Comparison of the projection methods shows that AI projection decreases the importance of the artifactual pixel of the "C" z-axis, while the MI projection increases its weight. However, in the processing protocol, the preceding use of a "Median Filter" with a radius of 1 eliminates most of those artefacts that are thus not present anymore at the projection step. Conversely, the AI projection of the "A" z-axis leads to loss of signal even if it is part of the object. Furthermore, with the AI projection, the contrast between object and background is weak, therefore the range for the appropriate threshold value is limited (Figure 3). After a MI projection, contrast is enhanced and thus threshold determination is easier for the experimenter, which helps limiting the experimenter bias. This is particularly important for signals with low contrast such as TUNEL. In our case, these two projection methods do not end up in drastically different results but, overall, MI projection presents more benefits than AI projection. Use of Custom Manual Thresholds Gives the Best Segmentation for Relevant Quantification The "Analyze Particles" function used for quantification requires the image to be binary. The transition from a greyscale image to a black and white image involves the setting of a threshold that defines an intensity value above which a pixel is turned to white and under which a pixel is turned to black. Ideally, this value should enable to get an image where white only corresponds to the signal of interest. Thresholding is the last step of segmentation and finally defines the objects of interest, which is critical for accurate quantification. Therefore, among the steps of image processing, it is the one that has the most dramatic effect on quantification accuracy, thus we dedicated particular attention to the threshold determination method. Threshold can be automatically set by algorithms that analyze specific features of the image intensity histogram to determine a threshold value using either simple indicators such as the mean, maximal, or minimal intensity values, or more complex formulas. Hence, algorithms appear as an unbiased method to obtain a specific threshold value per image. We thus wondered whether any of the 16 thresholding algorithms currently available in the menu Image > Adjust > Threshold of ImageJ/Fiji could be used to determine a threshold that properly segments apoptotic signal in our images. Using some randomly chosen images, after application of a "Median filter" with a radius of 1 and a Max Intensity projection, we visually checked if these algorithms could provide a threshold value allowing a relevant segmentation (i.e., consistent with apoptotic signal). Most algorithms did not pass the visual inspection step as they yielded unrealistic segmentation either by ignoring a great portion of the signal or by including artifactual signal. However, two of them, Otsu [15] and Moments [16], seemed capable of discriminating actual apoptotic staining from background. We then performed a more detailed analysis of the threshold values obtained with these algorithms by comparing them to the ones obtained by experimenters. To this end, for the 28 images of the vg > rbf1 genotype, three experimenters determined the threshold to use for each staining (TUNEL or anti-cleaved Dcp-1) by eye and in triplicate (see Supplementary Figure S1). The criteria for manual threshold determination were to set a threshold value that allows the positive signal to be consistent with the original image and located in the vestigial domain. Thresholds for these images were also determined using the 16 algorithms. As expected, algorithms inducing obvious unrealistic segmentation of the apoptotic signal yielded threshold values very far from the range of the ones determined by experimenters (Figure 4a,b; compare IsoData and Intermode with Exp. Data not shown). On the contrary, Moments, Otsu, and experimenter threshold values are in the same range (Figure 4a,b). From this global analysis, it could seem that algorithms can be as efficient as human eye for threshold determination (compare for instance Otsu and Exp in Figure 4b). However, visually, Otsu capability to determine a relevant threshold seemed irregular. We thus further deepened our data analysis and compared the threshold values obtained not globally but for each image. As shown in Figure 4c,d, the values obtained with Otsu, if they tend to be roughly the same on average than the ones obtained by experimenters, are actually most of the time out of the range of experimenters values. This is particularly striking for images obtained from TUNEL (Figure 4c) as Otsu's determined values are far higher or lower that the ones obtained by any experimenter. This would not be an issue as long as the threshold values obtained still allow a realistic segmentation of apoptotic signal and subsequent relevant quantification. However, such deviation of the threshold value results in an inappropriate segmentation (compare Figure 4e,f), that necessarily ends in a biased or most likely totally wrong quantification. When it comes to Moments, it provides threshold values that are usually lower than experimenters' ones ( Figure 4a,b), which means that using this algorithm tends to include some background noise to the quantification. The question resides then in determining whether this amount of background noise is important enough to alter quantification. In the case of TUNEL staining (Figure 4c), values are quite low, thus it certainly affects quantification rather significantly. By contrast, when images come from anti-cleaved Dcp-1 staining, the threshold values given by Moments are much closer to the ones obtained by experimenters, they actually seem very similar to the lowest values determined by experimenters (Figure 4d). Therefore, one could assume that the variability of threshold values between Moments and an experimenter is comparable to the one that exists between experimenters. We tested this by comparing the relationship between the two most distant experimenters' batch of measurements to the one between Moments and its closest dataset. As shown in Supplementary Figure S2B, if two experimenters do not determine exactly the same value for the threshold, their estimates remain consistent with each other (p = 10 −5 and R 2 = 0.53 for the most distant measurements), the difference can be more or less described as a given experimenter tending to set thresholds always lower than the other. This is a systematic error that should affect quantification only moderately. On the contrary, threshold values determined by Moments are not consistent with the values of experimenters (p = 0.8 for the closest in Supplementary Figure S2B). This indicates that Moments can set a low threshold value when all experimenters would have chosen a higher one, but it is not always the case, and most of all, the extent of this underestimation (i.e., the range of the difference between experimenters and Moments' threshold values) is variable. This is more problematic as the extent of background incorporation in the quantification will then vary and might alter quantification relevance. Contrary to algorithms, manual determination of the threshold values appears quite robust. Indeed, comparison of manually determined threshold values between experimenters for individual images shows a low variability and a good reproducibility both between several determinations of a given experimenter and between experimenters (Supplementary Figure S1). As all images from an experiment are acquired identically, with the same microscope settings and come from samples treated with the same solutions, at the same time, theoretically, the appropriate threshold value is expected to be the same for every image. Moreover, using the same unique threshold value for all images can be considered as more objective and unbiased. However, manually determined threshold values display some variability (Figure 4, Exp). This can justify using a distinct individual, manually determined, threshold value for each image as it might enable a more accurate segmentation and subsequent quantification. In order to assess to which extent these two thresholding methods (unique threshold value for every image or individual, manually determined threshold value per image) can affect quantification and detection of our biological effect, we tried both ( Figure 5). In the Manual condition, each image was binarized using its own manually determined threshold value (determined by experimenter 1, measure 3). From these individual threshold values, we calculated the median value per genotype and then the median of these medians. This latter value was used as the unique threshold value to binarize all images in the Total condition (1128 for TUNEL and 777 for anti-cleaved Dcp-1). We decided to use the median of the medians per genotype rather than the global median (calculated from the whole of the images independently of their genotype) to avoid giving more weight to a genotype (that might have a larger headcount for instance). For TUNEL stained images, both thresholding approaches give a workable quantification both for count and area readouts (Figure 5e,g). Indeed, a significant decrease in apoptosis between the two genotypes is detected in all cases. However, even if Total or Manual thresholding methods enable to detect the biological effect, we noticed that quantification is still somehow altered when a unique threshold is used (Supplementary Figure S3). As for anti-cleaved Dcp-1-stained images, the first observation is that the count readout was not usable. Indeed, we knew that apoptotic cells clusters might alter quantification as these clusters might be considered as a single object. Moreover, such underestimation is enhanced when the apoptosis rate rises, eventually leading to the flattening of the difference between two samples. However, we chose to keep this readout, as it was not possible to anticipate the extent of this phenomenon in our samples. As shown in Figure 5f, with this readout, the difference in the apoptosis rate between the two genotypes becomes undetectable. This indicates that the level of apoptosis induced by rbf1 generates apoptotic cells clusters frequently enough to significantly alter quantification, and this, whatever the thresholding method, thus prohibiting the use of the count readout. By contrast, when anti-cleaved Dcp-1 staining is quantified using the area readout, the difference between the two thresholding methods (unique versus individual thresholds) becomes obvious. As shown in Figure 5h, when a unique threshold value is used for all images (Total), the difference of apoptosis rate between the two genotypes is barely detectable (p = 0.033). Moreover, even with an individual, manually determined threshold value per image, all the quantification results show a noticeable variability ( Figure 5, Manual) due to differences between specimen, variation of the UAS/gal4 system intensity and small asynchrony in the development of the larvae. However, use of a unique threshold value to process every image tends to increase this variability and induces the appearance of extreme values compatible with an overestimation due to inadequate segmentation (see highest values for vg > rbf1, debcl E26 genotype in Figure 5h and also Supplementary Figure S3). On the contrary, the use of individual specific threshold values (Manual) enables to readily detect the difference of apoptosis rate between the two genotypes (p = 1.5 × 10 −5 ). In the end, this analysis shows that, in our case, using an individual threshold per image is more appropriate and turns out to be the safest option for accurate segmentation, and thus relevant quantification. > rbf1 (a,c) and vg > rbf1, debcl E26 (b,d) genotypes. White bar corresponds to a 50 µm scale. "Count" panel presents quantification of TUNEL (e) and anti-cleaved Dcp-1 (f) signal based on the counting of the number of objects according to the different thresholding methods (Manual and Total) for vg > rbf1 (in pink) and vg > rbf1, debcl E26 (in blue). "Area" panel presents quantification of TUNEL (g) and anti-cleaved Dcp-1 (h) signal based on the number of white pixels (stained area) according to the different thresholding methods (Manual, or Total) for vg > rbf1 (in pink) and vg > rbf1, debcl E26 (in blue). Manual indicates a custom dedicated threshold was used for each image whereas Total indicates that the same common unique threshold value was used to process all images. This unique threshold value is the median of the median per genotype of the manually determined values. p-values displayed above results were obtained using Wilcoxon tests. Quantifications of TUNEL or Anti-Cleaved Dcp-1 Stainings Do Not Have the Same Requirements TUNEL and anti-cleaved caspase stainings are widely used to assess the level of apoptosis in tissues. However, depending on the experimental set-up, the quantification step may become delicate. TUNEL appears as a quite robust apoptosis detection technique. Indeed, it allows to quantify apoptosis and to detect differences in apoptosis rate whatever the thresholding approach, and with both count and area readouts (Figure 5e,g). This was not totally expected since in our images, there was not a strong contrast between the apoptotic signal and the background (Figure 5a,b). However, as previously mentioned, TUNEL assay is costly, time consuming and lacks specificity as it also detects necrotic cells. On the contrary, using antibodies against cleaved caspase(s) is considered as a more specific and convenient staining of apoptotic cells. By contrast with TUNEL which labels nuclei, caspase staining covers the whole volume of the cell, raising the issue of adjacent apoptotic cells when the readout is counting cells. Indeed, counting the number of apoptotic cells stained with anti-cleaved caspase antibody is perfectly possible as long as apoptotic cells are sufficiently separated from each other. In our experimental set-up, it appeared that the apoptosis rate was too high to prevent the underestimation of the signal due to fusion in a single object of clustered apoptotic cells. When the stained area was measured, it revealed that images from anti-cleaved Dcp-1 stainings should be carefully processed because determination of the threshold value to use for binarization is particularly important. Indeed, even if the difference of apoptotic rate between the two genotypes was known and easily seen by eye ( Figure 5, compare (c) and (d)), its detection after quantification was not obvious. Actually, the decrease in apoptosis between the two genotypes is barely detected when a unique threshold value is used for the segmentation of all images (Figure 5h) whereas using a specific threshold value for each image enables to see it. Therefore, anticleaved Dcp-1 staining quantification is more affected by image processing than TUNEL and should be handled more carefully. Macro Explanation The macro consists of two parts described in Figure 6, the first part allows determination of threshold values, and the second part allows zone selection and quantification. image, thus, even if two objects (in our case two wing imaginal discs) are in the same field and can be captured in the same image, it is highly important to capture this field twice. The macro consists of two parts described in Figure 6, the first part allows determination of threshold values, and the second part allows zone selection and quantification. Figure 6. Fiji macro workflow. Giving our image processing protocol, the macro is divided in two major parts. Part 1 (left panel) is dedicated to manual threshold determination while Part 2 (right panel) is dedicated to zone selection and quantification after application of the previously determined threshold. In Part 1, images are opened, processed according to Figure 6. Fiji macro workflow. Giving our image processing protocol, the macro is divided in two major parts. Part 1 (left panel) is dedicated to manual threshold determination while Part 2 (right panel) is dedicated to zone selection and quantification after application of the previously determined threshold. In Part 1, images are opened, processed according to parameters set in the window presented in Figure 7 and presented to user for threshold determination. Once every image of a .lif file has been treated in Part 1, threshold results are recapitulated before starting Part 2. In Part 2, images are opened again and treated as in Part 1, chosen threshold is applied and the resulting image is presented to user for zone selection before quantification. When every relevant image has been treated, quantification results are superficially analyzed to yield mean, standard deviation, min, and max. before quantification. When every relevant image has been treated, quantification results are superficially analyzed to yield mean, standard deviation, min, and max. Once a .lif file has been chosen, the window presented in Figure 7 opens to set a few parameters such as selecting the channels of interest and the methods to use for background noise reduction and z-projection. A more detailed explanation of the macro is provided in the Supplementary Materials. Figure 7. Macro Part 1. The macro starts by collecting few parameters. Importantly, the chosen "Condition name" will end up in the name of the files associated with the .lif file. The default background noise reduction method is a "Median Filter" with a radius of 1 but user can apply its own protocol before and/or after z-projection if needed. As a thresholded image is in black and white, it can hardly be used to define region of interest when a zone selection is needed. Therefore, the macro offers the possibility to define region of interest (i.e., vestigial domain in our case) on another channel or an unthresholded version of channel of interest. If the whole image has to be analyzed, a "No selection needed" option is available. Concerning threshold determination, "A. Using an algorithm" and "B. Manual determination" options will lead to application of an individual threshold value per image. If use of a unique, representative, threshold value to treat every image of a .lif file is wanted, user can make its choice after treating every image (option "C"), 50% of the images (option "D"), 25% of the images (option "E") or 10% of the images (option "F"). If manual determination has already been carried out, the user may skip Part 1 with option "G". Once chosen, those parameters are recorded in the Log window which content is ultimately saved in a .txt file. Figure 7. Macro Part 1. The macro starts by collecting few parameters. Importantly, the chosen "Condition name" will end up in the name of the files associated with the .lif file. The default background noise reduction method is a "Median Filter" with a radius of 1 but user can apply its own protocol before and/or after z-projection if needed. As a thresholded image is in black and white, it can hardly be used to define region of interest when a zone selection is needed. Therefore, the macro offers the possibility to define region of interest (i.e., vestigial domain in our case) on another channel or an unthresholded version of channel of interest. If the whole image has to be analyzed, a "No selection needed" option is available. Concerning threshold determination, "A. Using an algorithm" and "B. Manual determination" options will lead to application of an individual threshold value per image. If use of a unique, representative, threshold value to treat every image of a .lif file is wanted, user can make its choice after treating every image (option "C"), 50% of the images (option "D"), 25% of the images (option "E") or 10% of the images (option "F"). If manual determination has already been carried out, the user may skip Part 1 with option "G". Once chosen, those parameters are recorded in the Log window which content is ultimately saved in a .txt file. Once a .lif file has been chosen, the window presented in Figure 7 opens to set a few parameters such as selecting the channels of interest and the methods to use for background noise reduction and z-projection. A more detailed explanation of the macro is provided in the Supplementary Materials. Once these parameters are set, the macro opens the first image (that should be a stack) and process it according to the chosen parameters for background noise reduction and zprojection. In our case, a "Median Filter" with a radius of 1 is applied and the Max Intensity method is used for z-projection. At this point, threshold can be manually determined on the processed images. Part 1 is over when all images have been processed and as it generates a table of the determined thresholds, Part 2 can be run right after or later. Part 2 begins with the opening of the window presented in Figure 8, which offers the possibility to use the threshold value previously determined in Part 1 and to apply additional limitations to quantification such as a size limitation. Once these parameters are set, the macro opens the first image (that should be a stack) and process it according to the chosen parameters for background noise reduction and z-projection. In our case, a "Median Filter" with a radius of 1 is applied and the Max Intensity method is used for z-projection. At this point, threshold can be manually determined on the processed images. Part 1 is over when all images have been processed and as it generates a table of the determined thresholds, Part 2 can be run right after or later. Part 2 begins with the opening of the window presented in Figure 8, which offers the possibility to use the threshold value previously determined in Part 1 and to apply additional limitations to quantification such as a size limitation. The one presented here corresponds to option "B. Manual determination". If option "A. Using an algorithm" was chosen, the window would start by asking which algorithm should be used. If any of options "C", "D", "E", or "F" were chosen, the window would start by asking which unique threshold value should be used. The following parameters (how to retrieve the results, objects' size, objects' circularity and range of series to analyze) are always present. Once chosen, those parameters are recorded in the Log window which content is ultimately saved in a .txt file. The one presented here corresponds to option "B. Manual determination". If option "A. Using an algorithm" was chosen, the window would start by asking which algorithm should be used. If any of options "C", "D", "E", or "F" were chosen, the window would start by asking which unique threshold value should be used. The following parameters (how to retrieve the results, objects' size, objects' circularity and range of series to analyze) are always present. Once chosen, those parameters are recorded in the Log window which content is ultimately saved in a .txt file. Depending on the study, the quantification needed can be more or less detailed. Indeed, one might need to compare objects size within an image whereas in our case the total stained area per image is sufficient. Both options are available on the macro and are not mutually exclusive. Once these settings are carried out, images are processed as in Part 1 except that the chosen threshold is applied. After thresholding, Part 2 will quantify but quantification can be restricted to a region of interest. This region can be drawn on the image of the channel indicated in the Part 1 window. If the thresholding is carried out using an algorithm, the zone selection is carried out before running the algorithm. Indeed, this prevents a highly illuminated artifact outside of the region of interest to weigh in the threshold value determination by the algorithm. To allow a wide range of application for this macro, we have chosen to ask the "Analyze Particles" function to quantify all the possible readouts. Conclusions Apoptosis quantification in a tissue is usually indirect as it generally relies on imaging techniques. There are many ways to analyze an image and once a readout has been chosen, many processing protocols are possible. Here, we describe a semi-automatic protocol running on Fiji for quantification of apoptosis on Drosophila wing imaginal discs after TUNEL or activated-caspase labelings. During the development of this protocol, we paid particular attention to the weight of specific steps to obtain a realistic segmentation, which underlies an accurate quantification. As with many image processing protocols, determination of the threshold for binarization turned out to be a critical step. In our case, none of the algorithms we tested was satisfying to determine relevant thresholds. We also considered using the same threshold value to process several images but, in the end, the best option for our data, was to use a specific threshold manually determined for each image. Indeed, this method proved to carry out a proper segmentation for all images resulting in valid quantification and subsequent detection of biological effects. Even if one could be concerned about the bias that might be induced by this approach, the bias is in fact limited as we observed that threshold values obtained by experimenters are actually highly consistent both for a given experimenter and between experimenters. Moreover, an appropriate processing of the images can facilitate this determination of a threshold value. In this sense, association of a "Median Filter" with a radius of 1 and a Max Intensity z-projection proved to be highly efficient. It would also be interesting to try the "Sum Slices" projection that adds up all pixels' intensity of a z-axis which should enhance contrast even more (particularly after a median filter) and thus facilitate threshold determination. The protocol presented here should not affect other readouts available in the "Analyze Particles" function such as: bounding rectangle, shape descriptors, centroid, perimeter, Feret's diameter, or stack position. Furthermore, we kept the options implemented in CASQITO macro rather open offering a possible use of this tool for a great variety of readouts, stainings, and biological questions. Figure 1 . 1Effect of different types of stainings on the "Count" readout. (a) and (b) scheme showing a virtual cluster of four apoptotic cells. In (a), green spots represent nuclei stained by TUNEL while in (b), red patches represent cytosols stained by anti-cleaved Dcp-1. (c) and (d) schemes present the result of image processing for these signals. Figure 1 . 1Effect of different types of stainings on the "Count" readout. (a,b) scheme showing a virtual cluster of four apoptotic cells. In (a), green spots represent nuclei stained by TUNEL while in (b), red patches represent cytosols stained by anti-cleaved Dcp-1. (c,d) schemes present the result of image processing for these signals. Figure 2 . 2Filters effect on background noise and segmentation. (A): The grid in (a) presents intensity values of a 3 × 3 pixels image. The grid in (b) depicts how a "Mean Filter" with a radius of 1 affects the central pixel of the original image (left) and the whole image (bottom right). In order to make intensity differences more visible, each boxes background color corresponds to the double of each pixel intensity value in greyscale. (B): (a) image of a portion of a TUNEL-labeled wing imaginal disc after a Max Intensity z-projection. (b) same as (a) after binarization using a manually determined threshold. (c) same as (b) but a "Median Filter" with a radius of 1 was applied to the image before Max Intensity z-projection. White bar corresponds to 10 µ m. Arrows with circled numbers 1, 2, and 3 target areas of interest. The number of visible objects in each panel was manually counted. Figure 2 . 2Filters effect on background noise and segmentation. (A): The grid in (a) presents intensity values of a 3 × 3 pixels image. The grid in (b) depicts how a "Mean Filter" with a radius of 1 affects the central pixel of the original image (left) and the whole image (bottom right). In order to make intensity differences more visible, each boxes background color corresponds to the double of each pixel intensity value in greyscale. (B): (a) image of a portion of a TUNEL-labeled wing imaginal disc after a Max Intensity z-projection. (b) same as (a) after binarization using a manually determined threshold. Figure 3 . 3Projection methods. The left panel presents different planes obtained by imaging a virtual object surrounded by a perfect background noise of 0. On the right, the upper panel presents the coordinates of three pixels: A, B, and C, and their respective intensity values along z-axis. Bottom panels show the resulting projection obtained either by an Average Intensity or a Max Intensity projection with respective intensity values obtained for A, B, and C pixels. For all representations, boxes background color corresponds to their pixel intensity in grey scale. Figure 3 . 3Projection methods. The left panel presents different planes obtained by imaging a virtual object surrounded by a perfect background noise of 0. On the right, the upper panel presents the coordinates of three pixels: A, B, and C, and their respective intensity values along z-axis. Bottom panels show the resulting projection obtained either by an Average Intensity or a Max Intensity projection with respective intensity values obtained for A, B, and C pixels. For all representations, boxes background color corresponds to their pixel intensity in grey scale. Figure 4 . 4Comparison of thresholding methods. Upper panel presents overall distribution of threshold values per image obtained with various thresholding methods for images of vg > rbf1 genotype. (a) and (b) Exp distribution (in yellow) corresponds to the whole of the threshold values determined by three experimenters in triplicate (raw data are presented in Supplementary Figure S1). Intermode (in light blue) and IsoData (in green) are examples of algorithms yielding inadequate values, very far from experimenters' distribution. Otsu (in brown) and Moments (in blue) are algorithms that seemed usable. (c) and (d) show a pairwise organization of the threshold values for each image obtained by Otsu (in brown), Moments (in blue) and experimenters (Exp, the black line corresponds to the mean of the nine values determined the experimenters and the yellow bars correspond to the range of the threshold values determined by the experimenters for each image). Images were ranked according to the mean of the experimenter values (black line). Bottom panel illus- Figure 4 . 4Comparison of thresholding methods. Upper panel presents overall distribution of threshold values per image obtained with various thresholding methods for images of vg > rbf1 genotype. (a,b) Exp distribution (in yellow) corresponds to the whole of the threshold values determined by three experimenters in triplicate (raw data are presented in Supplementary Figure S1). Intermode (in light blue) and IsoData (in green) are examples of algorithms yielding inadequate values, very far from experimenters' distribution. Otsu (in brown) and Moments (in blue) are algorithms that seemed usable. (c,d) show a pairwise organization of the threshold values for each image obtained by Otsu (in brown), Moments (in blue) and experimenters (Exp, the black line corresponds to the mean of the nine values determined the experimenters and the yellow bars correspond to the range of the threshold values determined by the experimenters for each image). Images were ranked according to the mean of the experimenter values (black line). Bottom panel illustrates the result of the binarization using the threshold values determined by experimenters (e,g), Otsu (f) and Moments (h). Black arrows of the middle panels target the image used for illustrations presented on the bottom panel. Importantly, these images were chosen as they are representative of the deviation between the algorithm and the experimenter average value (chosen images have a deviation equal to the median of the deviations). Figure 5 . 5Impact of thresholding on quantification. TUNEL and anti-cleaved Dcp-1 staining quantifications. Upper panel shows representative images of wing imaginal discs stained by TUNEL ((a) and (b)) or anti-cleaved Dcp-1 ((c) and (d)) for vg > rbf1 ((a) and (c)) and vg > rbf1, debcl E26 ((b) and (d)) genotypes. White bar corresponds to a 50 µm scale. "Count" panel presents quantification of TUNEL (e) and anti-cleaved Dcp-1 (f) signal based on the counting of the number of objects according to the different thresholding methods (Manual and Total) for vg > rbf1 (in pink) and vg > rbf1, debcl E26 (in blue). "Area" panel presents quantification of TUNEL (g) and anti-cleaved Dcp-1 (h) signal based on the number of white pixels (stained area) according to the different thresholding methods (Manual, or Total) for vg > rbf1 (in pink) and vg > rbf1, debcl E26 (in blue). Manual indicates a custom dedicated threshold was used for each image whereas Total indicates that the same common unique threshold value was used to process all images. This unique threshold value is the median of the median per genotype of the manually determined values. p-values displayed above results were obtained using Wilcoxon tests. Figure 5 . 5Impact of thresholding on quantification. TUNEL and anti-cleaved Dcp-1 staining quantifications. Upper panel shows representative images of wing imaginal discs stained by TUNEL (a,b) or anti-cleaved Dcp-1 (c,d) for vg Figure 8 . 8Macro Part 2. Starting window of Part 2 lightly differs according to the choice of threshold method made in Part 1. Figure 8 . 8Macro Part 2. Starting window of Part 2 lightly differs according to the choice of threshold method made in Part 1. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/biom11101523/s1, Figure S1: Individual experimenters' choice of threshold values, Figure S2: Statistical analysis of threshold values consistency, Figure S3: Pairwise comparison of thresholding methods, Supplementary information: User manual of the macro. Author Contributions: Conceptualization, J.d.N., J.C. and I.G.; methodology, J.d.N., J.C. and I.G.; software, J.d.N.; validation, J.d.N., M.H., J.C. and I.G.; formal analysis, J.d.N. and J.C.; investigation, J.d.N. and M.H.; writing-original draft preparation, J.d.N.; writing-review and editing, J.d.N., M.H., J.C. and I.G.; visualization, J.d.N., J.C. and I.G.; supervision, J.C. and I.G.; project administration, I.G.; funding acquisition, I.G. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by UVSQ, EPHE and private funding collected by the UVSQ Foundation. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Biomolecules 2021, 11, x FOR PEER REVIEW 17 of 19 Acknowledgments:We thank Vincent Scarcelli for his participation to thresholds determination, Aude Jobart Malfait for helpful discussions, Christine Wintz for technical assistance and Vincent Rincheval for his critical reading of the manuscript. Confocal microscopy was performed at the CYMAGES imaging facility (UVSQ/University of Paris-Saclay).Conflicts of Interest:The authors declare no conflict of interest.Data Availability Statement:The data that support the findings of this study are available from the corresponding authors, I.G. and J.C., upon reasonable request. . J G Nirmala, M Lopus, 10.1007/s10565-019-09496-2Cell Death Mechanisms in Eukaryotes. Cell Biol. Toxicol. 2020PubMedNirmala, J.G.; Lopus, M. 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Protoc. 13PubMedDaniel, B.; DeCoster, M.A. Quantification of SPLA2-Induced Early and Late Apoptosis Changes in Neuronal Cell Cultures Using Combined TUNEL and DAPI Staining. Brain Res. Protoc. 2004, 13, 144-150. [CrossRef] [PubMed] Quantification of TUNEL Assay in Hippocampus of Diabetic Rats by MAT LAB: Comparison with Stereological Method. S Ahmadpour, M A Y Heravi, 10.4172/2161-0681.1000108J. Clin. Exp. Pathol. 2Ahmadpour, S.; Heravi, M.A.Y. Quantification of TUNEL Assay in Hippocampus of Diabetic Rats by MAT LAB: Comparison with Stereological Method. J. Clin. Exp. Pathol. 2012, 2, 1-3. [CrossRef] An Open-Source Platform for Biological-Image Analysis. J Schindelin, I Arganda-Carreras, E Frise, V Kaynig, M Longair, T Pietzsch, S Preibisch, C Rueden, S Saalfeld, B Schmid, 10.1038/nmeth.2019Nat. Methods. 9PubMedSchindelin, J.; Arganda-Carreras, I.; Frise, E.; Kaynig, V.; Longair, M.; Pietzsch, T.; Preibisch, S.; Rueden, C.; Saalfeld, S.; Schmid, B.; et al. 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Efficacy and safety of a third-generation oncolytic herpes virus G47D in models of human esophageal carcinoma Shoh Yajima Division of Innovative Cancer Therapy Advanced Clinical Research Center Institute of Medical Science The University of Tokyo TokyoJapan Department of Gastrointestinal Surgery Graduate School of Medicine The University of Tokyo TokyoJapan Kotaro Sugawara Division of Innovative Cancer Therapy Advanced Clinical Research Center Institute of Medical Science The University of Tokyo TokyoJapan Department of Gastrointestinal Surgery Graduate School of Medicine The University of Tokyo TokyoJapan Miwako Iwai Division of Innovative Cancer Therapy Advanced Clinical Research Center Institute of Medical Science The University of Tokyo TokyoJapan Minoru Tanaka Division of Innovative Cancer Therapy Advanced Clinical Research Center Institute of Medical Science The University of Tokyo TokyoJapan Yasuyuki Seto Department of Gastrointestinal Surgery Graduate School of Medicine The University of Tokyo TokyoJapan Tomoki Todo Division of Innovative Cancer Therapy Advanced Clinical Research Center Institute of Medical Science The University of Tokyo TokyoJapan Efficacy and safety of a third-generation oncolytic herpes virus G47D in models of human esophageal carcinoma 10.1016/j.omto.2021.10.012Original Article Treatment options are limited for esophageal carcinoma (EC). G47D, a triple-mutated, conditionally replicating herpes simplex virus type 1 (HSV-1), exhibits enhanced killing of tumor cells with high safety features. Here, we studied the efficacy of G47D using preclinical models of human EC. In vitro, G47D showed efficient cytopathic effects and replication capabilities in all eight human esophageal cancer cell lines tested. In athymic mice harboring subcutaneous tumors of human EC (KYSE180, TE8, and OE19), two intratumoral injections with G47D significantly inhibited the tumor growth. To mimic the clinical treatment situations, we established an orthotopic EC model using luciferase-expressing TE8 cells (TE8-luc). An intratumoral injection with G47D markedly inhibited the growth of orthotopic TE8-luc tumors in athymic mice. Furthermore, we evaluated the safety of applying G47D to the esophagus in mice. A/J mice inoculated intraesophageally or administered orally with G47D (10 7 plaque-forming units [pfu]) survived for more than 2 months without remarkable symptoms, whereas the majority with wild-type HSV-1 (10 6 pfu) deteriorated within 10 days. PCR analyses showed that the G47D DNA was confined to the esophagus after intraesophageal inoculation and was not detected in major organs after oral administration. Our results provide a rationale for the clinical use of G47D for treating EC. INTRODUCTION Esophageal carcinoma (EC) remains a substantial cause of cancerrelated mortality worldwide. 1 Despite recent advances in diagnosis and multimodal treatments, the survival outcome of EC patients has improved only modestly over the past decades, with the 5-year survival ranging from 15% to 25%. 2,3 Esophageal squamous cell carcinoma accounts for the majority of the incident esophageal cancer cases each year, particularly in the high-incidence regions of eastern Asia and Africa, while esophageal adenocarcinoma is more prevalent in North America and Western Europe. 4 Radical esophagectomy with pre-or post-operative chemo-/chemoradiotherapy is the mainstay of treatment for resectable, locally advanced EC, and chemotherapy for unresectable or relapsed EC. [5][6][7] New modalities, such as taxane-based triplet regimens 8 and immune checkpoint inhibitors, [9][10][11] have recently been applied for treating advanced EC. Unfortunately, immune checkpoint inhibitors show low response rates in patients with gastroesophageal cancer. 12 EC treatment options are limited when the tumor progresses or the patient is intolerant to first-line standard chemotherapy. Therefore, there is a high need for developing novel therapeutic strategies for refractory EC. Oncolytic viruses exhibit antitumor activity through selective killing of cancer cells and induction of specific antitumor immunity, 13 and have emerged as promising immunotherapeutic agents for currently incurable malignancies. 14 Many oncolytic viruses have been tested in clinical trials, of which talimogene laherparepvec (T-VEC) was the first to be approved by the US Food and Drug Administration (FDA) for patients with malignant melanoma in 2015. 15,16 The clinical implementation of oncolytic viruses has been rather slow for patients with gastroesophageal cancer. [17][18][19] Oncolytic adenovirus, alone 20 or in combination with chemotherapeutic agents, 21,22 has been investigated in mouse models with subcutaneous EC tumors. Phase I and II clinical trials are ongoing using a telomerase-specific oncolytic adenovirus in combination with pembrolizumab or with radiotherapy. 23,24 G47D is a triple-mutated, third-generation oncolytic herpes simplex virus type 1 (HSV-1), which was developed by an additional deletion of the a47 gene from the genome of G207, a second-generation oncolytic HSV-1, to enhance the oncolytic activity and retain the immunogenicity of infected cancer cells. 25,26 In the course of selective cancer cell destruction, G47D facilitates the priming of the immune system with cancer neoantigens, serving as a platform to revert immunologically "cold" tumors into "hot" tumors. 25,27,28 G47D can be armed with therapeutic genes of interest to increase its antitumor potency. [29][30][31] The immunomodulatory features of G47D can potentially overcome tumor resistance to immune checkpoint inhibitors. 32 Notably, stereotactic injections with G47D provided excellent survival benefit for patients with glioblastoma in a phase II clinical trial (manuscript under preparation), and G47D awaits a governmental approval as a new drug in Japan. In preclinical studies, G47D has shown efficacy not only for brain tumors but also for a variety of cancers. [33][34][35][36][37] G47D has also been used in clinical trials for prostate cancer, olfactory neuroblastoma, and malignant mesothelioma. 38 Because of its versatile efficacy and the proven safety in the human brain, the clinical application of G47D should further expand in the near future. In this study, we explore the usefulness of G47D as a therapeutic agent for EC in preclinical models, including a clinically relevant orthotopic mouse EC model. Furthermore, we evaluate the safety of G47D for treating EC by direct inoculation or by oral administration in the esophagus of HSV-1 sensitive A/J mice. RESULTS Cytopathic effect and replication capability of G47D in vitro To characterize the oncolytic activities of G47D in esophageal cancer, we studied its cytopathic effects and replication capabilities in eight human esophageal cancer cell lines in vitro. In all cell lines examined, G47D at a multiplicity of infection (MOI) of 0.1 caused >60% cell destruction within 4 days after infection ( Figure 1A). TE8 cells (esophageal squamous cell carcinoma) and OE19 cells (esophageal adenocarcinoma) were especially susceptible to G47D, with >80% cell destruction by day 4 at an MOI of 0.01 ( Figure 1A). We further examined the replication capabilities of G47D in human esophageal cancer cells. Virus yields increased by 48 h after infection at an MOI of 0.01 in all cell lines tested, although the extent of replication varied among cell lines ( Figure 1B). These observations indicate that G47D can replicate well and exert efficient cell killing in human esophageal cancer cells, irrespective of tissue types. Efficacy of G47D in subcutaneous EC tumor models Next, we studied the in vivo efficacy of G47D using three mouse models with subcutaneous tumors of human esophageal cancer. Athymic mice harboring KYSE180, TE8, or OE19 tumors of approximately 6 mm in diameter were inoculated intratumorally with G47D (4  10 4 , 2  10 5 , or 1  10 6 plaque-forming units [ Figure 2). In all models, there was no significant difference among the three doses used, although there seemed to be a tendency for dose dependency. Generation of an orthotopic EC tumor model Subcutaneous xenograft tumor models do not fully reflect the clinical features of the tumors. 39 To evaluate the efficacy of G47D under a more clinically relevant condition, we attempted to generate a reproducible orthotopic EC tumor model that mimics the clinical situation of treating a tumor in its organ of origin. 40 We generated orthotopic tumors in athymic mice using eight human esophageal cancer cell lines ( Figure S1), and evaluated the change in body weight, the engraftment rate, and the survival rate. Most of the mice harboring orthotopic tumors did not show marked loss in body weight, irrespective of the type of implanted tumor cells ( Figure S2A). All tumor cell lines showed a high engraftment rate, four of eight cell lines being 100% ( Figure S2B). Most tumor models showed a rather long survival, except for the OE19 model, in which half the animals Figure 1. Cytopathic effects and virus yields of G47D in human EC cell lines (A) Cells were seeded onto six-well plates at 2  10 5 cells/well or onto 96-well plates at optimal cell density. After an overnight incubation, the cells were infected with G47D (MOI of 0.01 or 0.1 or mock). The cell viability was determined daily either by counting surviving cells with a Coulter Counter or by the CellTiter96 Aqueous Non-Radioactive Cell Proliferation Assay. The percentage of surviving cells is expressed as a percentage of mock-infected controls. G47D exhibited good cytopathic effects at an MOI of 0.1 in all human esophageal cancer cell lines examined. Data are presented as the mean of triplicates ±SD. One-way ANOVA followed by Dunnett's test was used to determine statistical significance (***p < 0.001; ns, not significant; versus mock-infected controls). (B) Cells were seeded onto six-well plates at 3  105 cells/well. Triplicate wells were infected with G47D at an MOI of 0.01. At 24 or 48 h after infection, cells were collected and progeny virus was titered on Vero cells. The dotted line shows the assumed titer of administered G47D per well. Vero cell line was used as a control. In all cell lines tested, G47D showed good replication capabilities by 48 h after infection. The results presented are the mean of triplicates ±SD. www.moleculartherapy.org Molecular Therapy: Oncolytics Vol. 23 December 2021 died within 90 days due to the progression of peritoneal dissemination ( Figure S2B). The orthotopic tumor model with TE8 cells (esophageal squamous cell carcinoma) exhibited a 100% engraftment rate and a consistent survival course without peritoneal dissemination, and therefore was considered the best suited among the tumor models evaluated for assessing the antitumor efficacy of G47D. To monitor the tumor growth using a noninvasive, semiquantitative bioluminescent imaging method, 41 we established TE8 cells that stably express luciferase (TE8-luc). When the TE8-luc cells were subcutaneously implanted into athymic mice, a significant and strong correlation was observed between tumor volume and the luciferase emission level (r = 0.945, p < 0.001, data not shown). Intratumoral G47D treatment inhibited the growth of orthotopic tumors The potential of G47D as a therapeutic means for esophageal cancer was studied using the orthotopic tumor model of TE8-luc. The experimental design is depicted in Figure 3A. Female athymic mice were inoculated with TE8-luc (2  10 6 cells) into the abdominal esophagus wall (on day À18). The mice were randomly divided into two groups 3 days later (n = 10 per group), at which time point there was no significant difference in the total photon counts of orthotopic tumors between the two groups. Eighteen days after the tumor implantation (on day 0), the established orthotopic tumors were intratumorally inoculated with G47D (1  10 6 pfu) or mock. In vivo imaging system (IVIS) for tumor-encoded luciferase revealed a significant reduction in tumor burden in mice treated with G47D compared with those treated with mock (p < 0.01 on day 13; Figure 3B). The changes of IVIS images of two treatment groups with time are shown in Figure 3C. The weight of harvested tumors from the G47D-treated mice on day 34 was significantly lower than that from the mock-treated surviving mice (median 32.1 mg versus 76.4 mg, p < 0.001). Hematoxylin-eosin (HE) staining of cross sections of the abdominal esophagus of mice with orthotopic tumors revealed that tumor cells invade extensively across the layers and into the lumen of the esophagus in mock-treated mice ( Figure 4A), whereas the layered structure of the esophagus was well preserved in G47D-treated mice ( Figure 4B). Immunohistochemical staining for HSV-1 in the same cross section indicated HSV-1 positivity, presumably depicting replicating G47D, localized within the tumor at 34 days after treatment ( Figure 4C). Safety evaluation of intraesophageal inoculation and oral administration of G47D To evaluate the safety of applying G47D in the esophagus, A/J mice were intraesophageally inoculated with mock, G47D (1  10 7 pfu), or wild-type HSV-1 strain F (1  10 6 pfu) (n = 5 per group). In another set of experiments, A/J mice were orally administered with mock, G47D (1  10 7 pfu), or strain F (1  10 6 pfu) (n = 5 per group). A/J is known to be one of the most susceptible mouse strains to HSV-1 infection. 42 Each mouse was monitored twice a week for clinical manifestations based on three parameters ( Figure 5A) and the body weight for 2 months. In both experiments, intraesophageal inoculation and oral administration, mice given either mock or G47D all survived, with only a few showing a transient and minor decrease in clinical scores in the beginning and without weight loss (mock, Figures 5B ). In contrast, many of the mice given strain F deteriorated within 10 days of administration and became moribund. With strain F, three out of five died by direct intraesophageal inoculation ( Figure 5B [right]) and four out of five died by oral administration ( Figure 5C [right]). These observations indicate that G47D is at least 10 times less toxic than strain F when applied to the esophagus in HSV-1-susceptible A/J mice. Biodistribution of G47D administered to the esophagus Before investigating the biodistribution of G47D, we sought to obtain data on the tendency of biodistribution using wild-type HSV-1. Strain F (1  10 7 pfu) was administered either intraperitoneally or orally to A/J mice, one mouse each was sacrificed daily until day 5, and the amount of viral DNA in nine organs was detected by qPCR. When strain F was administered intraperitoneally, the virus tended to spread widely to the organs of the peritoneum, then replicated in the adrenal glands, followed by replication in the spinal cord and the brain (Figure S3A). When strain F was administered orally, the virus was detected from organs of initial infection, the gastro-esophagus, and the lung (presumably from aspiration), then tended to enter and replicate in the brain ( Figure S3B). Two potential pathways have , or OE19 (lower left)) were generated in 6-week-old female athymic mice. Established tumors, 5-6 mm in diameter, were inoculated with G47D (4  10 4 , 2  10 5 , or 1  10 6 pfu) or mock on days 0 and 3 (n = 9-10 per group). G47D treatment caused a significant inhibition of tumor growth in all subcutaneous tumor models irrespective of the dose used. Each experiment was conducted at least twice with similar results. The results presented are the mean ± SEM (n = 9-10). One-way ANOVA followed by Dunnett's test was used to determine statistical significance (**p < 0.01; ***p < 0.001). been suggested for HSV-1 to spread after an intraperitoneal administration in mice: (1) from the adrenal glands via the spinal cord to the brainstem, or (2) from the myenteric plexus of the gut via the vagal nerves to the brainstem, 43 which supports the results of our preliminary study. Therefore, for the main studies with G47D, we chose the esophagus, the adrenal glands, the spinal cord, and the brain as major organs to investigate. To investigate the biodistribution of G47D after application in the esophagus, A/J mice were intraesophageally inoculated with G47D (1.0  10 7 pfu) or strain F (1  10 7 pfu) (n = 12 per group). Again, in another set of experiments, A/J mice were orally administered with G47D (1.0  10 7 pfu) or strain F (1.0  10 7 pfu) (n = 12 per group). Three mice per group were sacrificed on days 1, 3, 5, and 7, and the amount of G47D DNA in major organs was detected by qPCR. In A/J mice receiving a direct inoculation with G47D into the subserosa of the esophagus, G47D DNA was detected at high levels in the esophagus on day 1 (three out of three), then gradually decreased, Figure 3. Efficacy of G47D in an orthotopic EC model (A) Experimental design. Female athymic mice were inoculated with TE8-luc cells (1  10 6 cells) into the esophageal wall (day À18). After confirmation of the orthotopic tumors with IVIS 3 days after the implantation (on day À15), the mice were randomly divided into two groups (n = 10 per group), and were treated with an intratumoral inoculation with G47D (1.0  10 6 pfu) or mock on day 0. The photon counts of the tumors were calculated with IVIS every 3-4 days. On day 34, all surviving mice were euthanized and the tumor weight was compared between the groups. (B) The average of the logarithmic photon flux was plotted on a chart. G47D treatment significantly decreased the total bioluminescence from tumors (p < 0.01 on day 13). Each experiment was conducted at least twice with similar results. The results presented are the mean ± SEM (n = 10). The Welch's t test was used to determine statistical significance (**p < 0.01). (C) The changes of IVIS images with time of both treatment groups are shown. but could still be detected in all three mice on day 7 ( Figure 6A, top). G47D DNA was also detected in the adrenal glands (two out of three) on day 1, but not detected by day 7 (Figure 6A, top). G47D DNA was undetectable in the spinal cord and detected only in one out of three on two occasions in the brain ( Figure 6A, top). In marked contrast, in mice receiving a direct inoculation with strain F into the subserosa of the esophagus, the copy numbers of strain F DNA remained high throughout the time course in the esophagus and increased over time in the adrenal glands, the spinal cord, and the brain ( Figure 6A, bottom), indicating the replication of strain F in the nervous system. In A/J mice receiving an oral administration with G47D, G47D DNA was not detected in major organs throughout the time course, except for one out of three in the esophagus on day 1 ( Figure 6B, top). In contrast, in mice receiving an oral administration with strain F, strain F DNA was detected at high levels throughout the time course in the esophagus and markedly increased over time in the spinal cord and brain, indicating that orally administered strain F can enter and replicate in the central nervous system (Figure 6B, bottom). To summarize, G47D DNA distribution was confined mainly to the esophagus, the inoculated site, after a direct inoculation. G47D DNA was almost undetectable in the esophagus, and there was none in other major organs, after an oral administration. These observations further confirm the safety of G47D when applied to the esophagus. DISCUSSION Currently available therapies provide unsatisfactory outcomes for patients with EC, underscoring the need for innovative treatment strategies. 44 We demonstrate that all human esophageal cancer cell lines www.moleculartherapy.org tested are highly susceptible to G47D, irrespective of tumor histology. G47D exhibits remarkable efficacy in subcutaneous EC xenograft models and in a clinically relevant orthotopic EC tumor model. G47D proves to be safe when applied to the esophagus of HSV-1 sensitive A/J mice, either intraesophageally or orally. These findings indicate that G47D could be useful for improving the clinical outcome of EC patients. To our knowledge, the efficacy of an oncolytic virus has not been investigated in an orthotopic EC tumor model. Orthotopic tumor models reportedly characterize the cellular and biological features of tumors in a more clinically relevant way than subcutaneous tumor models, and therefore allow the study of tumor-stromal interaction, which is important to understand tumorigenesis and tumor responses to treatments. 39,40,45 In addition, our orthotopic tumor model enables accurate monitoring of tumor growth, without invasive procedures, through bioluminescence imaging. 41,46 Using the orthotopic tumor model, we find that an intratumoral inoculation with G47D is highly efficacious. Unlike in the mouse model, a direct intratumoral injection into esophageal cancer in patients is easily doable via endoscope, so it could become a principal means of treating EC in clinical settings. 47 We further show that application of G47D to the esophagus is safe, using HSV-1-sensitive A/J mice. An intraesophageal inoculation with a high dose of G47D does not cause a substantial clinical manifestation, and G47D DNA is detectable only locally within the esophagus. The safety studies with oral administration were performed on supposition of a leakage of G47D into the esophageal lumen or spraying of G47D to the tumor surface as a potential route of administration. The results support that G47D is safe even if it was administered orally. The safety features of G47D are not at all natural features of HSV-1 but are acquired by the well-designed, triple mutations within the genome. 25 In fact, we show that a wild-type HSV-1 strain F, not only when inoculated directly into the subserosa of the esophagus but also when administered orally, can enter the central nervous system and replicate, resulting in death of the majority of animals even at one-tenth of a dose of G47D. G47D DNA was detected in the brain of one mouse out of three that received a direct inoculation with G47D into the subserosa of the esophagus. The finding is presumably due to the neurotropic nature of HSV-1. 48 Although it is unknown whether G47D can become latent in sensory neurons, because G47D can replicate in cancer cells only, G47D DNA in normal cells in the brain should stay harmlessly as DNA without ever producing a virus. The replication capability of HSV-1 in normal cells is best demonstrated in brain tissue in vivo. A previous study showed that all of the A/J mice inoculated with strain F (2  10 3 pfu) in the brain deteriorated rapidly and became moribund within 7 days, clearly demonstrating the replication capability of strain F in the normal brains of HSV-1-susceptible A/J mice. 25 On the other hand, G47D did not cause any manifestation when inoculated into the brain of A/J mice at 2  10 6 pfu. Furthermore, the first-in-human clinical trial in patients with recurrent glioblastoma (UMIN000002661) proved the safety of G47D when injected into the human brain tumor. The subsequent phase II clinical trial in patients with glioblastoma (UMIN000015995) further confirmed the safety of G47D when injected repeatedly in the brain tumor for the maximum of six doses. This phase II trial led to recent approval of G47D as a new drug for malignant glioma in Japan. Besides glioblastoma, a phase I study in patients with castration-resistant prostate cancer (UMIN000010463) demonstrated the safety of G47D when injected into the prostate. The results from the present study and these preclinical and clinical studies indicate that G47D does not replicate in normal tissues. Because the biodistribution study was performed in normal A/J mice without tumors, the actual biodistribution of G47D in humans can only be observed in patients with esophageal cancer and therefore awaits a clinical trial. Furthermore, because the only natural host of HSV-1 is human, it is difficult to extrapolate the biodistribution data of mice to those in humans. However, previous preclinical studies demonstrated that intracerebral inoculation with high-titer G207 resulted in no viral distribution beyond the central nervous system at any time point after inoculation in non-human primates 49,50 and BALB/c mice. 51 The biodistribution of G207 in non-human primates did not differ from that in mice. The safety and biodistribution evaluation of G47D in HSV-1-susceptible A/J mice therefore at least suggests the safety and biodistribution profile in humans. As observed in preclinical safety studies in A/J mice, G47D was in fact safe when used in the human brain. Neoadjuvant therapy followed by surgery is a clinical paradigm for patients with advanced EC. Recent preclinical studies in mouse models have shown that a neoadjuvant use of oncolytic viruses has a potential of eradicating the microscopic presence of cancer cells. For example, replicating pox virus administered systemically prior to surgery could reverse surgical stress-induced natural killer cell suppression in metastatic breast cancer and melanoma. 52 Preoperative uses of Maraba virus and vaccinia virus proved to be useful for controlling postsurgical cancer recurrence via activation of innate immunity and specific antitumor immunity. 53,54 Furthermore, we recently demonstrated that G47D can be used as a neoadjuvant therapy to suppress tumor relapse after radiofrequency ablation. 34 Since neoadjuvant chemo-/chemoradiotherapy is standardly used for resectable EC patients, a neoadjuvant use of G47D for EC is a possibility that merits testing preclinically and clinically. A direct oncolysis with G47D alone may not be sufficient for a cure. We recently showed that G47D in combination with systemic administration of immune checkpoint inhibitors can cure half of the mice bearing murine EC tumors. 55 Arming of G47D with immunostimulatory genes has been shown to result in enhanced efficacy. 29 Human interleukin-12-expressing oncolytic HSV-1 with a G47D backbone (T-hIL12) is currently being tested in patients with melanoma (jRCT2033190086). In conclusion, G47D is efficacious in both subcutaneous and orthotopic EC tumors in mice. G47D is safe when a high dose is administered intraesophageally and orally. Intratumoral injections with G47D thus constitute a practical and useful therapeutic approach for human esophageal cancer. MATERIALS AND METHODS Cell lines Human esophageal squamous cell carcinoma cell lines KYSE70, KYSE180, KYSE220, T.Tn, and TE8, and human esophageal adenocarcinoma cell lines OE19, OE33, SKGT-4, and Vero (African green monkey kidney) cells were used. KYSE70, KYSE180, KYSE220, and T.Tn were obtained from the Japanese Collection of Research Bioresources (Osaka, Japan). OE19, OE33, and SKGT-4 were bought from the European Collection of Authenticated Cell Cultures (Porton Down, Salisbury, UK). TE8 and Vero cells were purchased from the Institute of Physical and Chemical Research (Riken, Saitama, Japan) and the American Type Culture Collection (Rockville, MD), respectively. Cells were cultured according to the directions provided by the suppliers. Luciferase-expressing TE8 (TE8-luc) cells were established with pre-made luciferase lentiviral particles expressing the firefly luciferase3 gene (#LVP326, GenTarget) according to the manufacturer's protocol. Clonal selection was performed in a medium containing 5 mg/mL blasticidin (Wako, Japan). All cell lines were used within 10 passages and confirmed to be mycoplasma free using the MycoAlert Mycoplasma Detection Kit. www.moleculartherapy.org Viruses G47D and a wild-type HSV-1, strain F, were grown, purified, and titered by plaque assay on Vero cells as described previously. 25,26 Cytotoxicity assays In vitro cytotoxicity studies were performed as previously described. 56 Briefly, cells were seeded onto six-well plates at 2  10 5 cells/well and incubated overnight at 37 C. The following day, the cells were infected with G47D (MOI of 0.01 or 0.1) or mock, and further incubated at 34.5 C. The number of surviving cells was counted daily with a Coulter Counter (Beckman Coulter, Fullerton, CA) and expressed as a percentage of mock-infected controls. Virus yield studies Cells were seeded onto six-well plates at 5  10 5 cells/well and incubated overnight at 37 C. The following day, triplicate wells were infected with G47D at an MOI of 0.01. At 24 and 48 h after infection, Figure 6. Biodistribution of G47D applied to the esophagus of A/J mice A/J mice were intraesophageally inoculated or orally administered with G47D (1  10 7 pfu) or wild-type HSV-1 strain F (1  10 7 pfu) (n = 12 per group). Three mice per group were sacrificed on days 1, 3, 5, and 7, and the amount of G47D DNA in major organs (esophagus, adrenal glands, spinal cord, and brain) was detected by qPCR. Each dot represents the viral DNA copy number of the specimen (n = 3). (A) In mice receiving a direct inoculation with G47D into the subserosa of the esophagus (top), high levels of G47D DNA were detected from the esophagus throughout the time course. G47D DNA was also detected in the adrenal glands early after inoculation, but not detected by day 7. In mice receiving a direct inoculation with strain F into the subserosa of the esophagus (bottom), the copy numbers of strain F DNA remained high throughout the time course in the esophagus and increased over time in the adrenal glands, spinal cord, and brain. (B) In mice receiving an oral administration with G47D, G47D DNA was not detected in major organs throughout the time course, except for one in the esophagus on day 1 (top). In mice receiving an oral administration with strain F, strain F DNA was detected at high levels throughout the time course in the esophagus and markedly increased over time in the spinal cord and brain (bottom). the cells were scraped into the medium and lysed by three cycles of freezing and thawing. The extracted viruses were titrated in Vero cells, and viral plaques were counted 3 days after infection as described previously. 56 Animal experiments All animal experiment protocols conformed to all relevant regulatory standards and were approved by the Committee for Ethics of Animal Experimentation and were in accordance with the Guideline for Animal Experiments in the University of Tokyo. Sixweek-old female athymic (BALB/c nu/nu) mice were purchased from Clea Japan (Tokyo, Japan). Mice were maintained under specific pathogen-free conditions and provided with sterile food, water, and cages. Subcutaneous tumor models Subcutaneous tumors of esophageal cancer were generated by inoculating 3  10 6 cells (KYSE180), 4  10 6 cells (OE19), or 5  10 6 cells (TE8) into the right flank of 6-week-old female athymic mice (BALB/c nu/nu). When tumors reached approximately 5-7 mm in diameter, the animals were randomized, and mock or G47D (4  10 4 , 2  10 5 , or 1  10 6 pfu) in 20 mL of phosphate-buffered saline (PBS) containing 10% glycerol was inoculated into the right-flank tumors twice (n = 9-10 for each group). Mock-infected extract was prepared from virus buffer-infected cells. 28 The tumor size was measured using a digital caliper (Mitutoyo) twice per week and the tumor volume was calculated using the following formula: tumor volume (mm 3 ) = length  width  height. Mice were euthanized when the maximum diameter of the tumor reached 24 mm. Each experiment was conducted at least twice with similar results. An orthotopic tumor model of esophageal squamous cell carcinoma Orthotopic implantation of TE8-luc cells (2  10 6 cells) was performed in 6-week-old female athymic mice as previously reported. 41 Briefly, a slanting 10-mm skin incision was made in the left upper abdomen under anesthesia. The stomach was pulled down with tweezers so that the abdominal esophagus could be seen. A 1-mL syringe with a 30-gauge needle (Nipro, Tokyo, Japan), in which TE8-luc cells were suspended in a 10-mL volume of RPMI medium and Matrigel (2.0  10 6 cells/20 mL), was inserted into the anterior wall of the abdominal esophagus. The tumor cells were then slowly injected so that they were not spilt on the peritoneal cavity. The appearance of a small swelling at the injection site indicated successful cell injection. The esophagus was then returned to the peritoneal cavity, and the abdominal wall and skin were closed with 5-0 polydioxanone (PDS). Three days after the tumor cell inoculation, orthotopic tumor volume was determined by bioluminescence imaging with an IVIS (IVIS Lumina Series III, SPI, Japan) and the mice were randomly divided into two groups (n = 10). Eighteen days after the tumor implantation (on day 0), the mice were inoculated intratumorally with mock or G47D (1  10 6 pfu) in 20 mL of PBS containing 10% glycerol. The total photon counts of orthotopic tumors were observed. On day 34, all mice were euthanized, the tumors were harvested, and the weight measurement and the histological examinations were performed. In vivo photon counting analysis was conducted twice a week on the IVIS with Living Image acquisition and analysis software (Living Image 4.4., Xenogen, United States) as previously described. 57 For detection of bioluminescence, the mice were anesthetized and intraperitoneally injected with D-luciferin (150 mg/kg; PerkinElmer, Waltham, MA). Each experiment was conducted at least twice with similar results. Histological and immunohistochemical analysis of orthotopic tumors Mice harboring orthotopic TE8-luc tumors were treated and euthanized 34 days after the treatment as scheduled in Figure 3A. Orthotopic tumors were harvested, fixed in 10% formaldehyde neutral buffer solution (Sigma-Aldrich, St Louis, MOSA) for 72 h, and embedded in paraffin. Sections (5-mm thick) were rehydrated using an alcohol gradient and subjected to heat-mediated antigen retrieval using target retrieval solution S1700 (Dako, Santa Clara, CA). Sections were mounted on silanized slides (Dako Cytomation, Glostrup, Denmark) and stained with HE. Sequential sections were subjected to immunohistochemical analysis to detect HSV-1. The sections were treated with peroxidase blocking solution (Dako) and Blocking One (Nacalai Tesque, Kyoto, Japan), incubated with a rabbit polyclonal anti-HSV-1 antibody (1:2,000 dilution, 3 mg/mL) (Dako Cytomation), rinsed, and incubated with an HRP-conjugated goat anti-rabbit IgG antibody (Nichirei Bioscience, Tokyo, Japan). The sections were developed with 3,3 0 -diaminobenzidine (DAB) Peroxidase Substrate kit (Vector laboratories, Burlingame, CA), and then counterstained with hematoxylin. A NanoZoomer Digital Pathology slide scanner (Hamamatsu Photonics K.K., Hamamatsu, Japan) was used to view slides. Safety evaluation of G47D applied to the esophagus G47D (1  10 7 pfu), strain F (1  10 6 pfu), or mock (PBS containing 10% glycerol) in a volume of 10 mL was injected into the subserosa of esophagus, or orally administrated into the lumen of esophagus using a feeding needle, in 6-week-old female A/J mice (n = 5 for each group). Cages were then blinded and mice monitored for changes in clinical manifestations and body weight twice a week for 2 months. Animal care was in accordance with institution guidelines. Virus biodistribution studies Six-week-old female A/J mice were given G47D (1  10 7 pfu) or strain F (1  10 6 pfu) by two different administration routes as described above; i.e., intraesophageal inoculation (n = 12 per group) and oral administration (n = 12 per group). Mice were sacrificed at 1, 3, 5, and 7 days after the administration (n = 3 for each day), and the amount of viral DNA in major organs (esophagus, adrenal glands, spinal cord, and brain) was quantified using semiquantitative realtime PCR. As a preliminary study, strain F (1  10 7 pfu) was administered either intraperitoneally or orally to 6-week-old female A/J mice, one mouse each was sacrificed daily until day 5, and the amount of viral DNA in nine organs (brain, spinal cord, gastro-esophagus, small intestine, colon, liver, lungs, kidneys, and adrenal glands) was quantified using semiquantitative real-time PCR. The organs were homogenized, and genomic DNA was extracted using the QIAamp DNA mini kit (Qiagen) according to the manufacturer's instructions. Absolute quantification of viral DNA was conducted using real-time TaqMan PCR (the 7500 Fast Real-Time PCR System, Applied Biosystems) using HSV-1 gB primers (forward primer, 5 0 -GGCACGCGGCAGTACTTT-3 0 ; reverse primer, 5 0 -CCATGCGCT CGCAGAGA-3 0 ; TaqMan probe, 5 0 -GGTCGACAGGCACCTACA ATGCCG-3 0 ). The standard plasmid containing the glycoprotein B sequence of strain F and G47D served as positive control, and the values were used to generate a standard curve from 10 to 1.0  10 6 copies. Statistical analysis All data were expressed as the mean ± standard deviation (SD) or the mean ± standard error of the mean (SEM). Correlations were analyzed graphically using scatterplots and by the Pearson correlation coefficient, with application of the appropriate significance test (t test). For cytopathic effect studies and subcutaneous tumor studies, one-way ANOVA followed by Dunnett's test was used, and, for others, Student's t test or the Welch's t test was used to analyze the statistical significance of differences. The survival curves were analyzed by the Kaplan-Meier method, and p < 0.05 was considered www.moleculartherapy.org as statistically significant using the log rank test. In the figures, standard symbols are used: *p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant. Statistical analyses were carried out with JMP 14.0.0 (SAS Institute, Cary, NC). SUPPLEMENTAL INFORMATION Supplemental information can be found online at https://doi.org/10. 1016/j.omto.2021.10.012. Figure 2 . 2Efficacy of G47D in subcutaneous EC tumor models Subcutaneous tumors of a human esophageal cancer cell line (KYSE180 (upper left), TE8 (upper right) Figure 4 . 4Histology and immunohistochemistry of orthotopic EC tumors treated with G47D Orthotopic EC tumors in athymic mice were treated with an intratumoral inoculation with G47D (1  10 6 pfu) or mock or G47D. Mice were euthanized 34 days after the treatments, orthotopic tumors excised, and the paraffin-embedded tissue sections stained with HE or immunostained with an anti-HSV-1 antibody. The red dotted circles indicate the extent of local tumor invasion (T, tumor; L, lumen; E, esophageal wall). Representative HE staining images of orthotopic EC tumors inoculated with mock (A) and G47D (B). (A) In orthotopic EC treated with mock, tumor cells invade extensively across the layers of the esophageal wall, narrowing the lumen of the esophagus. (B) In orthotopic EC treated with G47D, the tumor size remains small and the original structure of the esophageal wall is preserved. (C) The same tumor from (B) immunostained for HSV-1. HSV-1 positivity, presumably indicating replicating G47D, localizes within the tumor. Original magnification, Â30 (A) and Â40 (B, C), as indicated. Scale bars: 1 mm (A) and 500 mm (B, C). Figure 5 . 5Safety evaluation of G47D applied to the esophagus of A/J mice A/J mice were intraesophageally inoculated or orally administered with mock, G47D (1  10 7 pfu) or wild-type HSV-1 strain F (1  10 6 pfu) (n = 5 per group). Cages were then blinded, and each mouse was monitored twice a week for clinical manifestations and body weight for 2 months. (A) Clinical manifestation scores were estimated based on three parameters: appearance, activity, and response. Each parameter has a response on a five-point (0-4 points) ranging from normal (4) to death (0). Score 0 means the death of the mouse. (B) Time course changes in clinical manifestations (top) and body weight ratio based on the body weight on day 0 (bottom) in mice inoculated intraesophageally with mock (left), G47D (middle), or strain F (right). (C) Time course changes in clinical manifestations (top) and body weight ratio (bottom) in mice administered orally with mock (left), G47D (middle), or strain F (right). In both experiments (B and C), all mice treated with G47D survived without remarkable manifestations, but many of the mice treated with strain F deteriorated rapidly and became moribund. pfu]) or mock twice (days 0 and 3). Intratumoral inoculations with G47D caused a significant inhibition of tumor growth in all subcutaneous tumor models even at the lowest dose tested (p < 0.01 versus mock on day 30 [KYSE180], day 22 [TE8], and day 27 [OE19]; and 5C [left]; G47D, Figures 5B and 5C [middle] Molecular Therapy: Oncolytics Vol. 23 December 2021 ACKNOWLEDGMENTSAUTHOR CONTRIBUTIONSS.Y., K.S., and T.T. were involved with the conception and performance of experiments, statistical analysis, and writing the manuscript. M.I. assisted with some of the experiments. S.Y. and M.T. were involved with the conception and design of experiments. K.S. and T.T. participated in manuscript preparation. All authors reviewed and edited the manuscript.DECLARATION OF INTERESTST.T. owns the patent right for G47D in multiple countries, including Japan. R L Siegel, K D Miller, Jemal , A , Cancer statistics. 65Siegel, R.L., Miller, K.D., and Jemal, A. (2015). Cancer statistics, 2015. CA Cancer J. Clin. 65, 5-29. Oesophageal cancer. 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Comparative effectiveness and safety of laser, needle, and "quick fenestrater" in in situ fenestration during thoracic endovascular aortic repair 29 September 2023 Jiaxuan Feng Jia Hu Maruti Haranal Lefeng Qu qulefeng@smmu.edu.cn Xiaokai Wang Jianjin Wu Kangkang Zhi Sili Zou Jie Jin Jun Bai Second Military Medical University China Sichuan University China U N Mehta Institute of Cardiology and Research India Wolf-Hans Eilenberg Medical University of Vienna Austria Department of Vascular and Endovascular Surgery Second Affiliated Hospital of Naval Medical University ShanghaiChina Comparative effectiveness and safety of laser, needle, and "quick fenestrater" in in situ fenestration during thoracic endovascular aortic repair 29 September 20239B710AEFC83F37706949B1DEA6AD87C510.3389/fcvm.2023.1250177RECEIVED 29 June 2023 ACCEPTED 15 September 2023 Background: Special instruments are needed for the revascularization of aortic branches in in situ fenestration during thoracic endovascular aortic repair (TEVAR).This prospective study compared the effectiveness and safety of three currently used fenestraters: laser, needle, and Quick Fenestrater (QF).Methods: In all, 101 patients who underwent TEVAR for aortic disease (dissection, n = 62; aneurysm, n = 16, or ulcer, n = 23) were enrolled.All patients were randomly assigned to three groups: 34 were assigned to laser fenestration, 36 to needle fenestration, and 31 to QF fenestration.The epidemiological data, treatment, imaging findings, and follow-up outcomes were analyzed using data from the medical records.Results: The technical success rates of the laser, needle, and QF fenestration groups were 94.1%, 94.4%, and 100% ( p > 0.05).After correction of mixed factors such as age and gender, it was showed the average operative time (Laser group: 130.01 ± 9.36 min/ Needle group: 149.80 ± 10.18 min vs.QF group: 101.10 ± 6.75 min, p < 0.001), fluoroscopy time (Laser group: 30.16 ± 9.81 min/ Needle group: 40.20 ± 9.91 min vs.QF group: 19.91 ± 5.42 min, p < 0.001), fenestration time (Laser group 5.50 ± 3.10 min / Needle group 3.50 ± 1.50 min vs.QF group 0.67 ± 0.06 min, p < 0.001), and guide wire passage time after fenestration (Laser group 5.10 ± 1.70 min / Needle group 4.28 ± 1.60 min vs.QF group 0.07 ± 0.01 min, p < 0.001) were all shorter with QF fenestration than with the other two tools.The overall perioperative complication rates of the laser, needle, and QF fenestration groups were 5.9%, 5.6%, and 0% (p > 0.05): One case of sheath thermal injury and one case of vertebral artery ischemia occurred in the laser fenestration group; one case each of access site hematoma and brachial artery thrombosis were reported in the needle fenestration group.89 (88.1%, 89/101) patients were followed for a median of 12.6 ± 1.6 months.The overall postoperative complication rates of the laser, needle, and QF fenestration groups were 3.3%, 6.5%, and 0% (p > 0.05): In the laser fenestration group, there was one death due to postoperative ST-segment elevation myocardial infarction; in the needle fenestration group, one patient developed occlusion of the bridge stent; no complications occurred in the QF group.Conclusion: All three fenestration methods were effective in reconstructing supraarch artery during TEVAR.QF fenestration required less contrast agent, with a shorter surgery duration and fewer complications than laser and needle fenestration.KEYWORDS aortic arch, aortic disease, in situ fenestration, thoracic endovascular aortic repair (TEVAR), quick fenestrater (QF)TYPE Introduction Aortic aneurysm, aortic ulcer, and aortic dissection are the major thoracic aortic diseases.These diseases greatly affect public health (1,2).Thoracic endovascular aortic repair (TEVAR) is an effective method for the treatment of such diseases.However, aortic diseases involving supra arch branches are considered contraindications to TEVAR due to the retention of the supra arch branches (3)(4)(5).In situ fenestration can extend the sealing zone during TEVAR and has shown the potential for the revascularization of aortic branches (6)(7)(8).Three instruments are generally used in in situ fenestration during TEVAR-laser, needle, and "Quick Fenestrater" (QF) (9).In 2004, Mc Williams et al. (10) reported satisfactory clinical results with reconstruction of the left subclavian artery by in situ fenestration with a reversed end of a 0.018-inch guidewire.In 2009, Murphy et al. (11) first applied laser fenestration technology during TEVAR to preserve the left subclavian artery.There are increasing applications of in situ fenestration technology, and it is considered the most consistent technology with human anatomy and hemodynamics and is a safe and effective method (12,13). Both needle and laser in situ fenestration have limitations, such as injury to the contralateral aortic wall, long fenestration time, thermal injury, and invisible head end of the guide wire of laser fenestration (14).Therefore, our team pioneered a device, "Quick Fenestrater" (QF) specifically used for in situ fenestration of supra aortic vessels (Figure 1) (15).In our study, we showed that QF was a safe and effective method, but we did not compare the advantages and disadvantages of the three fenestration methods.In this study, we compared the effectiveness and safety of laser, needle, and QF in situ fenestration during the perioperative period and 1-year follow-up. Methods Patient enrollment In this prospective cohort study, 101 patients were enrolled from December 2016 to October 2019 at the Department of Vascular Surgery in Shanghai Changzheng Hospital.The inclusion criteria were (1) age above 18 years, (2) at least 1 supra-aortic branch encroached by thoracic lesions, (3) landing zone not long enough (≤15 mm) for fixation of the aortic endograft so coverage of the LSA or LCCA had to be performed, (4) aortic disease confirmed by at least 1 radiologic examination (e.g., computed tomography angiography [CTA], magnetic resonance angiography [MRA]), ( 5) patent supra-aortic branch, and (6) type I/II aortic arch.The exclusion criteria were (1) cardiopulmonary and renal insufficiency contraindicating general anesthesia (according to the anesthesiologist), (2) severe infection causing high fever or organ dysfunction, (3) allergy to contrast medium, (4) adverse cardiovascular or cerebrovascular event within 3 months before intervention, (5) occlusion or stenosis of the supra-aortic branch or severe twisting of the arteries to be fenestrated, (6) no appropriate peripheral access, (7) Stanford A aortic lesions, (8) type III aortic arch and (9) Marfan syndrome.The protocol for this study was approved by the participating hospital's Ethics Committee, and informed consent was obtained from each participant. Surgical procedure The enrolled patients underwent in situ laser, needle, or QF fenestration according to their order of admission.All interventions were performed by advanced interventional radiologists.The procedures were performed under general anesthesia.Two ProGlide vascular sutures (Perclose ProGlide, FIGURE 1 The structure and working principle of quick fenestrater (QF).Abbott Inc, USA) were preset for right femoral artery puncture, and a 12-F sheath was inserted.A 7-F sheath (pre-left carotid artery fenestration) was punctured and inserted via the left common carotid artery, and a 6-F 55 cm renal artery sheath (Flexor ® Check-Flo ® Introducer, Cook, Inc, USA) was punctured and inserted into the left brachial artery.A 5-F labeled pig-tail catheter was introduced into the sheath of the right femoral artery to the ascending aorta, and the aortic size was measured.A Lunderquist super-stiff guide wire was inserted, and the pigtail catheter was withdrawn to the ascending aorta through the left brachial artery or carotid artery sheath for real-time intraoperative angiography.After angiographic evaluation of the aortic lesions, aortic stents of appropriate size (Capitivia, Medtronic, USA/Ankura, Lifetech, China) were introduced along the super stiff guide wire, accurately positioned, and released to repair thoracic aortic lesions and cover the supra arch branch arteries.Advanced through the 0.035 inch support catheter, the needle (reversed end of a 0.018 inch guidewire) or intravenous laser catheter (LFK-SLT30, Rafcon, China) or QF (Suzhou Innomed Medical Device, China) was introduced along the supra arch branch artery and guided to the branch artery for stent graft fenestration.After fenestration, the needle, intravenous laser catheter, or QF was advanced into the aortic lumen.The fenestration instruments were withdrawn, and another angiogram was made to confirm that the needle, intravenous laser catheter, or QF was positioned in the aortic cavity, and a 0.035 inch guide wire was introduced along the catheter to the ascending or descending aorta.A balloon (Mustang, Boston Scientific Corporation, USA) of appropriate size to expand the fenestration was positioned.The stent deployed in the super arch artery had a 5 mm protrusion in the aortic arch, and the distal end in left subclavian artery did not cover the orifice of the vertebral artery.Post dilatation was carried out if necessary.Finally, aortography was performed to check that the intervention was carried out successfully. Outcomes After the intervention, follow-up was conducted at 1 month, 6 months, and annually thereafter.The following perioperative outcomes were assessed: success rate of fenestration; average durations of intervention, fluoroscopy, rupture, and guide wire passage after fenestration (from guide wire touching the membrane to passing through the hole); and amount of contrast agent used.Follow-up outcomes included 30-day mortality, type I and III endoleaks, retrograde dissection, mortality, stent patency rate, stent morphology, secondary re-interventions, and severe cardiovascular and cerebrovascular events.Computed tomographic angiography of the aorta was performed to evaluate the rates of branch stenting, internal leakage rate, and secondary intervention. Statistical analysis The data were analyzed with SPSS 22.0 software.The data were expressed as mean values ± SD.Continuous variables were presented as median and range in the case of nonparametric distributions, and comparisons were made using the Mann-Whitney test.Continuous variables are presented as means ± standard deviations in cases of parametric distributions, and comparisons were made using the independent t-test.Categorical variables were compared using the Chi-square test or Fisher's exact test and reported as frequencies (percentages).A p value of <0.05 was considered statistically significant. Results Patient demographics and presentation In all, 101 patients underwent in situ fenestration during TEVAR between May 2016 and October 2019 (85 males and 16 females; mean age, 67 ± 10 years; 71 acute onset).In all, 34 patients underwent 40 in situ laser fenestrations during TEVAR (aortic dissection: n = 21, aortic aneurysm: n = 5, aortic ulcer: n = 8); 36 patients underwent 43 in situ needle fenestrations during TEVAR (aortic dissection: n = 22, aortic aneurysm: n = 6, aortic ulcer: n = 8); and 31 patients underwent 37 in situ QF fenestrations during TEVAR (aortic dissection: n = 19, aortic aneurysm: n = 5, aortic ulcer: n = 7).The baseline characteristics, including risk factors and comorbidities, were not significantly different among the three groups (Table 1). Operative data TEVAR was technically successful in 94 patients (Figure 2).The technical success rates of the laser, needle, and QF fenestration groups were 94.1%, 94.4%, and 100%, respectively, showing an insignificant difference (p > 0.05).In the in situ laser fenestration group, one procedure failed due to an awkward fenestration angle of the laser guide wire.In the in situ needle fenestration group, one procedure failed because the needle had failed to puncture the membrane.After correction of mixed factors such as age and gender, variance analysis and Kruskal-Wallis test results showed the average operation, fluoroscopy, fenestration, and guide wire passage durations after fenestration were shorter in the QF fenestration group (p < 0.001).Moreover, the average amount of contrast agent in the QF fenestration group was less than that in the needle fenestration and laser fenestration group (p < 0.001) (Table 2). All the in situ fenestrated arteries were found to be patent on postoperative follow-up CTA imaging and clinical symptoms. Clinical follow-up The overall Perioperative complication rates of the laser, needle, and QF fenestration groups were 5.9%, 5,6%, and 0% (p > 0.05).In the perioperative period, no cerebral infarction, myocardial infarction, transient ischemic attacks, or other neurologic complications occurred.In the in situ laser fenestration group, one patient had a sheath injury due to laser heat (Figure 3A), and one patient presented with intraoperative vertebral artery ischemia.In the in situ needle fenestration group, one patient presented with an access-site hematoma, and one patient presented with a femoral arterial thrombus.No endoleak (include type I and type III), false lumen thrombosis, or subsequent remodeling of the aorta was evident.A total of 89 (88.1%, 89/101) patients were followed for a median of 12.6 ± 1.6 months.At the 1-year follow-up, the overall postoperative complication rates of the laser, needle, and QF fenestration groups were 3.3%, 6.5%, and 0% (p > 0.05).In the laser fenestration group, there was one death due to postoperative ST-segment elevation myocardial infarction, with no direct association with the intervention.In the needle fenestration group, one patient developed There were no complications in the QF group.No reverse tear or endoleak (type I and type III) was observed in any of the three groups (Table 3, Figure 4). Discussion The fenestration methods used in TEVAR include mechanical fenestration, represented by self-made guide wires or puncture needles, and thermal fenestration, represented by laser/radio frequency.Our center developed a special fenestration instrument, the "Quick Fenestrater" (QF), which has demonstrated safety and efficacy in a previous single-center clinical study (15).On analyzing the data of the QF, laser, and needle fenestration groups, we found that (1) all three methods were effective for in situ fenestration of the superior arch artery, each with a technical success rate of over 90%; the QF fenestration rate was 100% and (2) while there was no significant difference in the average durations of operation, fluoroscopy, fenestration, and guide wire passage after fenestration and volume of contrast agent used between the needle and laser fenestration groups, the lowest measurements were observed in the QF group (P < 0.001).(3) There were no significant differences in the endoleak, postoperative all-cause mortality, secondary surgical intervention, and branch stent patency rates among the three groups during follow-up. In situ fenestration during TEVAR is a safe and effective method for endovascular reconstruction of the branch arteries above the aortic arch (16)(17)(18)(19).In 2016, in a metaanalysis of 118 articles conducted by Crawford et al., the success rate of in situ fenestration was 96%, and the rate of comprehensive complications (death, stroke, and paraplegia) was 7% (20).In situ fenestration has the advantages of rapid fenestration, repeatability, minimally invasiveness, and few complications. Nevertheless, during the study of fenestration instruments, we found some undesirable features: needle fenestration requires manual or other types of puncture needles (e.g., biopsy needles); puncture needles have high rigidity and poor flexibility and cannot pass through the twisted subclavian artery, resulting in a relatively long fenestration time (21).Moreover, the direction of the puncture tip is difficult to adjust, and there is a risk of damaging the arterial intima and puncturing the opposite vessel wall (22)(23)(24).In this study, in the needle fenestration group, one patient developed an occlusion on the bridging stent at the 3-month follow-up; perhaps, because of the large torsion angle of the subclavian artery, the intima was damaged by the needle, resulting in a dissection.During recanalization, angiography showed that the stent itself was in good shape, and the dissection was seen at the tortuous subclavian artery at the distal end of the stent; the arterial dissection was repaired by balloon expansion and implantation of a bare stent.Laser in situ fenestration is invisible under fluoroscopy at the head end, and the direction of the head end is difficult to adjust, resulting in the risk of thermal damage (25, 26).In the laser group, thermal injury of sheath due to the supporting catheter damaged the intima of the subclavian artery and caused thrombosis.After laser fenestration, the laser fiber must be pushed out and the guide wire must be exchanged; in this process, because the fenestration window is small and invisible, exchanging the guide wire often takes time; if and when it is difficult to pass through, the membrane must be ruptured with laser again to enlarge the window (27,28).In the laser fenestration group, one patient, treated with local anesthesia, had sudden transient ischemia of the vertebral artery, and the symptoms resolved after the fenestration was completed.According to previous reports, the laser could lead to the formation of air bubbles, and the bubbles could enter cerebral blood vessels and cause a stroke, which could have occurred in our case (29). In contrast to other fenestraters, QF can fix the access point in the central area of the blood vessel through the support bracket at the front end and ensure the fenestration is relatively parallel to the vessel wall.QF uses a needle with adjustable strength and depth control to quickly fenestrate, thus preventing vascular damage.After successful fenestration, the guide wire, preset from the needle lumen, can quickly enter the aortic lumen to complete the fenestration process.The whole process is simple, smooth, and time saving.In summary, QF has significant advantages over the other two fenestration methods. The shape of the aortic arch and the angle of the branch blood vessels affect the success rate of the fenestration.Theoretically, fenestration perpendicular to the membrane has the highest success rate.For laser fenestration, the laser head must be placed on the stent graft.Given the arch type and angle of branch vessels, the laser head may slip off, resulting in failure of the fenestration.Needle fenestration faces the same problem.Moreover, the head ends of the needle and laser fibers are uncontrollable and their angles cannot be changed (24,30).Because of these disadvantages of needle and laser fenestration, we developed the QF to fix the access point in the central area of the blood vessel through the support bracket at the front and use the membrane-perforating needle with adjustable strength and controllable depth to quickly fenestrate and minimize the risk of vascular damage.By optimizing the operation and simplifying the operation steps, the success rate of fenestration is increased, and the operation time is reduced.In the present comparison of QF, needle, and laser fenestration, we found that by reducing the intervention time, QF fenestration can reduce the amount of contrast agent used and the operator's radiation exposure, so that complications in high-risk patients, such as renal insufficiency, are reduced. Conclusions Our comparative study of the effectiveness of the recently designed QF, needle, and laser as fenestration tools in endovascular aortic arch repair showed that the QF was associated with a shorter surgical duration, lower volume of contrast agent used, and better safety.At the follow-up, there were no complications in the patients who underwent endovascular aortic arch repair with the QF. FIGURE 2 2 FIGURE 2 Preoperative examination, fenestration instrument, fenestration, and balloon dilatation using three different fenestration methods.(A-D) Laser fenestration, (E-H) needle fenestration, (I-L) QF fenestration. FIGURE 3 3 FIGURE 3Complications in laser and needle fenestration groups.(A) In the laser fenestration group, one patient sustained sheath injury caused by laser ablation.(B-D) In the needle fenestration group, one patient developed subclavian artery occlusion after the intervention, but it was recanalized. FIGURE 4 4 FIGURE 4Postoperative computed tomography angiography of three different fenestration methods.(A) Laser fenestration, (B) needle fenestration, (C) quick fenestration. TABLE 1 1 Baseline characteristics of patients. Laser fenestrationNeedle fenestrationQF fenestrationPN = 34N = 36N = 31Average age65.767.268.1Male29 (85.3%)30 (83.3%)26 (83.9%)0.974Acute onset24 (70.6%)25 (69.4%)22 (71%)0.990Disease classificationThoracic aortic dissection21 (61.8%)22 (61.1%)19 (61.3%)0.998Thoracic aortic aneurysm5 (14.7%)6 (16.7%)5 (16.1%)0.974Thoracic aortic ulcer8 (23.5%)8 (22.2%)7 (22.6%)0.991Risk factorsHypertension16 (47.1%)17 (47.2%)14 (45.2%)0.983Hyperlipidemia10 (29.4%)11 (30.6%)10 (32.3%)0.969Diabetes mellitus5 (14.7%)6 (16.7%)5 (16.1%)0.974Smoking26 (76.5%)28 (77.8%)24 (77.4%)0.991ComorbiditiesSevere COPD3 (8%)5 (13.8%)4 (12.9%)0.800Cardiac dysfunction10 (29.4%)8 (22.2%)12 (38.7%)0.338Hepatic insufficiency01 (2.7%)01.000Renal insufficiency5 (14.7%)7 (19.4%)5 (16.2%)0.862 TABLE 2 2 Perioperative data for the three cohorts. Laser fenestrationNeedle fenestrationQF fenestrationPBranch artery revascularizedLSA28 (28/34)29 (29/36)25 (25/31)LSA + LCCA6 (6/34)7 (7/36)6 (6/31)Technical success rate94.1%94.4%100.0%0.545Intraoperative vascular injury2200.545Operation processAverage operation time (min)130.01 ± 9.36149.80 ± 10.18101.10 ± 6.75<0.001Average fluoroscopy time (min)30.16 ± 9.8140.20 ± 9.9119.91 ± 5.42<0.001Average fenestration time (min)5.50 ± 3.103.50 ± 1.500.67 ± 0.06<0.001Average contrast material (ml)152.02 ± 30.12180.21 ± 20.23100.01 ± 15.15<0.001Average guide wire passage time after fenestration (min)5.10 ± 1.74.28 ± 1.60.07 ± 0.01<0.001Patency rate of branch stent100%100%100%LSA, Left subclavian artery; LCCA, Left common carotid artery. TABLE 3 3 Follow-up of patients. frontiersin.org Frontiers in Cardiovascular Medicine 03 frontiersin.org Data availability statementThe original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.FundingThis work was supported by the National Natural Science Foundation of China (81870347, 82370498); Program of Shanghai Academic Research Leader (20XD1404900); Basic Research Cross Science Project of the Shanghai Science and Technology Commission's "Science and Technology Innovation Action Plan" (21JC1406500); and High-level Achievement Training Program of Changzheng Hospital (2020YCGPZ-205).Ethics statementThe studies involving humans were approved by Clinical Test Ethics Committee of Shanghai Changzheng Hospital.The studies were conducted in accordance with the local legislation and institutional requirements.The participants provided their written informed consent to participate in this study.Author contributionsLQ contributed to the study conceptualization and design.XW, JB, and JW performed the data analysis and interpretation.XW, JB, and SZ collected the data.SZ conducted the statistical analysis.XW, JW, and KZ drafted the article.JB and LQ made critical revisions to the article.All authors contributed to the article and approved the submitted version.Conflict of interestThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The handling editor JF declared a shared parent affiliation with the authors at the time of the review. 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Genetic Diversity and Differentiation of Juniperus thurifera in Spain and Morocco as Determined by SSR February 12, 2014 Helena Teixeira Susana Rodrı ´guez-Echeverrı ´a susanare@ci.uc.pt Cristina Nabais Centro de Ecologia Funcional Departamento de Cie ˆncias da Vida Universidade de Coimbra CoimbraPortugal University of Milano Bicocca Italy Genetic Diversity and Differentiation of Juniperus thurifera in Spain and Morocco as Determined by SSR February 12, 2014186226C826BD74BA16787E892320D8B510.1371/journal.pone.0088996Received October 10, 2013; Accepted January 14, 2014; Juniperus thurifera L. is an important tree endemic to the western Mediterranean basin that it is able to grow in semi-arid climates.It nowadays exhibits a disjunct distribution pattern, occurring in North Africa, Spain, France and the Italian Alps.The Strait of Gibraltar has acted as an efficient barrier against gene flow between African and European populations, which are considered different subspecies by some authors.We aimed at describing the intraspecific genetic diversity of J. thurifera in populations from the Iberian Peninsula and Morocco and the phylogeographical relationships among these populations.The ploidy level of J. thurifera was examined and eleven nuclear microsatellites (nSSRs) developed for J. thurifera were assessed for genotyping this species.Six nSSRs were polymorphic and subsequently used to assess the genetic diversity and structure of the studied populations.Genotyping of the tetraploid J. thurifera using nuclear microsatellites supports the separation of Moroccan and Spanish populations into two genetically differentiated groups that correspond to the proposed subspecies africana and thurifera.High values of within population genetic diversity were found, that accounted for 90% of the total genetic variance, while population structure was weak.The estimators of genetic diversity were higher in populations of Spain than in populations of Morocco pointing for a possible loss of genetic diversity during the spread of this species to Africa from Europe. Introduction Juniperus thurifera L. (section Sabina) is a dioecious tree endemic to the western Mediterranean basin [1].It is a long-lived species with individual trees lasting for several hundred years [2].Juniperus thurifera was likely widespread during cold stages of the Pleistocene but nowadays it exhibits a disjunct distribution pattern, occurring in North Africa (Morocco and Algeria), Spain, France (Pyrenees, Alps and Corsica) and in the Italian Alps [3,4].The most abundant populations are in Spain (.200,000 ha) and the Medium and High Atlas Mountains in Morocco (30,000 ha).Fragmentation of J. thurifera populations was initially driven by increased aridity and warmer climates, but it is currently caused by human activities such as intense wood removal and grazing in North Africa and land use changes in the Iberian Peninsula that lead to the colonization of Juniperus stands by pines or oaks [3,5].Juniperus thurifera is most often found in calcareous soils in the Iberian Peninsula and acidic soils in North Africa [3].It is an important component of woodland communities of cold and dry sites from 200 to 1800 m above sea level in the Iberian Peninsula, and in sites with subhumid cold winter climates mainly between 1800 and 3150 m above sea level in Morocco [3,6]. Juniperus thurifera is a morphologically variable species due to the long-term isolation of disjunct populations.The Strait of Gibraltar has acted as an efficient barrier against gene flow between Morocco and European populations for a long time [4].Consequently, two subspecies have been described: subsp.thurifera in Europe and subsp.africana in Africa.Juniperus thurifera subsp.africana (Maire) Romo et Boratyn ´ski differs from J. thurifera subsp.thurifera by its smaller cone diameter (5-6 vs. 7-12 mm), lower number of seeds per cone (only 1-2 vs. 2-5) and shorter leaf scales (1-1.5 vs. 2-2.5 mm) [3,7].Studies based on essential oils [8] and biochemical composition [9] supported the clear differentiation of populations from Europe and populations from Morocco.The existence of the two subspecies has been confirmed also by phylogeographic studies based on RAPD [10] and AFLP markers [4].However, not all authors agree upon the distinctiveness of subsp.africana.Farjon [11] considers that the biochemical composition is taxonomically uninformative and cannot be used to justify this distinction, and a clear separation of morphological traits is not always clear.Maire [12] cited specimens from Algeria with bigger cone diameter (9 mm) and material from Spain with short leaves similar to the African populations.Also, populations from Algeria do not cluster with African populations but are genetically intermediate between Spanish and Moroccan populations [4]. A number of PCR-based techniques can be used to detect polymorphisms for studies of genetic diversity, being AFLP (Amplified Fragment Length Polymorphism) and SSR (Simple Sequence Repeat or microsatellites) the most popular molecular markers used [13].Some authors have compared the data produced by AFLP and SSR and showed that both markers have comparable efficiency [14,15].However, SSR could be more informative because they are co-dominant markers (unlike AFLPs that are dominant markers), and the heterozygosity of polymorphic loci for SSR is greater than for AFLP because replication slippage occurs more frequently than single nucleotide mutations and insertion/deletion events [15].In addition, using SSRs makes possible the analysis of problematic samples, like dry leaves, because the targeted DNA fragments have a smaller size than those in AFLP. In the present study, we used nuclear microsatellites (nSSRs) to assess the genetic structure of J. thurifera populations from the Iberian Peninsula and Morocco.Specifically, our aims were (1) to analyse the ploidy level of J. thurifera in both sides of the Strait of Gibraltar, (2) to describe the intraspecific genetic diversity of J. thurifera in populations from the Iberian Peninsula and Morocco, and (3) to study the phylogeographical relationships among these populations. Materials and Methods Plant material Fresh leaves were collected from 20-30 individuals selected randomly in 11 natural populations of Juniperus thurifera.Five populations were sampled in Morocco and six in Spain.A total of 261 individuals were included in the study.Table S1 gives the details of the geographical location of populations and number of sampled individuals per population.Special permissions for sampling were not required since this is not a protected species, sampling was conducted outside protected areas and leaf collection does not represent a threat for the sampled individuals. DNA extraction The leaves collected from each individual were dried at 30uC overnight before DNA extraction.Genomic DNA was extracted from 20 mg of leaves using the DNeasy Plant Mini Kit (Qiagen).DNA extraction followed the manufacturer's protocol with a few modifications: instead of 200 ml of buffer AE we only added 150 ml and the time of incubation was extended to 10-15 min.The quality of the extracted DNA was checked on 1% TBE agarose gels stained with RedSafe.DNA concentration was estimated using NanoDrop 2000c (Thermo Scientific, Wilmington, USA) and samples were stored at 220uC. Flow cytometry analysis The genome size of J. thurifera was inferred using flow cytometry following Castro et al. [16].Nuclear suspensions were obtained by chopping leaf tissue of J. thurifera, J. phoenicea and Vicia faba with a razor blade in a Petri dish containing 1 ml of WPB buffer (0.2 M Tris-HCl, 4 mM MgCl2?6H2O, 1% Triton X-100, 2 mM EDTA Na2.2H2O, 86 mM NaCl, 10 mM metabisulfite, 1%PVP-10, pH adjusted to 7.5).Vicia faba was used as the internal reference standard (2C = 26.90pg DNA).The nuclear suspension obtained was filtered with a nylon filter and 50 mg/ml of propidium iodide was added to stain the DNA.50 mg/ml of RNAse was added to avoid staining of double stranded RNA.All samples were analyzed in a Partec CyFlow Space flow cytometer and the results were checked using Partec FloMax software v2.4d (Partec GmbH, Mu ¨nster, Germany).At least 5000 particles were analysed per sample.For each sample, the DNA index was calculated and the nuclear DNA content of J. thurifera was estimated by multiplying the DNA index by the known genome size of the V. faba, using the formula 1 pg = 978 Mbp. Microsatellites amplification Due to the larger number of alleles per locus, the discriminative power of a locus is potentially higher for polyploids than for diploids and the successful fingerprinting of polyploids requires substantially fewer loci [17].An initial set of putative polymorphic 11 nuclear microsatellite (nSSR) loci developed for J. thurifera by Genoscreen (Lille, France) was tested (Table 1).Six out of these 11 primer pairs amplified successfully the targeted loci and showed polymorphism.These six polymorphic primers pairs were therefore used in this study (Table 1).These primers were developed during this study and have not been tested for crossamplification with other species. Multiplex reactions were prepared using primers with similar annealing temperature that amplify fragments of different size.Polymerase chain reaction (PCR) was performed in a total volume of 20 ml, containing 20 ng of DNA template, 26 QIAGEN Multiplex PCR Master Mix and 1 mM of each forward (F) and reverse (R) primers and RNase-free water.All PCR were run in a Gene Amp 9700 (Applied Biosystems) using the following program: Initial denaturation step for 15 min at 95uC, followed by 40 cycles of 30 s at 94uC, 90 s at locus-specific annealing temperature (see Table 1) and 60 s at 72uC and a final extension step for 30 min at 60uC and cooling down to 4uC.PCR products were checked on 2% TBE agarose gels stained with RedSafe.PCRs that did not produce bands or that had different size were repeated.The amplification products were combined with formamide and a size standard (GeneScan-500 LIZ, Applied Biosystems) and separated on a 3730 ABI automated sequencer.Sample profiles were scored manually using GeneMarker v2.4 (Applied Biosystems). Data analysis The quantification of genetic diversity in polyploids can be problematic because of the difficulties in assigning the correct allele dosage for each loci and individual [18][19][20].Although it has been proposed that allele dosage could be estimated in tetraploids on the basis of electropherogram peak height [19], this was not possible for the studied species.Fixed heterozygosity is common in polyploidy species and homozygote individuals are rare due to the presence of at least two allele variants associated with different isoloci [19].Due to these deviations from diploid meiotic behaviour, indices such as expected heterozygosity (H e ), cannot be used to study genetic diversity in polyploids such as J. thurifera [19,21,20].Also, the common karyotype homogeneity of gymnosperms impedes to know if J. thurifera is an autopolyploid or allopolyploid species.The effective number of alleles per locus (Ae9) and heterozygosity within each population (Hs) were calculated using GenoDive, a software that allows analysing polyploids with unknown dosage of alleles [22].For every incomplete marker phenotype (e.g.AB) it is possible to calculate the likelihood of observing that phenotype given some set of allele frequencies, by combining the likelihoods of the underlying genotypes (AAAB, AABB, or ABBB).This correction uses a maximum likelihood method based on an algorithm that calculates for each locus and population the likelihood of different allele frequencies for all observed phenotypes until convergence is reached. The multilocus data was transformed to a binary matrix of presence/absence of each allele for each individual [18,19,23], which was used for further analysis with GenAlex 6.5 [24].Total number of alleles, genetic diversity and Shannon's information index for each population, and the number of private alleles per population and region were calculated using this approach.The genetic differentiation between populations was determined using phiPT, a measure that allows intra-individual variation to be suppressed and is therefore ideal for comparing codominant and binary data, with 10,000 permutations [25].Analysis of molecular variance (AMOVA) among and within populations and geographical regions was performed using GenAlex [24,26].A Mantel test, as implemented in GenAlex, was performed using genetic and geographical distances to test for isolation by distance [27].Finescale genetic structure was investigated using spatial autocorrelation analysis in GenAlEx 6.5 [24].The spatial autocorrelation coefficient (r) was computed using the geographic distance and the multilocus genetic distance between individuals.Tests for statistical significance were conducted by random shuffling (999 times) of individual geographic locations to define the upper and lower bounds of the 95% confidence interval for each distance class, and estimating 95% confidence intervals around mean r values by bootstrapping pair-wise comparisons within each distance class (1000 repeats).Analyses were performed using the variable distance classes option based on the geographical distances estimated in each population. The original genotypic data matrix was used to perform a PCA using the Bruvo distance to explore the genetic similarity between samples.This analysis was performed with PolySat [28], an R package for polyploid microsatellite analysis in ecological genetics. The genetic structure of each population was inferred with the BAPS software [29] following a model-based Bayesian assignment that allows mixed ancestry of individuals (admixture model). Results Ploidy level in Juniperus thurifera Flow cytometry from individuals of the two Juniperus species resulted in clear fluorescence peaks.Juniperus phoenicea, a known diploid species, had 2C of <20 pg, while J. thurifera had 2C of <40 pg, revealing that the genomic size of J. thurifera is double than that in J. phoenicea.Knowing that J. phoenicea is a diploid species, the value observed for J. thurifera would correspond to a tetraploid species.The 2C values obtained were the same for all J. thurifera samples, meaning that ploidy levels did not experience variation among regions or populations.The values obtained were similar to those published by Romo et al. [30] for J. thurifera and correspond to a tetraploid species. Genetic diversity A total of 153 alleles were amplified from the 6 microsatellite loci.All 6 loci were found to be highly polymorphic and displayed up to 4 alleles per sample, in agreement with the ploidy level detected by flow cytometry.The number of different alleles found within each population (An) was generally higher in the populations from Spain than in Morocco, while the average number of effective alleles per locus was similar in all populations (Table 2).No significant differences between regions were found for any of these diversity estimators (Wilcoxon test, P = 0.12).All populations showed also similar high values of heterozygosity (Table 2).Private alleles were found in all populations from Spain and in two populations from Morocco (Tizi Techt and Azzaden Oussem).The number of private alleles per population found in each region was significantly higher in the Iberian Peninsula (Wilcoxon test, P = 0.01).Significantly higher values were also found in the Iberian Peninsula for the Shannon's information index (I) and the estimator of genetic diversity (H) (Table 2, Wilcoxon tests, P = 0.05 and P = 0.04 respectively). From the 153 alleles observed, there were 105 alleles (69%) shared by all populations.The remaining alleles were exclusively found in populations of Spain or in populations of Morocco.The number of private alleles found in Spain was higher than in Morocco (Table S2).40 private alleles (26%) were found only in populations from Spain while only 8 private alleles (5%) were found exclusively in the populations from Morocco.From the six loci used in this survey, JT33 was the locus with the highest number of private alleles, with 12 private alleles in Spain and 4 in Morocco.The locus JT04 was the only one that did not display private alleles in Spain.In Morocco, the loci JT01 and JT40 did not display private alleles and the loci JT04 and JT30 just had one private allele (Table S2). Population differentiation and genetic structure Pairwise PhiPT genetic distances ranged between 0.004 (Azzaden Oussem/Matat) and 0.127 (Azzaden Oussem/Luna B).The PhiPT distances between Azzaden Oussem and Matat (0.004) and between Tizi Techt and Matat (0.012) were not statistically significant (Table 3).The Analysis of Molecular Variance (AMOVA) based on PhiPT values indicated that most of the genetic diversity occurred within populations (90%) while the variability among populations and among regions contributed 4% and 6%, respectively, to the observed genetic diversity (Table 4).The Principal Component Analysis (PCA) separated samples from Spain and Morocco into two groups along the first axis, although there was some overlap between them (Figure 1).The Mantel test based on Euclidean distances among populations revealed a weak but significant correlation among genetic and geographic distances (R 2 = 0.048, P = 0.01).Spatial autocorrelation was detected only for the smallest distance class (Figure S1). Bayesian analysis using BAPS showed the existence of 2 ancestral groups (K = 2) that were dominant in each of the two regions, Morocco and Spain (Figure 2).The two clusters were easily distinguished although there were a few migrants in both groups (2% of individuals in Morocco and 9% of individuals in Spain).Genetic admixture between the two ancestors was not detected. Discussion The analysis of 11 populations of J. thurifera using 6 polymorphic nSSR revealed high values of genetic diversity within populations and a clear separation between the populations of Morocco and Spain.This is the first study of the polyploid J. thurifera using nuclear microsatellites.Six primer pairs were developed that are useful for genotyping both J. thurifera subspecies.Our results show some geographical differentiation following the current division of this species into two different subspecies separated by the Strait of Gibraltar: J. thurifera subsp.africana in Africa and J. thurifera subsp.thurifera in Europe [9,10,7,4], although some genetic flow was detected between both regions. Polyploidy is a rare event in gymnosperm species.The genus Juniperus is the gymnosperm genus with most number of cases of natural polyploidy.There are three known cases of natural tetraploidy (J.chinensis, J. sabina and J. thurifera) and others cases of non-natural triploidy and tetraploidy (J.chinensis, J. sabina, J. squamata, J. virginiana).But J. thurifera is the only species exclusively polyploidy in the genus [30].All the analysed J. thurifera individuals had the same genome size and no differences in genome size were found between J. thurifera subsp.thurifera and J. thurifera subsp.africana.These results are in accordance with the survey published by Romo et al. [30] and suggest that polyploidy in J. thurifera happened early in its evolutionary history. The high values of within population genetic diversity found in J. thurifera are in accordance with other studies in conifers [31,32,10,4,33].Wind-pollination and dioecy might contribute largely to this high level of genetic diversity within populations. The two studied regions accounted for 6% of the variability, while between populations variation only accounted for 4% of the total genetic diversity.Weak spatial genetic structure of individuals was revealed by spatial autocorrelation analysis, likely due to the high within population genetic diversity observed for this species.The PhiPT values also showed a weak genetic differentiation between populations within each of the studied regions as previously shown by Terrab et al. [4] using AFLPs.This lack of genetic differentiation between populations within regions might be explained by active seed dispersal by animals.Thrushes (Turdus spp.), rabbits (Oryctolagus cuniculus), red foxes (Vulpes vulpes), stone martens (Martes foina) and even domestic sheeps (Ovis aries) consume J. thurifera fleshy fruits and can be effective seed dispersers for this species at medium and long distances [34].A larger distribution of J. thurifera during cold periods of the Pleistocene that allowed gene flow between the currently fragmented populations might also have contributed to the low genetic structure detected within regions [35,4]. We did find evidences of genetic differentiation between the Moroccan (subsp.africana) and Spanish (subsp.thurifera) populations of J. thurifera, likely due to the geographical barrier imposed by the Strait of Gibraltar [4,36].However, this is not an impervious barrier and the PCA indicates not only isolation by distance but also a possible gene flow between populations of both sides of the Strait of Gibraltar.This might happen through seeds transported by migrant thrushes, which are the main seed disperser of J. thurifera [37,34]. J. thurifera populations were more diverse in Spain than in Morocco as shown by the significantly higher genetic diversity and the higher number of private alleles found in this region.The higher number of private alleles found in Spain might reflect the existence of glacial refugia, as it has been shown for other conifers [38,39] or a higher historical, or contemporary, gene flow in Europe [4].Also, the lower genetic diversity in Morocco might be explained by the colonization history of J. thurifera, which is a species that originated in Europe and spread to North Africa from the Iberian Peninsula [10].Our results indicate a possible loss of genetic diversity during migration due to a bottleneck effect, environmental filters in North Africa or genetic drift.Posterior isolated evolution of the migrant genotypes would lead to the current genetic differentiation between Morocco and Spain. To sum up, genotyping of J. thurifera using nuclear microsatellites supports the separation of Moroccan and Spanish populations into two differentiated genetic groups that correspond to the proposed subspecies africana and thurifera.A high number of private alleles were found in Spain, which might indicate that these populations were part of genetic refugia during glaciations or reflect the longest isolation of the Moroccan populations.Within population genetic diversity was high in both regions while population structure was weak, which might reveal an active transport of seeds between populations and also across the Strait of Gibraltar. Supporting Information Figure 1 . 1 Figure 1.Principal Component Analysis (PCA) of the 261 individuals based on genotyping with 6-microsatellite loci.Red: samples from Morocco, blue: samples from Spain.doi:10.1371/journal.pone.0088996.g001 Figure Figure S1 Correlogram from spatial autocorrelation analysis using the correlation coefficient r by Smouse & Peakall (1999), and variable distance classes.95% confidence error bars for r were estimated by bootstrapping over pairs of samples; dashed lines (U, L) represent upper and lower bounds of a 95% CI for r generated under the null hypothesis of random geographic distribution.(DOCX) Table 1 . 1 Characteristics of the 11 primers tested for J. thurifera genotyping: locus name, forward and reverse primer sequences, motif, size, annealing temperature and fluorescent dye used. LocusAllele size rangeAnnealingnameForward primer sequenceReverse primer sequenceMotif(bp)T (6C)JT_01AATCCATCACATGCCATCTTTCCCTCATAAGAATCAATGAGATCCTA 6120-18257JT_04CCAAGGAATGATCTAACCTTTGAATGGGATGCATATCTTATCTTCCTAGA 7174-21955JT_30AATCCCCTATCCTTGCCAGTTCAACAATATCAGCAAGTAATGAGATCT 10128-22057JT_33GAGCTTCCTTTGTAGATTTTGGGGTAAGAAGACACCACTCAGTCGATCT 11177-28557JT_40GGCCGCATGATCCATTACTTCGTAACGTAATGACATGTATAGTGCCA 2098-15054JT_46TGAGATCACCTACTTCCTAGTGGACCACCAAGGGCATAGAGTTCAGG 7174-23754JT_02TTCTTCCTCACTTTTGTTGCCAGGTGCTAGGGGTGCTTGTAGAA 814855JT_03ACCCTCTATAAGGATGCTACCATGAAAAAGATAGATTGATAAGTTGAAAGGGCTT 814055JT_34CATGCATGGGTTATAATAATAGAGATATGGGCACAAATTTTAGTGTAATGAG 1211055JT_37GATGTTTGTATCATATCCTTGATTGGTCCACACCTATCGGGTTCATGT 1412355JT_38CCAACAAGCCTCCACCCTATCAAGTTTGGAAAGTGTGGTCAAC 1411455Above: loci used in genotyping, below: loci that did not show polymorphism.doi:10.1371/journal.pone.0088996.t001 Table 2 . 2 Diversity of the studied populations of Juniperus thurifera obtained from the analysis of 6 nuclear microsatellite loci. CountryPopulationNAnAe9PAHsIHMoroccoOukaimeden 119886.27200.8020.2130.129Tizi Techt22805.23010.7930.1900.115Oukameiden 220715.58000.7920.1810.112Matat19735.65100.8000.1890.117Azzaden Oussem31825.01910.7730.1870.113SpainLunaA21746.07530.8110.1930.120LunaB301016.67240.8220.2250.135Abejar24886.14610.7950.2110.129Cabrejas241006.69330.8020.2220.134Lanaja27905.75310.7890.2060.124Monegros24805.41940.7760.1970.120N = number of individuals, An = number of alleles in each population, Ae9 = average number of effective alleles per locus, PA = number of private alleles perpopulation, Hs = heterozygosity within populations, I = Shannon's Information Index, H = genetic diversity.doi:10.1371/journal.pone.0088996.t002 Table 3 . 3 Pairwise PhiPT genetic distances between the studied populations. Oukameiden 1 Tizi Techt Oukameiden 2 Matat Azzaden Oussem LunaA LunaB Abejar Cabrejas Lanaja MonegrosOukameiden 10.0010.0040.001 0.0010.0010.0010.0010.0010.0010.001Tizi Techt0.0700.0010.118 0.0070.0010.0010.0010.0010.0010.001Oukameiden 20.0300.0430.003 0.0030.0010.0010.0010.0010.0010.001Matat0.0660.0120.0510.2820.0010.0010.0010.0010.0010.001Azzaden Oussem0.0420.0220.0260.0040.0010.0010.0010.0010.0010.001LunaA0.0840.1150.0990.091 0.0970.0010.0010.0010.0010.001LunaB0.1050.1250.1220.112 0.1270.0320.0010.0010.0010.001Abejar0.0730.0940.0880.077 0.0850.0460.0560.0080.0040.002Cabrejas0.0870.0840.0960.075 0.0980.0610.0420.0220.0030.002Lanaja0.0680.0900.1060.074 0.0860.0550.0580.0230.0260.012Monegros0.0840.0760.1060.068 0.0930.0650.0480.0340.0290.019PhiPT values are shown below diagonal. Probability based on 1000 permutations is shown above diagonal. doi:10.1371/journal.pone.0088996.t003 Table 4 . 4 Analysis of molecular variance (AMOVA) showing the partitioning of genetic variation within and between regions of J. thurifera.(df= degree of freedom, SS = sum of squares, MS mean squares, Est.var.= estimate of variance, % = percentage of total variation, P-value is based on 1000 permutations).doi:10.1371/journal.pone.0088996.t004 Source ofEst.variationdfSSMSVar.%PAmong Regions199.89899.898 0.6315.80,0.001Among Population9172.09319.121 0.3943.60,0.001Within Population2502451.678 9.8069.80690.50 ,0.001 PLOS ONE | www.plosone.org February 2014 | Volume 9 | Issue 2 | e88996 AcknowledgmentsWe thank all colleagues that helped with field sampling and flow cytometry analysis.We also wish to thank Professor Mohamed Alifriqui for helping with the selection of field sites and sampling in Morocco and Dr Cristina Garcı ´a for her advice on a previous version of the manuscript.Data archiving statementThe raw data of this work (genotype at each locus for every individual) are available in the TreeGenes Database under accession TGDR011 (https://dendrome.ucdavis.edu/tgdr/index.php).This work was supported by the project MEDIATIC (PTDC/AAC-CLI/103361/2008) funded by the Fundac ¸a ˜o para a Cie ˆncia e Tecnologia (FCT) from the Portuguese Government.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Author Contributions R P Adams, Juniperus of the World: the genus Juniperus. 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Neurodegenerative disease: models, mechanisms, and a new hope Aaron D Gitler Paraminder Dhillon James Shorter Neurodegenerative disease: models, mechanisms, and a new hope 10.1242/dmm.030205EDITORIAL SPECIAL COLLECTION: NEURODEGENERATION Neurodegeneration is a feature of many debilitating, incurable diseases that are rapidly rising in prevalence, such as Parkinson's disease. There is an urgent need to develop new and more effective therapeutic strategies to combat these devastating diseases. Models from cell-based systems, to unicellular organisms, to complex animalshave proven to be a useful tool to help the research community shed light on the mechanisms underlying neurodegenerative diseases, and these advances have now begun to provide promising therapeutic avenues. In this themed issue of Disease Models & Mechanisms, a special collection of articles focused on neurodegenerative diseases is introduced. The collection includes original research articles that provide new insights into the complex pathophysiology of such diseases, revealing candidate biomarkers or therapeutic targets. Some of the articles describe a new disease model that enables deeper exploration of key mechanisms. We also present a series of reviews that highlight some of the recent translational advances made in studies of neurodegenerative diseases. In this Editorial, we summarize the articles featured in this collection, emphasizing the impact that modelbased studies have made in this exciting area of research. Introduction Neurodegenerative diseases represent a major threat to human health. These age-dependent disorders are becoming increasingly prevalent, in part because the elderly population has increased in recent years (Heemels, 2016). Examples of neurodegenerative diseases are Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, frontotemporal dementia and the spinocerebellar ataxias. These diseases are diverse in their pathophysiologywith some causing memory and cognitive impairments and others affecting a person's ability to move, speak and breathe (Abeliovich and Gitler, 2016;Canter et al., 2016;Taylor et al., 2016;Wyss-Coray, 2016). Effective treatments are desperately needed but will only come with a deep understanding of the causes and mechanisms of each disease. One way to learn about how a disease works is to develop a model system that recapitulates the hallmark characteristics of the disease. Powerful experimental model organisms such as the mouse, fruit fly, nematode worm, and even baker's yeast have been used for many years to study neurodegenerative diseases and have provided key insights into disease mechanisms (Link, 1995;Krobitsch and Lindquist, 2000;Boillee et al., 2006;Bruijn et al., 1998;Yamamoto et al., 2000;Auluck et al., 2002;Outeiro and Lindquist, 2003;Cooper et al., 2006;Gitler et al., 2008Gitler et al., , 2009Elden et al., 2010;Couthouis et al., 2011;Armakola et al., 2012;Jovičićet al., 2015;Becker et al., 2017). The more recently acquired ability to generate inducedpluripotent stem cells (iPSCs) from differentiated human cells has made it possible to generate patient-specific cell lines in a tissue culture dishgenerating human models of human disease (Han et al., 2011). Recently, there have been technological innovations that allow for these cells to be cultured in three dimensions, to produce organoids that represent various human tissues, even the brain (Marton and Paşca, 2016;Paşca et al., 2015). These threedimensional brain organoid systems permit cell-cell interactions and complex cyto-architecture to be modeled and studied in greater detail and in more physiological contexts than traditional tissue culture models with isolated cells. Furthermore, accruing evidence suggests that many neurodegenerative diseases are not merely diseases of dying neurons. Non-neuronal cells in the brain, such as glial cells, which are even more abundant in the brain and the central nervous system than neurons, play major roles in disease progression (Ilieva et al., 2009). Incorporating these neuron-glial interactions into such 3D brain organoid models will empower the elucidation of cell non-autonomous disease mechanisms. We anticipate that these 3D brain organoid systems will be a powerful addition to the disease modeler's experimental arsenal. Remarkable advances in genome sequencing technology have made it possible to read genomes of individual patients to discover causes of both rare and common genetic diseases. But which sequence variants are damaging to gene function and which are benign? Model organisms will also be powerful tools to test the effects of candidate gene variants discovered by human genome sequencing. One highly inspirational success story is the development of a therapy for spinal muscular atrophy (SMA). SMAa neuromuscular disease caused by loss-of-function mutations in the SMN1 geneis the most common genetic killer of babies. Pioneering studies of the molecular mechanisms of the disease and the development of animal models (Hua et al., 2010(Hua et al., , 2011 laid the foundation for the recent clinical trials testing antisense oligonucleotides (ASOs) as a therapeutic strategy to correct a splicing defect and restore functional SMN protein. Studies in animal model systems revealed that this therapeutic strategy could work (Hua et al., 2010(Hua et al., , 2011 and two recent clinical trials in children with SMA demonstrated that the strategy does work. In a spectacular advance, infants that received the ASO drug showed substantial improvement in motor function compared with children who did not receive the drug (Finkel et al., 2016). At the end of 2016, the United States Food and Drug Administration approved this therapy, making it the first disease-modifying treatment for SMA. A remarkable and game-changing win for model systems and especially for patients and their families. These stunning results augur well for ASO drugs that are being developed for testing in several other neurodegenerative diseases, including Huntington's disease and amyotrophic lateral sclerosis (ALS). We now have a new hope and a clear path forward for effective therapies for neurodegenerative diseases. It truly is an inspiring and hopeful time to be a researcher in this field. Our goal in compiling this special Disease Models & Mechanisms (DMM) collection, 'Neurodegeneration: From Models to Mechanisms to Therapies', is to convey the sense of excitement and hope in the neurodegenerative disease research field as well as to acknowledge the challenges ahead. To launch the collection, we present, in this issue, a number of new reviews and research articles that demonstrate the translational impact of studies involving neurodegenerative disease model systems. The collection also includes some of DMM's most-read neurodegenerationfocused research and review content from recent years. Below, we summarize the collection so far and provide a preview of what's to come in upcoming issues of the journal. Reviews: trends in neurodegeneration research We begin this neurodegeneration-focused issue with an exclusive interview with Huda Zoghbi, a professor in the Departments of Pediatrics, Molecular and Human Genetics, Neurology and Neuroscience at Baylor College of Medicine. Huda is renowned for her many groundbreaking discoveries in neurological disease research, not least her contributions to elucidating the genetic causes and molecular mechanisms underlying spinocerebellar ataxia and Rett syndrome. These insights have also shed light on the complex mechanisms at play in common neurodegenerative diseases. In this interview, Huda describes her journey from the clinic to the bench, highlighting the experiences, mentors and collaborations that helped to shape her research interests (Zoghbi, 2017). One of the founding editors of DMM, Huda has always been an advocate of the use of model systems in human disease research, and her interview particularly emphasizes the power of animal models for exploring brain disorders. Previous DMM 'A Model for Life' interviewees whose research is also focused on neurodegenerative diseases include David Rubinsztein and Rick Morimoto (all interviews can be found at: http://dmm.biologists.org/content/by/section/A MODEL FOR LIFE). Next, in the review section of this issue, Edward Lee and colleagues present an 'At a Glance' overview of RNA metabolism in neurodegenerative diseases (Liu et al., 2017). In recent years, altered RNA processing has emergedlargely through studies involving model systemsas a key contributing factor in several neurodegenerative diseases. This article and its accompanying poster illustrate how impaired RNA metabolism can underlie the pathophysiology of neurodegeneration, as well as discussing potential therapeutic interventions to target these processes. An earlier DMM 'At a Glance' poster, by Julie Valastyan and Susan Lindquist, provides a snapshot of protein-level mechanisms that have long been implicated in neurodegenerative and related disorders (Valastyan and Lindquist, 2014). Another of the new reviews published in this issue focuses in on the role of calcium signaling in Parkinson's disease. As a major global health problem, Parkinson's disease has been the focus of a high proportion of model-based research in recent years, and many such studies have indicated that disruption of calcium homeostasis is a key pathological feature. This review, written by Gabriela Caraveo and colleagues, discusses the current evidence for dysregulation of calcium-dependent pathways in this disease and potential for these findings to guide drug development (Zaichick et al., 2017). Rounding up the review section, Wim Robberecht and colleagues provide a comprehensive discussion of model systems used to study ALS, from cell-based systems to fruit flies to rodents (Van Damme et al., 2017). Thanks to the diverse range of models available to study this devastating diseaseemphasized by the many ALSrelated research papers also published in this issue (see below)many of the genes and pathways involved have been unveiled. The ALS research community is now poised to translate these findings to the clinic. A related DMM review published in 2014 highlighted the impact that zebrafish models in particular have made in improving our understanding of the pathogenic mechanisms underpinning ALS and related motor neuron disorders (Patten et al., 2014). New research: models, mechanisms and more In addition to the review content, this Special Collection showcases original research articles that impact our understanding of neurodegenerative disease mechanisms. These papers span different diseases, from common ones such as Parkinson's disease and Alzheimer's disease to rare neurological disorders. Some focus on modeling human gene defects in animals, whereas others develop biomarkers to track disease progression. In each case, they show how diverse model systems can be powerfully used to explore the mechanisms of neurodegeneration. Below, we highlight the key findings and translational implications of a subset of the new research papers published in this issue. In the first of ten new research papers, Javier Fernández-Ruiz and colleagues extend previously published observations from a mouse model of familial ALS to a canine model of the disease (Fernández-Trapero et al., 2017). Dogs can develop a type of neurodegenerative disorder called degenerative myelopathy, which can be caused by mutations in the same gene that is frequently mutated in human ALS: superoxide dismutase 1 (SOD1). The observation that cannabinoid receptor type-2 (CB 2 ) expression is upregulated in human ALS and in a mouse model of ALS caused by SOD1 mutations led the authors to test for similar effects in the dog model. Consistent with previous results, they found that CB 2 receptor expression is upregulated in the spinal cord of dogs affected by degenerative myelopathy (Fernández-Trapero et al., 2017). These results lend weight to the putative neuroprotective effect of CB 2 receptor modulation in ALS, and further support the use of canine degenerative myelopathy as a relevant model to study the pathophysiology of this disease. The dog model represents the first spontaneous animal model of ALS, offering several advantages over models that rely on transgenic overexpression. Also in this issue, Paulo Ferreira and colleagues contribute to the exciting new concept that impairments in nucleocytoplasmic transport can underlie neurodegenerative diseases (Cho et al., 2017). Using a conditional knockout approach in mice, the authors inactivated RANBP2, a regulator of the Ran GTPase cycle, which is known to power nuclear import. Selective loss of RANBP2 function from motor neurons led to ALS-like symptoms, including progressive neuron loss, weakness, and fatal paralysis. At the cellular level, nucleocytoplasmic transport and proteostasis of substrates involved in motor neuron homeostasis were disrupted, supporting the involvement of the Ran GTPase cycle in ALS pathophysiology. The new conditional mouse model generated here can be used for further mechanistic exploration (Cho et al., 2017). Loss of appetite and weight loss are common features of neurodegenerative diseases such as ALS. In another new paper, Sabine Cordes and colleagues use mouse genetics to explore the molecular basis for regulating appetite and focus their studies on a role of the tyrosine receptor kinase 3 (Tyro3) gene in regulating the neural circuitry that governs appetite . They provide evidence that Tyro3 can act as a modifier for the anorexia mutation in mouse. In addition to shedding light on the neuropathological basis of anorexia, their findings could help suggest strategies to prevent or slow weight loss that accompanies neurodegenerative disorders. The neuromuscular junction is a remarkable structure that is particularly vulnerable to neurodegenerative disease. However, it possesses powerful ways to resist injury and to regenerate. In their new DMM paper, Michela Rigoni and colleagues use an in vitro cellular model of the degenerative disease Miller Fisher syndrome to define the Schwann cell and neuron signaling events, components and cross-talk required to promote nerve regeneration in the face of peripheral neuropathies (Rodella et al., 2017). The peripheral nervous system (PNS) is also a target of neurodegenerative disease. A group of disorders called hereditary sensory and autonomic neuropathies (HSANs) are caused by PNS dysfunction. One such disorder, familial dysautonomia, is caused by mutation of the IKBKAP gene. Reported in this issue, Frances Lefcort and colleagues generate the first mouse model to study the consequence of loss of IKBKAP specifically in neurons (Chaverra et al., 2017). The mutant mice develop both autonomic and nonautonomic neuronal deficits, suggesting a role for IKBKAP beyond the PNS, to CNS development and function. Parkinson's disease (PD) is one of the most common age-related neurodegenerative disorders worldwide. The ability to predict and detect the disease presymptomatically on the basis of biomarkers is likely to give therapies the best chance of working. Non-invasive biomarkers, such as blood or imaging-based markers are ideal, because they can be analyzed longitudinally and could even be used to track the efficacy of a proposed treatment. In one of two new PDfocused studies in this issue, Georg Auburger and colleagues analyzed the blood samples of a large Turkish family affected by a PD-causing duplication of the α-synuclein gene (SNCA) (Lahut et al., 2017). They detected significant downregulation of complexin-1 (CPLX1) mRNA in the blood of presymptomatic heterozygotes at risk of PD based on prodromal features, and performed functional studies to provide insight into the role of CPLX1 in PD. Their study provides a new blood biomarker with the potential to be used in early screens for PD risk. Defining intrinsic cellular defects that characterize a particular disease state can provide mechanistic insight but could also serve as a biomarker to predict disease based on analysis of patient cells. Miguel Weil and colleagues discovered that human mesenchymal stem cells (hMSCs) isolated from the bone marrow of sporadic ALS patients have an altered response to DNA-damaging agents compared with hMSCs from healthy subjects (Wald-Altman et al., 2017). The authors connect this defect to autophagy and show that autophagy regulation is perturbed in ALS-patient cells. These findings reinforce a role for autophagy dysregulation in ALS and also provide a potential cellular biomarker of the disease. Last but not least, Laura Calzà and colleagues examined the vulnerability of neurons from a transgenic mouse model of Alzheimer's disease (AD) to oxygen-glucose deprivation (an in vitro model of hypoxia) (Baldassarro et al., 2017). This is an important question in AD research as vascular dysfunction is a feature of the disease, but the link with amyloid pathology remains unclear. Calzà and co-authors demonstrate that intraneuronal amyloid-β (Aβ) accumulation in neurons increases vulnerability to cell death upon oxygen-glucose deprivation, and interestingly, exposure of neurons to astrocyte-conditioned culture media confers protection. These results highlight important cell non-autonomous interactions between neurons and glia in Alzheimer's disease. Greatest hits Beyond the papers featured in this thematic issue, there have been several exciting papers focused on neurodegeneration published in DMM in the past year. We cannot highlight all of them because of space limitations, but we would like to mention a couple of standout papers from 2016. First, in a study examining the convoluted issue of mitochondrial dysfynction in PD, Paul Fisher and colleagues reported that patient-derived immortalized lymphoblasts show enhanced respiratory activity compared with control cells (Annesley et al., 2016). The authors propose that this trait could provide a reliable biomarker of the disease. The second study of note is by Ellen Sidransky and colleagues, and focuses on Gaucher disease. Mutation of GBA1, which encodes the lysosomal enzyme glucocerebrosidase, causes Gaucher disease. In one of the most exciting recent developments in the neurodegenerative disease field in the last several years, it has been appreciated that mutations in GBA1 are also a risk factor for Parkinson's disease. How Gaucher disease connects to Parkinson disease at the molecular and cellular level is an area of intense interest (Abeliovich and Gitler, 2016). Sidransky and her colleagues have generated a mouse neuronal cell model with nearly complete loss of glucocerebrosidase activity, mimicking the situation in Gaucher disease (Westbroek et al., 2016). This model provides the field a powerful tool to study not only Gaucher disease but also to explore potential connections with PD pathophysiology. Keep an eye out for a review by Sidransky and co-authors on iPSC models of lysosomal storage disorders in an upcoming issue of the journal. Concluding remarks We hope you enjoy this issue and the overall collection, which will be added to over the coming months. All articles can be freely accessed via this dedicated page: http://dmm.biologists.org/ collection/neurodegenerative-disorders. We dedicate this collection to Susan Lindquist, our beloved mentor, friend, and founding editor of DMM who sadly passed away in October, 2016. Among several moving tributes (Chernoff, 2017;Serio, 2017;Bevis, 2017;Morimoto, 2016;Hartl, 2016;Shorter and Gitler, 2016;Whitesell and Santagata, 2016;Fuchs, 2016;Khurana et al., 2017), a particularly touching obituary written by Vivian Siegel was published in DMM (Siegel, 2017). Sue was a visionary pioneer who was unsurpassed in her ability to harness model systems to explore fundamental biology and elucidate mechanisms of human disease. Her fearlessness led her to champion the humble budding yeast as a simple, yet powerful new model to study the cell biology underpinning neurodegeneration. This pioneering strategy allowed Sue to quickly arrive at mechanism, which could then be rapidly translated to more complex model systems. We suspect that these insights will soon reach the clinic. This article is part of a special subject collection 'Neurodegeneration: from Models to Mechanisms to Therapies', which was launched in a dedicated issue guest edited by aron Gitler and James Shorter. See related articles in this collection at http://dmm. biologists.org/collection/neurodegenerative-disorders. © 2017. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2017) 10, 499-502 doi:10.1242/dmm.030205 AcknowledgementsWe would like to thank the authors, reviewers and editors who helped to compile this special issue and ongoing collection. Particular thanks are due to Monica Justice for valuable advice, and to Steven Clapcote for handling many of the research articles published in this issue. 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Human adipose-derived mesenchymal stem cells attenuate collagen antibody-induced autoimmune arthritis by inducing expression of FCGIIB receptors Hyoju Yi Kwi Young Kang Youngkyun Kim Hyerin Jung Yeri Alice Rim Narae Park Juryun Kim Seung Min Jung Sung-Hwan Park Ji Hyeon Ju Human adipose-derived mesenchymal stem cells attenuate collagen antibody-induced autoimmune arthritis by inducing expression of FCGIIB receptors 10.1186/s12891-015-0634-yR E S E A R C H A R T I C L E Open Access Background: Adipose-derived stem cells (ASCs) are mesenchymal stem cells (MSCs) derived from adipose tissue. MSCs have multiple properties including anti-inflammatory and immunomodulatory effects in various disease models and human diseases. However, the mechanisms underlying this wide range of effects need to be explored.Methods: Collagen antibody-induced arthritis (CAIA) is a unique model in which arthritis is rapidly and strongly induced. ASCs were intraperitoneally infused into CAIA mice before or after arthritis induction. The serum levels of various cytokines, adipokines, and chemokines were measured. The expression of FC gamma receptors (FCGRs) was investigated in peritoneal macrophages ex vivo. RAW264.7 cells and ASCs were co-cultured to elucidate the direct and indirect role of ASCs on FCGR expression.Results: ASCs attenuated arthritis in CAIA mice. Serum levels of tumor necrosis factor α, interleukin (IL)-15, resistin, and leptin were reduced in ASC-treated CAIA mice, whereas serum levels of IL-6 and adiponectin were not affected. In peritoneal macrophages isolated from ASC-treated mice, expression of FCGRIIB, which is immunoinhibitory, was higher than that of FCGRI. Co-culture of ASCs with RAW264.7 cells modulated the expression of FCGRs. The expression patterns and timings of peak expression differed among FCGRs. Expression of FCGRIIB was higher and peaked earlier than that of FCGRI. FCGRIII expression was not affected by this co-culture.Conclusions: This is a study to show that ASCs have anti-arthritic effects in CAIA mice. Modulation of FCGRs by ASCs might be a therapeutic mechanism in this antibody-associated arthritis model. Background Mesenchymal stem cells (MSCs) are cells of a stromal origin that can self-renew and differentiate into various lineages of mesenchymal tissues. Furthermore, MSCs exert profound immunosuppressive effects that are superior to those of all other immunosuppressive cell types [1]. The effects of MSCs on immune cells have mostly been studied using bone marrow-derived MSCs (BM-MSCs). BM-MSCs have widespread effects on innate and adaptive immune cells [2]. BM-MSCs can inhibit CD4+ T-cell proliferation and B-cell differentiation and induce the differentiation of regulatory T-cells (T-reg) [1,[3][4][5]. In relation to innate immune cells, BM-MSCs suppress the generation of dendritic cells from monocytes [6], reduce the expression of CD80 and CD86 co-stimulatory molecules on antigen-presenting cells (APCs), and reduce the production of pro-inflammatory cytokines, such as interleukin (IL)-2, interferon-γ, and tumor necrosis factor α (TNFα), by APCs [7]. Adipose-derived MSCs (ASCs) and BM-MSCs can both differentiate toward multiple mesodermal tissue types, including bone, cartilage, and adipose tissue, are both immunosuppressive, and have similar surface protein marker expression [8][9][10][11]. However, ASCs senesce later than BM-MSCs, which may be beneficial for the treatment of chronic or persistent conditions. ASCs have a multilineage differentiation capacity and elicit immunosuppressive effects on activated immune cells [12,13]. ASCs release growth factors that are important for wound healing, modulate the immune system, decrease inflammation, and home to injured tissues [14]. ASCs may be of great clinical utility in regenerative therapies for Parkinson's disease, Alzheimer's disease, spinal cord injury, heart diseases, and rheumatoid arthritis (RA). The immunosuppressive effects of ASCs are well known. In vitro, ASCs inhibit the proliferation of activated lymphocytes via cell-cell binding and paracrine signaling [15]. Expanded ASCs have immunosuppressive properties in mice, thereby alleviating graft-versus-hostdisease and colitis [16,17]. ASCs also have antiinflammatory effects via inducing immune tolerance in a RA mouse model, namely, type II collagen-induced arthritis (CIA) mice [16]. The immunosuppressive effects of ASCs in CIA were explained by Th1/Th17 suppression and T-reg induction [16,18]. RA involves a multicellular inflammatory process, including the infiltration of lymphocytes and granulocytes into articular cartilage, proliferation of synovial fibroblasts and macrophages, and neovascularization of the synovial lining of joints. Many cellular components (macrophages, dendritic cells, neutrophils, T-cells, and B-cells), cell surface molecules, signaling components, and humoral components interact and aid the progression of RA [19]. CIA is the most commonly used arthritis model and is induced by type II collagen treatment. Collagen antibodyinduced arthritis (CAIA; induced by anti-type II collagen antibodies (anti-COL II)) is another widely used mouse model [20]. The actions of anti-collagen antibodies are initiated by direct binding to their antigens and involve immune complex formation, immune complex deposition, and activation of complement and Fc receptors [19]. Type II collagen-specific antibodies induce arthritis in the absence of T-and B-cells [21]; therefore, CAIA is considered to be a T-cell-and B-cell-independent arthritis model, in contrast to CIA. The suppressive effects of ASCs on arthritis are related to the T-cell balance in CIA mice, and the effects of ASCs on other immune cells, aside from T-cells, have not been investigated. This study investigated the effects of human ASCs on autoimmune arthritis in CAIA mice, which is not related to the T-cell immune response, and analyzed the influence of ASC treatment on serum levels of adipokines in vivo. Methods Animals and ethics All procedures involving animals were in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Guidelines and Policies for Rodent Experimentation provided by the Institutional Animal Care and Use Committee of the School of Medicine of The Catholic University of Korea. The study protocol was approved by the Institutional Review Board of The Catholic University of Korea (CUMC-2012-0033-01). Female DBA1/J mice (7-weekold; OrientBio, Korea) were purchased and housed in specific pathogen-free conditions under approved institutional guidance. Induction of CAIA and injection of ASCs Seven-week-old female DBA1/J mice (five mice per group) were intravenously injected with anti-CII (2 mg; Chondrex, WA, USA). After 3 days, 50 μg of lipopolysaccharide (LPS; Chondrex) was administered intraperitoneally to mice. As a control, the same volumes of normal saline were injected into wild-type mice instead of anti-CII and LPS (WT group). For the ASC-pre-injected group (ASC pre), 1 × 10 7 cells/mouse (in 500 μl of saline) were injected intravenously three times every other day starting 3 days prior to immunization with anti-CII. For the ASC-postinjected group (ASC post), ASCs were injected identically starting 4 days after immunization with anti-CII. Evaluation of disease severity The incidence and severity of arthritis were monitored and scored as described previously [22]. The incidence was calculated as the percentage of mice in which one or more paws were swollen. Histological evaluation of arthritis Mice were sacrificed 14 days after immunization with anti-CII. A hind limb of each mouse was removed and fixed in 10 % formalin. After decalcification in 10 % (w/ v) EDTA, the samples were embedded in paraffin. The 4 μm-thick sections were stained with hematoxylin and eosin (H&E) or toluidine blue. The inflammation score and joint destruction score were determined. Briefly, the presence of synovial hyperplasia and leukocyte infiltration in H&E-stained samples were graded in a blinded fashion by three independent observers; the sum of the two values was used as the inflammation score. The presence of pannus formation and cartilage erosion were scored and the sum of the two scores was used as the joint destruction score. Assessment of serum levels of cytokines and hormones Venous blood (300 μl) was obtained from mice on the day of sacrifice. Blood samples were incubated at room temperature and then centrifuged for 10 min at 4°C to obtain serum. Serum samples were used to assess cytokines and hormones. Resistin, gastric inhibitory polypeptide (GIP), glucagon-like peptide 1 (GLP-1), ghrelin, leptin, IL-15, IL-1α, IL-6, IL-7, TNFα, and adiponectin were analyzed using Procarta kits from Affymetrix (Fremont, CA, USA) according to the manufacturer's protocols. All specimens were assayed in triplicate. Multiplex immunoassays were performed using the Luminex 100 IS System (Luminex Corp., Austin, TX, USA). Sample concentrations were calculated from standard curves using Bio-Plex Manager 4.1.1 (Bio-Rad Laboratories, Hercules, CA, USA). Cells The murine macrophage cell line RAW264.7 was cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10 % fetal bovine serum (FBS; Gibco) and 1 % penicillin/streptomycin solution (P/S solution; Gibco). ASCs (passage 2-3) were purchased from Catholic MASTER Cells (Seoul, Korea) and maintained in DMEM supplemented with 10 % FBS and 1 % P/S solution. Isolation of peritoneal cells Five milliliters of phosphate-buffered saline containing 3 % FBS was injected per mouse into the peritoneal cavity using a 10-ml syringe. After gentle massage, the peritoneal fluid was collected using the same syringe and centrifuged at 1500 × g for 5 min. The isolated cells were used for RNA purification. Reverse-transcription PCR Total RNA was purified from cells using TRIzol reagent (Invitrogen) following the manufacturer's instruction. A Revertaid ™ First Stranded cDNA Synthesis Kit (Fermentas) was used to synthesize cDNA from RNA. PCR was performed using an iTaq DNA Polymerase Kit (iNtRON biotechnology) following the manufacturer's guidelines. The primer sequences used for PCR are as follows: 5'-TGCGGAACCAGAGCAGGGGT-3' and 5'-TTTGGGC CAGTGTTCCCGCC-3' for FCGRI (NM_010186.5), 5'-TGGGAGGTCCATCCGGAGCC-3' and 5'-TCAGGAG GATTGTCTGGAACCTGC-3' for FCGRIIB (NM_0101 87.2), 5'-TTCCACCACTGACAATTCTGCTGCT-3' and 5'-GGCCCGTGTCCACTGCAAACA-3' for FCGRIII (NM_010188.5), and 5'-GCCAAACGGGTCATCATCTC-3' and 5'-GACACATTGGGGGTAGGAAC-3' for GAPDH (NM_008084.2). Statistical analysis Statistical analysis was performed using Student's t-test. p < 0.05 was considered significant. Results ASCs ameliorate arthritis in CAIA mice To investigate the effects of ASCs on CAIA, 1 × 10 7 ASCs were injected three times into CAIA mice. The timing of ASC injection might be important; therefore, we divided ASC-injected mice into two groups: ASC pre and ASC post. In the ASC pre group, ASCs were injected before arthritis symptoms arose. In the ASC post group, ASCs were injected after LPS boosting. Both ASC-injected groups had an ameliorated disease severity score and exhibited delayed disease incidence ( Fig. 1a and b). Fig. 1 Injection of adipose-derived mesenchymal stem cells (ASCs) ameliorates arthritis in collagen antibody-induced arthritis (CAIA) mice. CAIA mice were injected with ASCs three times every other day starting 3 days prior to (ASC pre group) or 4 days after (ASC post group) immunization with an anti-collagen type II antibody. As controls, wild-type mice were injected with saline (WT group) and CAIA mice were similarly immunized but not injected with ASCs (CAIA group). Each group comprised 5 mice. The disease severity score (a, mean ± standard error of the mean) and incidence of arthritis (b) were calculated at various numbers of days following immunization ASCs prevent inflammation and bone destruction The effects of ASCs were examined histologically by H&E and toluidine blue staining (Fig. 2a). The histological inflammation and bony destruction scores were lower in the ASC-injected groups than in control CAIA mice (Fig. 2b and c). TRAP staining showed that multinucleated TRAP-positive osteoclasts (>3 nuclei) were more prominent in the joints of control CAIA mice than in the joints of mice in the ASC pre and ASC post groups (Fig. 2d). The number and area of osteoclasts were higher in the joints of control CAIA mice than in the joints of mice in the ASC pre and ASC post groups ( Fig. 2e and f ). Serum levels of TNFα, IL-15, resistin, and leptin are decreased in ASC-injected mice To understand the mechanism underlying the effects of ASCs on CAIA, we examined the serum levels of five cytokines (IL-15, IL-1α, IL-6, IL-7, and TNFα), five diabetes-related hormones (resistin, GIP, GLP-1, ghrelin, and leptin), and adiponectin using multiplex immunoassays. The serum levels of IL-15, resistin, and leptin were decreased in the ASC pre and ASC post groups (Fig. 3d, e, and f ). The serum level of TNFα was reduced in the ASC pre group, but not in the ASC post group (Fig. 3a). IL-6 and adiponectin did not significantly differ among the experimental groups ( Fig. 3b and c). ASCs induce the over-expression of Fc gamma receptors (FCGRs) in peritoneal cells T-cells and B-cells are rarely involved in disease pathogenesis in CAIA models; therefore, the immune complex is regarded as a potential player in this system. The immune complex usually signals via FCGRs. The relationship between FCGRs and stem cells has not been explored. The mRNA levels of FCGRs found on macrophages were investigated. Peritoneal cells of ASC-injected mice expressed FCGRI, FCGRIIB, and FCGRIII (Fig. 4a). Expression of FCGRIIB, which is a well-known anti-inflammatory signal mediator, was higher than that of FCGRI (Fig. 4b). The conditioned media of ASCs induces the overexpression of FCGRI and FCGRIIB in macrophages To simulate the effects of ASCs on FCGRs, ASCs and RAW264.7 cells were co-cultured. Co-culture with ASCs increased the expression of FCGRI and FCGRIIB in a time-dependent manner (Fig. 5a). Although FCGRI expression increased concomitant with that of FCGRIIB, expression of FCGRIIB increased more than expression of FCGRI, which has a pro-inflammatory role (Fig. 5b). The conditioned media of ASCs increased expression of FCGRI and FCGRIIB in RAW264.7 cells (Fig. 5c). FCGRI and FCGRIIB levels were highest at 12 hr after stimulation with this conditioned media. Thereafter, expression of FCGRs decreased in a time-dependent manner. In conclusion, expression of FCGRI and FCGRIIB on macrophages was associated with ASCs or ASC-secreted molecules. The higher expression of FCGRIIB than of FCGRI is an interesting phenomenon that may be linked with the anti-inflammatory properties of ASCs (Fig. 5d). Discussion The present study demonstrates that human ASCs can induce expression of inhibitory FCGRs on macrophages in CAIA mice. This is a novel mechanism via which ASCs elicit immunosuppressive effects. There are several mechanisms through which ASCs suppress immunity. ASCs secrete transforming growth factor β1, a growth factor that promotes premature immune tolerance [8]. Furthermore, ASC treatment increases expression of IL-10, which might enhance T-reg function, in mice with experimental colitis. In vitro, human ASCs suppress the collagen-specific response of T-cells from patients with RA. Human ASCs inhibit the proliferative response of collagen-activated T-cells, as well as the production of inflammatory cytokines by these cells, and increase the number of IL-10-producing T-cells and monocytes. Human ASCs also stimulate the generation of T-reg [23]. Human ASCs have immunosuppressive effects in CIA mice [16,18]. Systemic infusion of human ASCs reduces the incidence and severity of CIA, decreases collagen-specific Th1/Th17 cell differentiation, and induces T-reg generation. CIA is an immunologically mediated disease involving T-cells, B-cells, and inflammatory cells that infiltrate joints, whereas CAIA is a T-cell-and B-cell-independent arthritis model [21]. In the present study, ASC treatment in CAIA mice significantly decreased the severity of arthritis, but had little effect on the Th17/T-reg balance, which is a mechanism via which ASCs elicit anti-arthritic effects in CIA. This may be related to the T-cell-independent characteristic of CAIA. Our data elicited a novel mechanism in which ASCs elicit immunomodulatory effects by regulating FCGRs. FCGRs play a crucial role in antibody-mediated autoimmune diseases. FCGRs can mediate various functions, such as antibody-dependent cellular cytotoxicity and phagocytosis. FCGRs are considered to be important immune regulators linked to autoimmunity. The actions of FCGRs are mediated by activating and inhibitory receptors that bind to the same Fc portion of IgG. FCGRI and FCGRIII are activating receptors. FCGRIIb1 and FCGRIIb2 contain an immunoreceptor tyrosine-based inhibition motif that is responsible for mediating inhibitory, rather than activating, functions [24]. FCGRIIB is widely expressed on cells of the innate immune system, including mast cells, eosinophils, basophils, monocytes, neutrophils, dendritic cells, and macrophages. FCGRIIB can modulate the activities of innate immune effector cells [25]. Inhibitory FCGRIIB contributes to immune protection in two ways: (1) by down-regulating effector cell responses, and (2) by maintaining peripheral tolerance [26]. In RA, the balance between activating and inhibitory FCGR signaling controls inflammation and tissue damage [27,28]. FCGRIIB-deficient mice display a strongly augmented IgG anti-collagen II humoral response that causes a more severe arthritis phenotype than that observed in control mice [29]. Antibodies mediate pro-and anti-inflammatory activities via engagement of their Fc fragment with FCGRs. Mice lacking the common FCGR chain are highly resistant to CAIA. The absence of FCGRIIB in mice exacerbates arthritis [30]. Expression of FCGRs is dysregulated in macrophages of arthritic mice; (See figure on previous page.) Fig. 2 Injection of adipose-derived mesenchymal stem cells (ASCs) prevents inflammation and bone destruction. Collagen antibody-induced arthritis (CAIA) mice were injected with ASCs three times every other day starting 3 days prior to (ASC pre group) or 4 days after (ASC post group) immunization with an anti-collagen type II antibody. As controls, wild-type mice were injected with saline (WT group) and CAIA mice were similarly immunized but not injected with ASCs (CAIA group). Each group comprised five mice. a Hind limbs were histologically evaluated by staining with hematoxylin and eosin (H&E; scale bar, 400 μm) or toluidine blue (scale bar, 200 μm) at 14 days after immunization. From this, the inflammation score (b) and joint destruction score (c) were determined. d Hind limbs were histologically evaluated by staining with TRAP at 14 days after immunization. From this, the percentage of TRAP-positive cells (e) and the area of these cells (f) were determined. All quantitative data show the mean ± standard error of the mean. *** p < 0.001 using Student's t-test Fig. 3 Serum levels of various cytokines/hormones are reduced in adipose-derived mesenchymal stem cell (ASC)-injected mice. Collagen antibody-induced arthritis (CAIA) mice were injected with ASCs three times every other day starting 3 days prior to (ASC pre group) or 4 days after (ASC post group) immunization with an anti-collagen type II antibody. As controls, wild-type mice were injected with saline (WT group) and CAIA mice were similarly immunized but not injected with ASCs (CAIA group). Each group comprised five mice. Serum levels of tumor necrosis factor α (TNFα, a), interleukin (IL)-6 b, adiponectin c, IL-15 d, resistin e, and leptin (f) were evaluated using multiplex immunoassays at 14 days after immunization, the day on which mice were sacrificed. Data show the mean ± standard error of the mean. * p < 0.05, ** p < 0.01, and *** p < 0.001 using Student's t-test expression of activating FCGRI and FCGRIII are prolonged and expression of inhibitory FCGRII is downregulated, resulting in chronic inflammation and severe cartilage destruction [27]. Our data suggest that ASCs elicit inhibitory effects on autoimmune arthritis by controlling FCGR expression on macrophages. Inhibition of macrophages by ASCs reduced the secretion of inflammatory cytokines, such as IL-15, IL-6, and TNF-α. IL-15 is a pro-inflammatory cytokine that is over-expressed in RA and is produced by differentiated macrophages, dendritic cells, and bone marrow stromal cells [31]. IL-6 and TNF-α are wellknown pro-inflammatory cytokines that are important to the pathogenesis of RA. Suppression of these pro- Fig. 4 Adipose-derived mesenchymal stem cells (ASCs) increase expression of Fc gamma receptors (FCGRs) in peritoneal cells. Collagen antibody-induced arthritis (CAIA) mice were injected with ASCs three times every other day starting 3 days prior to (ASC pre group) or 4 days after (ASC post group) immunization with an anti-collagen type II antibody. As controls, wild-type mice were injected with saline (WT group) and CAIA mice were similarly immunized but not injected with ASCs (CAIA group). Each group comprised 5 mice. Peritoneal cells were isolated at sacrifice time. Total RNA was extracted from these cells and reverse-transcription PCR was performed with primers designed to amplify FCGRI, FCGRIIB, FCGRIII, and GAPDH (a). The intensities of the bands, relative to those of GAPDH, were determined (b, mean ± standard error of the mean) Fig. 5 Adipose-derived mesenchymal stem cell (ASCs) conditioned media increases Fc gamma receptor (FCGR) expression in macrophages. RAW264.7 murine macrophages were co-cultured with commercially purchased ASCs (a and b) or with the conditioned media of these cells (ASC-CM, c and d) for various numbers of hours. Total RNA was extracted from the cells and reverse-transcription PCR was performed with primers designed to amplify FCGRI, FCGRIIB, FCGRIII, and GAPDH (a and c). The intensities of the bands, relative to those of GAPDH, were determined (b and d, mean ± standard error of the mean) inflammatory cytokines by ASCs decreased the severity of arthritis in mice. This is the first report of the inhibitory effect of ASCs on CAIA via increased expression of FCGRIIB. However, the mechanisms underlying the induction of FCGRs by ASCs were not addressed in this study. It remains to be established whether ASCs act directly on FCGR expression. The results of the present study suggest that treatment with ASCs did not increase the serum levels of proinflammatory adipokines. The expression of most proinflammatory adipokines, including leptin, is increased in obesity and this contributes to a "low grade inflammatory state", which causes a variety of metabolic aberrations that affect joints and bone [32]. Leptin is an adipokine with pleiotropic actions that regulate food intake, energy metabolism, inflammation, and immunity [33]. A recent study reported that leptin is involved in promoting the pathogenesis, development, and/or progression of RA [34]. Resistin is also associated with increased inflammation and joint destruction in RA patients [22]. Although ASCs reduced the severity of arthritis, they could potentially cause chronic inflammation if they increase the generation of pro-inflammatory adipokines in vivo. Our data showed that ASCs attenuate arthritis without increasing the serum levels of adipokines. Conclusions In summary, the present study shows for the first time that human ASCs attenuate autoimmune arthritis in CAIA. They induce the expression of inhibitory FCGRs on macrophages and inhibit the secretion of proinflammatory cytokines without affecting the serum levels of pro-inflammatory adipokines. The regulation of FCGRIIB by ASC treatment will be the focus of therapeutic approaches to treat RA. Fig. 2 ( 2See legend on next page.) Yi et al. BMC Musculoskeletal Disorders (2015) 16:170 Abbreviations Anti-COL II: Anti-type II collagen antibody; APC: Antigen-presenting cell; ASC: Adipose-derived mesenchymal stem cell; ASC post: Adipose-derived mesenchymal stem cell-post-injected group; ASC pre: Adipose-derived mesenchymal stem cell-pre-injected group; BM-MSC: Bone marrow-derived mesenchymal stem cell; CAIA: Collagen antibody-induced arthritis; CIA: Collagen-induced arthritis; DMEM: Dulbecco's modified Eagle's medium; FBS: Fetal bovine serum; FCGR: Fc gamma receptor; GIP: Gastric inhibitory polypeptide; GLP-1: Glucagon-like peptide 1; H&E: Hematoxylin and eosin; IL: Interleukin; LPS: Lipopolysaccharide; MSC: Mesenchymal stem cell; P/S solution: Penicillin/streptomycin solution; RA: Rheumatoid arthritis; T-reg: Regulatory T-cell; TNFα: Tumor necrosis factor α. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Competing interestsThe authors declare that they have no competing interests.Authors' contributions HY, YK, NP, JK carried out the molecular studies. HJ carried out the tissue staining and immunostaining. HJ, YAR carried out animal experiments. JHJ, KYK, SHP, SMJ participated in the design of the study and performed the statistical analysis. HY, KYK, JHJ conceived of the study. KYK, HY helped to draft the manuscript. All authors read and approved the final manuscript. Human mesenchymal stem cells modulate B-cell functions. 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Retinitis Pigmentosa: Pathogenesis, Diagnostic Findings, and Treatment 10/30/2023 Saakshi P Kamde saakshikamde@gmail.com Forensic Medicine Jawaharlal Nehru Medical College Datta Meghe Institute of Higher Education and Research WardhaIND Forensic Medicine Jawaharlal Nehru Medical College Datta Meghe Institute of Higher Education and Research WardhaIND Anil Anjankar Forensic Medicine Jawaharlal Nehru Medical College Datta Meghe Institute of Higher Education and Research WardhaIND Retinitis Pigmentosa: Pathogenesis, Diagnostic Findings, and Treatment 10/30/202328FA621B788CB0A88826B6DB354B514710.7759/cureus.48006Received 07/11/2023PathologyForensic MedicineOphthalmology retinitis pigmentosa (rp)retinal dystrophyretinal diseasesdiabetic retinopathyfundus examinationtreatmentgene therapycell therapytherapeutic landscape Retinitis Pigmentosa (RP) is an inherited retinal dystrophy (IRD) that causes progressive visual loss.Patients suffering from RP have a substantial influence on their everyday activities, social contacts, and jobs, lowering their quality of life.Frequent referral delays, as well as the lack of a standard therapy for the majority of patients, contribute to the significant unmet demand for RP.Any retinal injury has the potential to result in total blindness and visual impairment.Despite the fact that there is no cure for RP, people can manage it using rehabilitation programs and low-vision gadgets.The purpose of this research is to characterize the expanding treatment landscape for RP, as well as the justification for advanced therapy medicinal products (ATMPs).Vitamin A supplements can help prevent the sluggish visual loss caused by a prevalent kind of RP.The presence of visual purple in the rods and the underlying vascular choroid causes the retina to look purplish red.The major portion of the retina damaged is the rod photoreceptor electric cell; the development of diverse diseases is progressive.Because of the retina's accessibility, immunological privilege, and compartmentalization, hereditary retinal diseases are amenable to cell and gene therapy.Therapeutic techniques that attempt to rescue photoreceptors (gene therapies) require the existence of non-functional target cells, but other therapies (cell therapies) do not require the presence of live photoreceptors.To provide successful therapy choices for RP patients at all disease phases, the development pipeline must be continually diversified and advanced, as well as ongoing efforts to encourage early patient identification and quick diagnosis.Future research will focus on avoiding vision loss in genetic eye illnesses and assisting patients in regaining their eyesight.Retinal implants, cell therapies, supplementary medications, and gene therapies may become common treatments for reducing vision loss in the future. Introduction And Background Retinitis Pigmentosa (RP) is a hereditary retinal dystrophy that causes progressive visual loss.The most prevalent inherited retinal dystrophy (IRD) is RP, which affects over 1.5 million people globally.RP is classified into two types: syndromic and non-syndromic.Non-syndromic RP begins as night blindness, develops into vision loss, and eventually falls into the peripheral vision range.Retinal dystrophies are defined depending on the location of the retina, the kind of cells damaged (rods, cones, or both), the retinal pigment epithelium (RPE), the inner retina, and whether the progression is stagnant or progressive.RP is a degenerative condition that causes individuals to lose their night vision first, followed by decreased visual acuity and a gradual narrowing of the visual field.By the age of 40, the majority of patients are legally blind.Increased difficulty completing everyday tasks and decreased autonomy are connected with worsening symptoms.Anxiety and depression levels are higher, social isolation is greater, and overall quality of life is lower.As a result, RP has a severe impact on all aspects of the patient's life, imposing a huge cost on patients, families and caregivers, and society. Progressive dystrophy only related to cones is called cone dystrophy.Only one copy of a disease allele is required for a person to be susceptible to expressing the phenotype in autosomal dominant (AD) inheritance.Two copies of a disease allele are necessary for an individual to be at risk of manifesting the phenotype in autosomal recessive (AR) inheritance.For an X-linked dominant disease (XD) in the AD inheritance, just one copy of a disease allele on the X chromosome is necessary.For a person with two X chromosomes (a female) to have an X-linked recessive disease (XR), two copies of a disease allele on the X chromosome are necessary.This is similar to AR inheritance.The electric response of the light is delayed or received in less amount in RP, as the result is given by electroretinogram (ERG) with the characteristic resemblance of 'bone spicules' in the retina [1].Usher syndrome is the most common form of syndromic RP, accompanied by loss of hearing.In non-syndromic RP, the histology findings show dilution of an outer nuclear layer as rods and cones photoreceptor cells die, an inherited retinal dystrophy.The brain interprets vision as rod and cone photoreceptors in the retina transform light into electrical impulses.Typically, weak light affects rods, resulting in night vision and peripheral vision degeneration.When cones are compromised in RP, the symptoms are central vision dimensions, color perception, and visual acuity.Genetic factors are important since this illness is caused by mutations in molecular genes.Tunnel vision is a condition in which the visual field compresses continually until just a small fraction of the center vision remains.[2][3][4].The RPE performs the primary job of maintaining the photoreceptor layer.RP can be X-linked or recessive, dominant, or part of another condition, depending on the kind of molecular variant present.Recognizing the indicators of RP at the early stage is critical for effective medical care, as it allows for optimal management.In contrast to macular degeneration in RP, central vision is retained, but night vision and peripheral vision are lost [5,6]. The study delves into the genetic heterogeneity of the disease, diagnostic methods such as electroretinography (ERG) and optical coherence tomography (OCT), and potential treatments, including gene therapy, stem cell therapy, and vitamin supplements.The overall results of generic reporting aspects of reviews applicable to this systematic review are presented in Table 1.Search terms, years searched, inclusion or exclusion criteria, description of intervention, and categories of studies included were characteristics that were given by all reviews (more than 80%).Number of 34 reviews reported irrelevant in terms of duplication and other language of selection criteria.Infrequently reported items for results included using the PRISMA diagram (59 percent) and a description of other diseases along with the intended study is (43 percent). Reporting Item Total N=32 n (%) Pathogenesis The majority of patients have symptoms of diminished peripheral vision on both an upper and lower visual field, as well as night vision loss.The dark pigmented spots are the pigments of melanin.Deoxyribonucleic acid (DNA) testing, visual field testing (VFT), ocular coherence tomography (OCT), and electroretinography (ERG) are some supportive testing methods used to determine the gene accountable for a particular type of RP called an inherited retinal dystrophy.Night blindness and peripheral vision are caused by rod cell degeneration.The eye is a non-digital camera with many elements, such as the lens, retina, and cornea.Any injury to these structures might lead to visual loss [7,8].Cyclic guanosine monophosphate (cGMP) is greatly abundant in photoreceptor cells in the dark.This cGMP causes the entrance of positive ions such as sodium and calcium, resulting in depolarization.Depolarization causes glutamate to be released in the dark.The cell hyperpolarizes as the photoreceptor moves into the light.Light enters the eye, travels to the photoreceptors, and triggers a conformational change in opsin, a unique protein.This modification stimulates a G-protein known as transducin, which subsequently activates a protein known as phosphodiesterase (PDE).The major purpose of the phosphodiesterase is to convert cGMP into 5-GMP, the inactivated form of cGMP.The cGMP-dependent cell may be a therapeutic target for hereditary photoreceptor degeneration.When exposed to light, there is a drop in the quantity of cGMP, which causes the ion channels to close and inhibits positive ions from entering.The transfer of an electric signal from the retina to the processing center located in the occipital lobe of the cerebral cortex alters the potential of photoreceptor cells.Multiple genes that get mutated produce multiple defective proteins, resulting in the illness RP.It is a hereditary disorder that disrupts the normal physiology of phototransduction, that is, the conversion of light into a change in the electrical potential across the cell membrane.AD-inherited RP is caused by mutations in pre-mRNA splicing.The X-linked RP is caused by mutations in the RPGR and RP2 genes at their respective loci.Multiple mutations result in photoreceptor cells degenerating [9][10][11]. RP is mostly caused by mutations in numerous rod-specific genes, which then cause cone degeneration and alterations in the retinal pigment epithelium and ganglion cells.The fundus examination reveals the loss in retinal blood vessels in addition to the peripheral bone-spicule deposits.ERG is the prosperous diagnostic standard, showing the reduction in the rod and the cone response that are united with a delay in their timing.The visual field (VF) loss is a crucial indicator since it shows the disease's course and the effectiveness of treatment, ranging from patchy loss of the peripheral visual region to tunnel vision, ring scotoma, and eventually leading to total blindness.With the patterned advance of retinal degeneration in disease and the development of effective medical care of retinal and choroidal vasculature the imaging information from the high-resolution, optical coherence tomography angiography (OCTA), a valuable imaging technique, has become necessary.The lyonization is termed as turned off or inactivated development of one of the X chromosomes during development; the genes present on that chromosome are usually inactivated.To date, around 260 hereditary causal genes of genetically heterogeneous blinding diseases have been identified.The photoreceptor genetic modifications lead to the secondary degeneration of cones and the primary degeneration of rods.Rod photoreceptors begin to deteriorate in blinding disorderliness, progressing to cone photoreceptor death [12,13]. The causes of vision loss are affected retinal ganglion cells (RGC) and retinal pigment epithelium (RPE), changed inner retinal vascular supply, and damage to the outer retina as a result of these alterations.The microglias accumulated in the retina's plexiform layers are principally responsible for the continual monitoring of microenvironmental homeostasis.The movement of various photoreceptor triggers quickly degenerates microglia, which finally destroys the layers and transforms into phagocytes.In addition to phagocytosis, a considerable number of pro-inflammatory neurotoxic substances are secreted when interacting with invading blood cells.Other variables, such as hemodynamics and retinal vasculature, are linked to RP.The parasympathetic and sympathetic nervous systems control the choroidal tissue, whereas the retinal blood vessels can be regulated.The trigger for blood vessel autoregulation might be hypoxic or hyperoxic, which helps maintain retinal homeostasis.Vascular malfunction, rather than the presence of photoreceptors in the retina, may be related to mortality in RP [14].According to histology, the primary cause of the illness is a deficiency in the rod and cone photoreceptors.According to pathological findings of an enucleated eye with autosomal recessive RP, two types of pigmented cells were observed in the retina: macrophage-like cells that contain melanin and veritable RPE cells that are transmigrating away from the retinal pigment epithelium layer.Photoreceptor involvement was assumed to be a reactionary response to photoreceptor injury.The outside segments, like the inner component, are poorly structured, truncated, or missing in autosomal dominant RP.The histopathology findings in RP are the perinuclear cytoplasmic membranous swirls and Inclusion bodies.The toxic level of cyclic guanosine monophosphate is raised due to a defect in cGMP phosphodiesterase [15]. When RP is advanced, there are no distinguishing features of rod and cone degeneration.The boundary that separates RP from congenital retinal sightlessness is also blurry.Congenital retinal vision impairment is difficult to identify since the ophthalmologist does not know how early and severe the visual loss must have happened in childhood.The loss of photoreceptors is measured by the ERG amplitudes and quantitative measures.The optic nerve atrophy followed by arteriolar attenuation and the changes in the pigment are the characteristics of RP.The particular genetic defect determines if rod cells are involved or the cone cells.The most common being the rod cell photoreceptor defect, cone cell dystrophy is rarely present.More than 80 genetic abnormalities and various other inheritance patterns have been identified.In the Usher syndrome, visual decline advances more rapidly than sensorineural deafness does.External ophthalmoplegia and heart block are also symptoms of Kearns-Sayre syndrome.Mental impairment is linked to the Bardet-Biedl and Laurence-Moon disorders.There is loss and breakdown of the photoreceptor cell.Arteriolar attenuation, retinal pigmentary alterations (either hypopigmentation or hyperpigmentation in the form of bone spicules and pigment clumping), and waxy disc pallor are the typical clinical trials of RP.The atrophy of the optic disc and the optical field failure are also characteristics of open-angle glaucoma.In the pathogenesis of the open-angle glaucoma (OAG), there are specific genetic variants and elevated intraocular pressure.There is the same cone vulnerability in the pathways of RP and OAG, which includes S and M cone loss.There is a similarity between the vascular dysfunction in OAG and the reduction in the blood flow in RP [16,17]. The retina's thin covering on the back of the eye is the primary source of vision and acceptance of a light beam.The retinal cells are neuroectoderm, the early embryonic ectoderm component that gives rise to the central and peripheral nervous systems.They are divided into seven layers and contain six different kinds of neuronal cells.The layers are the retinal pigment epithelium, photoreceptor cell layer, inner plexiform, nuclear region, and ganglion cell region.The role of the ganglion cell layer cells is to send visual signals to the optic nerve.The photoreceptor cell layer comprises the retina's light-excitable neurons.Cone and rod cell nuclei with synaptic connections are found in the outer nuclear and outer plexiform regions [18].The retinal pigment epithelium is the last area that lacks neuronal cells; it promotes the preservation and growth of the photoreceptor cell layer, and the effect is mitigated by soaking up additional light.Several genes become the source of the disease of the same phenotype because of genetic heterogeneity.Phenotypic heterogeneity is seen when a particular mutation happens in the identical gene, which is a different disease; for example in autosomal dominant RP, the mutation in the rhodopsin produces autosomal recessive RP.Despite its intricacy, finding RP genes and mutations has advanced significantly.The allelic heterogeneity has been seen in cases where various diseases produce mutations in each gene.Syndromic and nonsyndromic are the two types of RP.The identified 56 gene mutations become the source of non-syndromic RP.The mutated 23 genes are the AR and AD RP contains 36 mutated genes and X-linked RP has three mutated genes.Ushers syndrome is caused in the case of systemic form, with mutation observed in the 12 genes and Bardiet-Biedl syndrome is seen when the mutation occurs in 17 genes [19].The places where the RP is disseminated include the macula, fovea, mid-periphery of the retina, and the cardinal retina.The disease's clinical symptoms include night blindness, or both night blindness and a progressive loss of peripheral vision known as "tunnel vision," which leads to total blindness in maturity.Numerous diseasecausing mutations have been identified in the genes that cause RP in an individual.The discovery of genes and mutations has simplified the etiology of AD RP.The genes involved in AD RP and their functional roles are depicted in Table 2 (Adapted from [20,21]).The genetic mutation in the DNA and also in the rods and cone cells are the major cause of RP.Rod and cone cell dystrophy are the other side problems in patients suffering from RP [22]. Sr. No. Symbol Chromosome Functional Role of Gene/protein Symptoms and diagnosis The longer to adjust to the darkness is the first symptom because rod cells are affected first also known as night blindness.There is also a loss of peripheral vision at night, as well as a reduction in eyesight and tunnel vision.When you can't see items to the side without rotating your head, you have tunnel vision. Cones are frequently impacted later in the disease's progression.There is photophobia, which is an excessive sensitivity to light, and it may be difficult to see in strong lighting since the cone cells die first.A thorough history, as well as a neurologic and neuro-ophthalmic examination that takes into account visual fields, are required for the treatment of photophobia; it is a condition that causes bursts of light to shimmer or flicker [23].The majority of the symptoms manifest themselves in early adulthood or youth.The most visible sign of the condition is loss of vision.Because progressive RP is gradual, most people never lose their vision entirely.Night blindness, loss of peripheral vision, difficulty adapting to changes in light or dim light, and difficulty seeing in bad weather are the most common symptoms.The existence of peripheral vision reduces the size of the tunnel vision.Colors such as green and blue are difficult to perceive.Patients with RP have clumsiness as well as visual loss, which can be partial or full.Cataracts with fuzzy vision are one of the symptoms of the illness that might create problems in the latter stages of RP.Visual field testing is used to assess peripheral vision, visual acuity measures how well you can see progressively smaller objects, a dark adaptometery test measures how well you can see in the dark, and the ERG measures the electrical response of the retina to a light stimulus [24]. The initial indication of the condition is trouble seeing in bright or low light, as well as poor night vision. People suffering from RP also lose their visual acuity and side vision.Tunnel vision occurs when the outer vision is dark and the center of vision is contracting.An ophthalmoscope is required to check the illness since it allows the physician to view into the eye via the pupils.The orange or orange-red fundus is observed by the physician in normal eyes.The appearance of black or dark-brown spots in the fundus indicates that the person has RP.The ERG is used to confirm the diagnosis of RP; in this test, lights of various colors and intensity levels are scarcely flashed into the patient's eyes through an enormous reflecting globe.Ensure the proper functioning of the photoreceptors in the retina since there is less electric activity in that area [25].Ocular diseases or prechiasmal optic neuropathy within the visual pathway are commonly responsible for monocular or binocular visual impairment.RP symptoms include difficulties seeing details, glares, reading problems, restricted peripheral vision, and poor night vision.To assess the degree of the eyesight loss.Visual tests can be performed to determine the disease's genetic mutation [26]. Retinal rods are damaged, resulting in poor night vision that generally begins in maturity but can occur as early as childhood.The development of the ailment is early in the severe types, whereas it may occur in the fifth or sixth decades of life in the lesser variants.The narrowing peripheral vision loss is followed by tunnel vision.The condition causes a ring pattern of vision loss apparent by visual field tests, in which the peripheral ring scotoma gradually spreads, and central vision may be compromised as well.Furthermore, macular dystrophy becomes more active and can lead to blindness; patients suffer from RP report of little shimmering of flickering lights and flashes of light.Hearing loss is one of the symptoms of Usher syndrome and is related to RP in certain people.Funduscopy, in conjunction with electroretinography, is used to diagnose suspected patients with poor night vision [27]. Treatment In terms of treatment for RP, the ERG, which shows how the retina responds to vision and optical coherence tomography are used.Light mobility is used in tomography to provide detailed pictures of the retina.The technique is known as fundus autofluorescence imaging, and it involves the physician photographing the retina using blue light.The heterogeneity of RP determines the vast range of onset of symptoms.The importance of the Luxturna® results may be seen in the increased number of gene-based clinical research focusing on specific retinal degenerative diseases.The formal treatment is given to the patient; symptoms frequently intensify as they develop to the advanced stage.Luxturna is now the sole approved treatment for RP.It is an approved medication for people with mutations to overcome RPE65 deficiency.Vitamin supplements, protection from visible radiation, and visual assistance can provide the greatest supportive care.Night blindness caused by RP symptoms can be treated with vitamin A supplementation.RPE65 is required for the production of 11-cis-vitamin A during the retinal visual cycle.There is presently no cure or effective medication available to delay or stop the progression of the condition [28]. In more severe instances, the therapy is primarily focused on preventing cone photoreceptor degeneration, which prevents blindness, as well as vision improvement, photophobia reduction, and peripheral vision improvement.Inhibiting the visual cycle is a currently under-investigation strategy for treating retinal degenerative diseases.Furthermore, the night blindness associated with this medication may significantly impair patients' ability to drive at night, among other serious problems [29].Clinical trials for the novel therapy are currently underway, and persons with RP are receiving auto serum injections.The vision rehabilitation procedure and occupational therapy may aid in illness healing.In pharmaceutical innovations, voretigene neparvovec is only accessible for a small percentage of RP patients with the particular RPE65 mutation; there is no viable curative treatment for the vast majority of RP patients, with significant unmet demand.Formal therapies, such as vitamin A supplements, visual assistance, sun protection, and surgical procedures, may contribute to delaying the progression and alleviating the symptoms of RP.Vitamin A and vitamin E supplementation have been demonstrated to be useful.Night blindness and tunnel vision are symptoms that can be cured with devices such as magnifying glasses and infrared night vision.Although ultraviolet radiation might aggravate the illness, wearing sunglasses can help to protect it [30]. The correct method of capturing history can provide information on the type of rapid representation.Genetic counseling and testing can determine a child's risk for the condition.The advancements made to reduce the risk of this condition have been based on a better knowledge of the pathophysiological pathways and scientific developments in these disorders [31].Gene therapy methods for dominantly inherited disorders may face more challenging obstacles.With gene therapy and stem cell technology research into the genes causing RP and in upcoming clinical trials, there is hope for treatment for inherited RP.Stem cell transplantation-based approaches have had some success in recent years, particularly the transplantation of bone marrow-derived mesenchymal stem cells into retinal cells and the integration of cells to replace apoptotic or injured retinal cells.There is encouraging progress in clinical trials where marrow-derived mononuclear cells were injected into the vitreous of RP patients.Together, technological advances in gene and cell therapy, promising early-stage human research outcomes, and possible crucial trials suggest that some of these previously unimaginable procedures are now a predictable, near-term reality [32].Summary of all the articles included in the review is listed in Table 3. Authors Year Country Findings Poornachandra et al., [1] 2022 India Patients with retinal dystrophies may benefit from improved care in the future, as new genetic diagnostic techniques and clinical studies show encouraging results. Kannabiran C, [2] 2019 Singapore The rod and cone-dominated diseases are classified as either stationary or progressing.These disorders are further grouped based on their manner of inheritance. Fahim A, [3] 2018 USA Recent developments in imaging technologies, DNA sequencing, gene therapy, and stem cell biology are currently under investigation and stem cell therapeutics. Ferrari et al., [5] 2011 Italy The genetics of RP is complicated by the vast variation of the condition.It is discussed which genes are implicated in the formation of RP and how mutations may lead to retinal degeneration. Michaelides et al., [6] 2003 UK Enhanced understanding of the mechanics of hereditary macular dystrophy, which aided in the refinement of diagnosis, illness categorization, and prognosis, as well as enhanced genetic counselling Prelich G, [8] 2012 USA The many mechanisms of overexpression employed for reagents accessible in numerous species, as well as the significance of overexpression to human illness, are discussed. Tolone et al., [9] 2019 Europe It discusses how major development efforts would go into translating new drugs or biomarkers based on cGMP signaling, along with the drug delivery technology that goes with them. Megaw et al., [10] 2015 USA Key models used to test and treat RPGR illness and imply that gene augmentation treatment.Cell replacement treatment based on retinal progenitor cells is a longer-term promise claim. Veltel et al., [11] 2009 UK RPGR and RP2, the two gene products, were discussed, and an effort was made to link the genetic information of the patients with the functional information of the corresponding proteins. Lu et al., [12] 2021 USA Vascular alterations were shown to be significantly affected by OCTA in contrast to healthy controls.The abnormalities were associated with both choroidal atrophy and loss of central vision. Scalabrino et al., [13] 2022 UK Cone and rod medicine are examined because gene remedies for rod degenerative illnesses are likely to prolong cone-mediated vision even in the event that cone shape and density change. Lang et al., [14] 2019 USA The onset of RP has been connected to vascular abnormalities.Although PR degeneration is not primarily caused by vascular abnormalities in the fovea and parafovea Kukreja et al., [15] 2012 USA There is a discussion on the significance of the cGMP pathway.A key intracellular second messenger that controls a variety of tissue and cellular reactions. Rivolta et al., [16] 2002 USA Clinically and genetically, retinitis pigmentosa (RP) and related disorders differ.A large number of genes connected to various diseases are addressed. Natarajan S, [17] 2011 USA Concerns have been expressed concerning the possible repercussions of injecting a virus into the eye, as well as the safety of vectors. Becherucci et al., [18] 2023 USA The expanding number of current clinical trials investigating the feasibility and effectiveness of various strategies in treating RP heralds a new era in the treatment of uncommon disorders. Daiger et al., [19] 2013 USA Existing strategies for RP gene identification and mutation detection risks and outstanding issues. Wright et al., [20] 2010 USA The genetic and mechanical causes of PR cell death-induced retinal degeneration.The discovery of RPE65's isomerohydrolase fills a critical gap for understanding of the visual cycle. Hamel CP, [22] 2007 USA Management seeks to delay the degenerative process, cure problems, and assist patients in coping with the social and psychological consequences of blindness. Digre and Brennan, [23] 2012 USA Clinical features and diseases related with photophobia, the anatomy and physiology of this condition, and a practical approach to diagnosis and therapy. Hartong et al., [24] 2006 USA The results of controlled studies of dietary interventions to decrease illnesses, Newly discovered genes, research, and urgent cures are all greatly awaited.Sugawara et al., [25] 2010 USA The significant relationship between peripheral visual field loss and vision-related quality of life score determines the degree of visual field loss. Raharja and Whitefield, [26] 2022 USA A thorough first investigation is essential in determining the urgency of the recommendation. Muniz et al., [27] 2007 USA Underlines the importance of understanding how cone photoreceptors maintain ability to detect light Maguire et al., [28] 2021 USA Considerations for Luxturna administration were discussed, as well as how the Luxturna experience may lead to future gene-based therapeutics for blindness. Palczewski K, [29] 2010 USA Pharmacological vision recovery holds great potential for the development of improved therapies for those who are on the verge of blindness or have lost all of their sight. Wu et al., [30] 2023 USA This included categorization, epidemiology, clinical symptoms, prognosis, and traditional treatments. Sahel et alo., [31] 2014 USA Progress toward IRD therapeutic strategies, as well as advancements in the pathophysiological processes and technology breakthroughs. MacLaren et al., [32] 2016 USA Stem cell-derived transplants were evaluated to relieve, reverse, and restore disease processes. TABLE 3: Summary of all the articles included in the review Conclusions This comprehensive review investigates the complexities of retinitis pigmentosa (RP), an inherited retinal disorder.The article explores the pathogenesis, symptoms, diagnosis, and treatment options associated with RP.It discusses the genetic heterogeneity of the disease, the diagnostic methods like clinical symptoms, the functional test used to identify it, and the current treatment approaches, including gene therapy.While RP remains a challenging condition with no known cure, advancements in research, such as Luxturna® and ongoing clinical trials, offer hope for potential treatments.The preliminary findings are favorable, and we anticipate great growth in the coming years.Understanding the multifaceted nature of RP, from its genetic origins to clinical manifestations, is critical for both medical professionals and patients.For therapy, researchers examined the condition and developed visual aids, sunglasses to reduce contact with UV radiation, and dorzolamide or acetazolamide eye drops, as well as vitamin A and E supplements. As further studies and innovations unfold, the quest to mitigate the impact of this condition continues, and individuals affected by RP can find some solace in the ongoing pursuit of effective treatments.This article serves as a foundation for grasping the intricate landscape of RP and highlights the importance of ongoing research to improve the lives of those living with this condition. FIGURE 1 : 1 FIGURE 1: PRISMA methodology used in the study PRISMA: Preferred Reporting Items for Systematic Reviews and Meta-Analyses TABLE 1 : Reporting of articles searches, study selection, and results in systematic reviews 1 TABLE 2 : Genes mutation causes autosomal dominant retinitis pigmentosa and functional role. 2BEST1: bestrophin 1; CA4: carbonic anhydrase 4; CRX: cone-rod homeobox protein; FSCN2: fascin actin-bundling protein 2; GUCA1B: guanylatecyclase activator 1B; HK1: hexokinase 1; IMPDH1: inosine monophosphate dehydrogenase 1; KLHL7: kelch like family member 7; NR2E3: nuclearreceptor subfamily 2 group E member 3; NRL: neural retina leucine zipper; PRPF3: pre-mRNA processing factor 3; PRPF4: pre-mRNA processing factor4; PRPF6: pre-mRNA processing factor 6; PRPF8: pre-mRNA processing factor 8; PRPF31: pre-mRNA processing factor 31; PRPH2: peripherin 2;RDH12: retinol dehydrogenase 12; RHO: rhodopsin; ROM1: retinal outer segment membrane protein 1; RP1: RP1 axonemal microtubuleassociated; RP9: RP9 Pre-MRNA Splicing Factor; RPE65: retinal pigment epithelium-specific 65 kD protein; SEMA4A: semaphorin 4A; SNRNP200: smallnuclear ribonucleoprotein U5 subunit 200; TOPORS: TOP1 binding arginine/serine rich protein, E3 ubiquitin ligase Kamde et al. Cureus 15(10): e48006. DOI 10.7759/cureus.48006 Additional Information DisclosuresConflicts of interest:In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work.Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work.Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. Inherited retinal dystrophies-clinical profile & management. B Poornachandra, I Acharya, S Sangai, S Thomas, B V Priya, N K Yadav, J Vis Sci. 2022 Genetics of eye diseases: an overview. C Kannabiran, 10.1007/978-981-13-7146-2?utm_medium=email&utm_source=transactionNature. 2019Springer . 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S Natarajan, 10.4103/0301-4738.83608?utm_medium=email&utm_source=transactionIndian J Ophthalmol. 592011 The new era of therapeutic strategies for the treatment of retinitis pigmentosa: A narrative review of pathomolecular mechanisms for the development of cell-based therapies. V Becherucci, G M Bacci, E Marziali, A Sodi, F Bambi, R Caputo, 10.3390/biomedicines11102656?utm_medium=email&utm_source=transactionBiomedicines. 2023 Genes and mutations causing retinitis pigmentosa. S P Daiger, L S Sullivan, S J Bowne, 10.1111/cge.12203?utm_medium=email&utm_source=transactionClin Genet. 842013 Photoreceptor degeneration: genetic and mechanistic dissection of a complex trait. A F Wright, C F Chakarova, Abd El-Aziz, M M Bhattacharya, S S , 10.1038/nrg2717?utm_medium=email&utm_source=transactionNat Rev Genet. 112010 RPE65 is the isomerohydrolase in the retinoid visual cycle. 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Department of Plant Pathology Califonlia. Dr. Reichle's present address is Department of Botany University of California Berkeley Berkeley. Dr. Alexander's present address is Department of Biology University of California Long Beach State College Long BeachCalifornia 9FE92471EB8D5B57CF855DF1EA276776 INTRODUCTION The first report of pores in fungus septa in filamentous fungi was made by de Bary (6) in 1866. Between 1866 and about 1902, a number of papers were published dealing with fungus septations and their formation.Among these papers are reports by Woronin (23), Wahrlich (22), Ternetz (21), and Strasburger (19).Woronin (23) also reported the presence of small bodies associated with septations.This report was verified by Ternetz (21) (pp. 278, 279).Bullet (4) reviewed the early literature thoroughly and confirmed the above cited observations.The limit imposed by the resolution of the light microscope did not permit a more detailed investigation until the advent of the electron microscope.Girbardt (10), in 1958, was the first to show a septum in a Basidiomycete, which differed from the classical concept of a single-pored, simple, disk-like structure.The details of the Basidiomycete septum were later carefully worked out by Bracker and Butler (3).Septations in Phycomycetes vary greatly.Some lack septa or have them only at the base of sporangia, while others appear to have fairly complex septa when observed with the light microscope.Buller (4) reports that Rhizopus nigricans has an imperforate septum.Barrett (1) first described and illustrated the so called "pseudo-septa" in Allomyces in 1912.Coker (5), in 1930, published a photograph of such a septum.Published light and electron micrographs of Ascomycete and Deuteromycete septa show no essential difference from the earlier concept of a single-pored septum in these fungi (2,7,8,13,17).Exceptions to this are a number of reports of septa with multiple pores in the fungus component of lichens by Poirault (14), Meyer (12), Kienitz-Gerloff (11), and Strasburger (20) between 1896 and 1902, and in a short review by Smith (18, p. 51). The purpose of this light and electron microscope study is to describe multiperforate septa, in Fusarium hyphae, and spherical Woronin bodies which form plugs in the septal pores.These plugs apparently serve as safe W valves for the fungal ceils. MATERIALS AND METHODS The following species of Fusarium were used in this study: Fusarium solani f. phaseoli; F. solani f. cucmbitae (perfect stage: Hypomyces), Fusarium rigidiuscula (perfect stage: Calonectria).Both perfect stages are in the Hypocreales of the Ascomycetes.For sectioning and observation under the electron microscope, the fungus was grown on a cellophane paper disk placed on a layer of Potato Dextrose Agar (PDA) in a petri dish.Small sections of the mycelium were fixed in two ways: 1. Cold (4°C) KMnO4 for 3 minutes.2. 6 per cent glutaraldehyde in 0.1 M phosphate buffer, pH 6.8, for 4 hours in the cold, followed by thorough washing in cold buffer and fixation in 2 per cent OsO4 overnight.Both fixations 1 and 2 were followed by acetone dehydration.Uranyl nitrate in 70 per cent acetone was used as a postfixation stain.The material was embedded in Epon 812.Sections were cut on a Porter-Blum microtome and viewed in an RCA-EMU-3F. Fungus septa were isolated from mycelium and conidia in the following way: mycelium grown on PDA-agar slants was boiled in water to remove the agar, and placed in 23 M KOH, following the technique for chitin analysis described by Fuller (9).After autoclaving the KOH mixture for 3 hours, it was washed in water, neutralized, washed with 2 per cent acetic acid, and sonicated.A drop of the diluted suspension was placed on formvar grids, dried, carbonstabilized, shadowed with uranium, and examined with the electron microscope. Fungus mycelium and conidia were also degraded enzymatically.The enzyme preparation consisted of a sterile, crude filtrate obtained from a 7-day-old shake culture of Streptomyces sp.The culture was grown on a salt medium, described by Reynolds (15), to which unpurified chitin had been added as a carbon and nitrogen source.The spores and mycelium were incubated in the filtrate at 37°C for 2 to 4 days.They were then washed and placed on grids for viewing with the electron microscope. Light microscope pictures were taken with a Zeiss automatic photomicroscope, using oil-immersion, phase contrast lenses.Both live hyphae as well as isolated septa were examined.The latter were stained with acid fuchsin in lactic acid for greater contrast. RESULTS MULTIPERFORATE SEPTATIONS: The classical Ascomycete and Deuteromycete septum is a simple disk with a central pore.The septum is formed as an ingrowth from the lateral wall.Fig. 1 is a longitudinal section of a Fusarium hypha showing the pore.In Fig. 3 isolated single-pored septa from macroconidia are shown, and the pore is seen in face view.Figs. 6, 9, and l0 show similar septa in conidia where they are still attached to the lateral walls.The thickened outer rim and the central pore area are also visible under the light microscope (Fig. 7).The diameter of these septations can vary greatly depending on the part of the conidium from which they originated. In the sectioning of large hyphae, one often finds septa of the type shown in Figs. 2 and 17. Here the cross-wall is perforated a number of times.The pores are located toward the edge of the septum (Fig. 17).Figs. 4 and 5 show an isolated multiperforate septum and a septal fragment.It is interesting to note that the dense outer rim found in the single-pored septum is missing.The diameter of the pores in multiperforate septa is about the same as that of the pore of singlepored septa.The absence of the heavy rim thickening can also be seen under the light microscope (compare Fig. 7 with 8).Fig. 11 is believed to be a multiperforate septum in a large hypha which is branching.One septum and its hyphal branch are visible to the upper left coming from the larger, main hypha below.On the upper right of this main hypha one can see the face-view of a number of septal pores with the outline of the septal rim around it.The second branch hypha has been digested away above the septum by the KOH treatment.The distinct thickening of the septal rim, as in the left septum and in the septa found in conidia shown in Fig. 3, 6, 7, 9, and 10, is missing.From isolated septa it appears that in both the single-pored and multiperforate septations the area immediately surrounding the pore is somewhat more electron opaque or thicker. WORONIN BODIES: In the species of Fusarium which we have sectioned, we have found an electron-opaque body (Woronin body) which is round or oval in shape.It is generally associated with septa and especially septal pores (Figs.I, 2, 14 to 17).We have also found these bodies in Phymatotrichum omnivorum.From two to four of these bodies are close to each septal pore in electron micrographs.The number visible depends on the All illustrations are from Fusariura soIani f. phaseoli.The magnifications indicated are approximate. I~GUaE 1 Longitudinal cross-section through septal pore and one Woronin body.KMnO4.X 40,000.FrGURE 9 Same as Fig. 1.Three septal pores can be seen.KMnO4.)< !~l,000.FIGURE 3 Isolated septa from macroconldia.Note central septal pore and dense outer rim of septum.Uranium shadow.> 1~,000.FmURE 4 Isolated multiperforate septation printed as a negative to show pores in higher contrast.Note absence of outer rim of septum which has been lost during isolation.Uranium shadow.X ~0,000. B 1~ ~ E V N O T E S way in which the section passes through the pore and its adjacent areas.These bodies show little internal structure, and a distinct membrane surrounds them (Figs.15,16).Woronin bodie~ can also be readily seen with the phase-contras!microscope in young, non-vacuolated hyphae (Figs.12,13).The size of these dark structures under the light microscope corresponds approximately to the size of the structures found in electron micrographs.Woronin bodies, although much less discernible, are also visible in the light microscope without phase-contrast.They rarely move about and are closely associated with the septum. From one to six of these bodies are seen, under phase, adjacent to a septum.No attempts were made to stain them with nucleophilic stains as did Ternetz (9l) and Schrantz (16).Two other types of bodies, of similar density, were observed with the phase-contrast microscope.One is of the same size as the Woronin body and moves within a cell of the hypha, occasionally coming in contact with the cell's septa.The other is twice as large in diameter, and also moves around, rarely associating itself with the septum.A number (six to eight) of bodies which are of the same size as Woronin bodies were observed around newly forming septa in hyphal tip cells.S~PTAL PLUGS: In hyphae which have degenerated or are in the process of degeneration, or which have been damaged, a plug forms in the septal pore.This plug is of the same electron density as the Woronin bodies and has a single membrane surrounding it (Figs.14,16). DISCUSSION MULTIPERFORATE SEPTA: The reports of multiperforate septa in the fungal components of lichens at the turn of the century (11,12,14,20) have largely been ignored, except in reviews by Smith (18) and Buller (4).The pores in multiperforate septations which we have found in Fusarium are arranged in a circular pattern near the outer edge of the septum, with one or more pores in the center.The pore sizes of septa isolated by three separate methods (sonication alone, enzymatic digestion, and KOH-treatment) were approximately the same, regardless of the method of isolation.The size of pores in multiperforate septa coincides with that of pores of single-pored septa. The majority of the single-pored septa which we have found seem to have come from conidia.These septa are less readily degraded by the various isolation procedures than are the multiperforate septa.We think this is due to the fact that the conidia, from which many single-pored septa originate, are much more solidly constructed than hyphae.This may be the reason that the isolated multiperforate septum which originates in the hypha is in a much more advanced state of disintegration after isolation than the single-pored septum.Furthermore, this explains FIGURE 5 Fragment of isolated multiperforate septum, printed as a negative to show pores better.Uranium shadow.X 83,500.I~GUBE ll Face view of multiperforate septum in a large branching hypha.Branch hypha belonging to this septum has been digested away by KOH treatment.Note absence of dense rim around septum.Second branch hypha to the upper left with included septation (dark area).Uranium shadow.X 4,800. 492 BRIEF NOTES why we have had considerable difficulty in finding isolated muhiperforate septa, whereas they could be found quite easily in sectioned material.We have never observed a muhiperforate septum in a conidium, either in isolated or in sectioned material. The precise location of the muhiperforate septa in the hyphal thallus is a matter of conjecture.We have only found them in large hyphae.A possible location for such muhiperforate septum would be a conidiophore in a sporodochium in which a large amount of material has to be translocated for the manufacture of the large conidial spore masses.We have no indication as to how these septa are formed.The pores could be formed by a digestion process, in which parts of the septal wall are removed to facilitate greater translocation of materials.That wall digestion can take place is evidenced by the anastomoses of hyphae, and crozier formation in ascogenous hyphae.Another mode of formation could be the one described for the "pseudo-septa" in Allomyces by Barrett (1) in which the wall grows inward in a wagon wheel spoke-like fashion, leaving empty spaces for septal pores. SEPTAL PLUGS AND WORONIN BODIES: Buller (4) and Zalokar (94) have demonstrated that when a hyphal cell is ruptured or otherwise disturbed its pores in the end walls will close almost instantaneously, sealing off the adjacent cells very effectively.This appears to be an important device for the self-preservation of the fungus.How this mechanism functions is not clear.The electron micrographs of Shatkin and Tatum (17), Moore and McAlear (13), and Dickson (8) show that a dark plug is formed in the septal pore.The plug appears uniformly dense and is surrounded by a membrane (Fig. 16) (Dickson, Fig. 7).The plug shown by Shatkin and Tatum (17, Fig. 7) has 5 distinct layers.Our interpretation of the picture is that the section passed through the edge of the plug and pore, skimming the edge of the septal pore slightly.This accounts for the light central area in this picture.Furthermore, this light central area is clearly continuous with the septal wall to the left of the plug (Fig. 16 shows the same thing).Median sections through plugs are uniformly dark in appearance. Associated with almost all septa which we have found are round or oblong bodies.Similar bodies can be seen in electron micrographs of other authors (2,7,8,13,16).They have been interpreted as lipid bodies (7,13) and as bodies of an unknown nature associated with pores and plugs (2,8,16).They resemble septal plugs in their density and in being bounded by a membrane (Fig. 15) (2,8,16).The constant close association of these bodies with pores would make them appear to be the logical source of the plugs.When an adjacent cell is ruptured, the pressure in the cell should force one of these balls into the pore, plugging it.Several balls per pore only increase the safety factor.In a recent paper Schrantz (16) has come to the same conclusion, although he l~Gui~n 1~ Phase uficrograph of young hypha and septum of Fusarium solani f. phaseoli. Note two dark bodies (Woronin bodies) adjacent to center of septum.)< 4,800.I%GUUE 13 Same as Fig. 1~.Showing four Woronin bodies associated with septum.X 4,~00. FIGUaE 14 Septum showing two dense bodies (Woronin bodies) on each side of septum and a membrane-bounded central plug in the septal pore.Note central area of plug is lighter because the section probably does not pass exactly through the center of the pore.Glutaraldehyde-OsO4. X 58,000.shows no plugs in his pictures.Zalokar (24) reports that centrifuged hyphae of Neurospora recover within about 1 hour after centrifugation, resuming growth and protoplasmic streaming.It is not clear, though, from his paper whether this streaming takes place through pores formerly plugged or through newly formed septa.Bracket (2) assumes that septa in haustorial necks of Erysiphe graminis may be plugged at times by these balls. We believe that the round bodies are the same structures as those first described by Woronin (23) nearly one-hundred years ago.Their location and behavior are similar.Buller (4) also observed similar structures.His description differs from our observations in that he claims that these bodies move around in the vacuole of the fungus.With the light microscope, we have observed structures of the same size and appearance as the Woronin bodies, which move in the manner described by Buller.We believe, though, that they are quite separate entities, since according to our observations the Woronin bodies remain confined to the immediate vicinity of the septum.BIBLIOGRAPHY FIGVEE 6 FtGURE 8 FmuuE 9 689 FIGVEE 6 Phase micrograph of macroconidium containing folded septations, and three isolated septa (arrows).X 1,800.FIQ~'nE 7 Phase micrograph of isolated single-pored septum.Note dense rim of septum.X 850.FtGURE 8 Phase micrograph of isolated multiperforate septum.Note absence of rim around septum, similar to electron micrograph in Fig. 4. X 1,700.FmuuE 9 Part of macroeonidium showing folded septations with dense rims.X ~,300.I~GVRE 10 Part of macroconidium containing septations and isolated septa.)< 2,600. FIGURE 15 Two 15 FIGURE 15 Two Woronin bodies close to central septal pore.Note membrane surrounding balls.KMnO4.X 67,000. FmuaE 16 16 Septum showing two Woronin bodies and plug in septal pore.KMnO4.X 71,000. FIGURE 17 17 FIGURE 17 Longitudinal section through a septum at outer edge of a hypha.Note multiple pores in this area as well as dark Woronin bodies.KMnO4.× 48,000. B R I E F N O T E S B R I Z F r¢ O T r S The authors are indebted to Dr. A. H. Gold, Department of Plant Pathology, for his technical advice and for reading the manuscript, and to Dr. W. C. Snyder for providing the Fusarium cultures; to Dr. W. A. Jensen and Dr. M. S. Fuller, Department of Botany, for advice and criticism of the manuscript.The senior author would also like to thank Dr. C. E. Bracker, Purdue University, for technical advice and interesting discussions.This work was supported by the National Science Foundation Grant No. G-17599.Received for publication, July 6, 1964.SUMMARYThree structures of Fusarium hyphae are illustrated and discussed: multiperforate septa, Woronin bodies, and septal plugs.Septations with multiple perforations were found in large hyphae.Multiple perforations were demonstrated in sectioned septa, as well as in chemically and enzymatically isolated septa, under the electron microscope and also under the light microscope.Woronin bodies, which are small spherical to oblong structures, are found closely associated with septa.It is suggested that they function as safety valves, which protect the hyphal cell content by the plugging of septal pores upon injury of an adjacent cell. . J T Barrett, But, Gaz, 1912353 C E Brac:Ker, Jr, D Ph, Thesis, The Ultrastructure of Host-Parasite Relationships in the Powdery Mildew Disease of Barley. 1964University of California . C E Bracker, E E Jr, Butler, Mycologia. 351963 Researches on Fungi 5, Longmans, Green and Co. A H R Buller, 1933London; New York, Toronto . W C Coker, Journal of the Elisha Mitchell Scientific Society. 461171930 Bary De, A , Handbuch der Physiologischen Botanik. LeipzigVerlag Wilhelm Engelmann1866Flechten nnd 1Vtyxomyceten . C Delay, Compt. rend. Acad. sc. 47211963 . M R Dickson, New Zealand J. But. 1963, 1, 381 . M S Fuller, Am, But, 1960838 . M Gmeardt, Arch. Mikr. 2551958 . Kiemtz-Gerloff , F , Ber. deutsch. But. Ges. 931902 . A Meyer, But. Ztg. 1391902 . R T Moore, . H Mcalear, Am. d. But. 861962 . G Poirault, Bull. Soc. Mycol. France. 101311894 . D M Reynolds, J , Gen. Micr. 1501954 . J P Schrantz, Compt. rend. Acad. sc. 33421964 . A J Shatkm, E L Tatu~, J. Biophysic. and Biochem. Cytol. 64231959 . A L Smith, Lichens, 1921Cambridge University PressLondon . E Strasburoer, Jena Das Botanische Practicum, Gustav Verlag, Fischer, 1887 . E Strasburoer, Jahrb. wissensch. But. 4931901 . C Ternetz, Jahrb. wissensch. But. 2731900 . W Wahrlmh, Script. But. Hurt. Univ. Imp. Petropolitanae. 1893, 4, 101 . M Woronin, Abhandl. Senkenbergischen Naturforsch. Ges. 1866, 5,333 . M Zalokar, Exp, Cell Research. 191141960

J ; H Hinton Recording Secretary 41 West 32d StreetNew York New York Academy of Medicine.-At a Stated Meeting of the New York Academy of Medicine, held in New York March 20, 1862, the fol lowing preamble and resolutions were presented by Dr. A. H. Stevens, and unanimously adopted: Whereas, during the present unhappy war many of our professional brethren in service among the combatants have risked their lives, or gone into involuntary captivity, rather than desert their sick and wounded, and have exercised their skill alike on friend and foe ; .therefore, Be it Resolved, That in such conduct this Academy recognizes the true spirit -which should ever animate the ministers of humanity, and in testimony whereof, It further Resolve's, To welcome t-o its sittings those who have acted under these self-sacrificing and generous impulses.

BMC Genomics Allele-specific expression assays using Solexa Bradley J Main bradleyjmain*-bmain@usc.edu Section of Molecular and Computational Biology Department of Biological Sciences University of Southern California 90089Los AngelesCaliforniaUSA Ryan D Bickel Section of Molecular and Computational Biology Department of Biological Sciences University of Southern California 90089Los AngelesCaliforniaUSA Lauren M Mcintyre laurenmmcintyre-mcintyre@mgm.ufl.edu Departments of Molecular Genetics and Microbiology and Statistics University of Florida 32610-1399GainesvilleFloridaUSA Rita M Graze Departments of Molecular Genetics and Microbiology and Statistics University of Florida 32610-1399GainesvilleFloridaUSA Peter P Calabrese peterpcalabrese-petercal@usc.edu Section of Molecular and Computational Biology Department of Biological Sciences University of Southern California 90089Los AngelesCaliforniaUSA Sergey V Nuzhdin sergeyvnuzhdin-snuzhdin@usc.edu Section of Molecular and Computational Biology Department of Biological Sciences University of Southern California 90089Los AngelesCaliforniaUSA BMC Genomics Allele-specific expression assays using Solexa 10.1186/1471-2164-10-422BioMed Central Page 1 of 9 (page number not for citation purposes) Methodology article * Corresponding author Background: Allele-specific expression (ASE) assays can be used to identify cis, trans, and cis-bytrans regulatory variation. Understanding the source of expression variation has important implications for disease susceptibility, phenotypic diversity, and adaptation. While ASE is commonly measured via relative fluorescence at a SNP, next generation sequencing provides an opportunity to measure ASE in an accurate and high-throughput manner using read counts.Results:We introduce a Solexa-based method to perform large numbers of ASE assays using only a single lane of a Solexa flowcell. In brief, transcripts of interest, which contain a known SNP, are PCR enriched and barcoded to enable multiplexing. Then high-throughput sequencing is used to estimate allele-specific expression using sequencing counts. To validate this method, we measured the allelic bias in a dilution series and found high correlations between measured and expected values (r>0.9, p < 0.001). We applied this method to a set of 5 genes in a Drosophila simulans parental mix, F1 and introgression and found that for these genes the majority of expression divergence can be explained by cis-regulatory variation.Conclusion:We present a new method with the capacity to measure ASE for large numbers of assays using as little as one lane of a Solexa flowcell. This will be a valuable technique for molecular and population genetic studies, as well as for verification of genome-wide data sets. Background Genotype-phenotype mapping is a fundamental aim of biological science. This is critical for many goals such as understanding of how genetic architecture shapes phenotypic variation and adaptation [1][2][3], and more specific aims such as deciphering how genetic variation in humans may affect response to treatment [4,5]. Many genetic variants resulting in phenotypic differences are mediated through changes in gene expression. Thus, analyzing gene expression allows us to better understand gen-otypic variation. Variation in gene expression can be due to polymorphisms both at the gene locus (cis) and in other genes that influence its expression (trans), as well as the non-additive interactions between the two (cis-bytrans) [6]. Furthermore, epigenetic mechanisms [7], chromatin conformation [8], copy number variation [9,10] and microRNA [11] all play important roles in the transcription of a given gene. By partitioning regulatory variation into cis, trans, and cis-by-trans, we can identify their respective contributions to changes in gene expression and potentially how expression levels evolve within the genomes of complex organisms [12,13]. Allele-specific expression (ASE) studies have introduced a creative method to uncover the respective contributions of cis-and trans-regulatory variation [12,[14][15][16][17]. First, total expression differences are measured from a pooled sample of two homozygous lines. Then, cis-regulatory variation is estimated from the allelic imbalance (unequal expression of alleles) in the corresponding F1 heterozygote, where each allele is regulated by the same trans-factors [18]. Trans can then be inferred from the total expression differences that are not explained by cis. Of course, these inferences about cis-and trans-regulatory variation can be complicated if cis-elements and trans-factors interact non-additively [17,19,20]. Allelic imbalance in non-imprinted genes has been shown to be common in mice, maize and humans [18,21,22]. Also, a few studies have investigated cis-and trans-regulatory variation within and between species of Drosophila. Measuring ASE, Wittkopp et al. reported that cis-regulatory variation plays a predominant role in divergent gene expression between D. melanogaster and D. simulans [15]. Allele-specific expression has been measured using various targeted approaches including reverse transcription-PCR (asRT-PCR; [23]), and pyrosequencing [15,24]. Genome-wide approaches have also been used including serial analysis of gene expression (SAGE) [25,26], massively parallel signature sequence (MPSS) [27,21], and microarray-based methods [22,28]. Here, we introduce a simple approach to ASE assays that combines a targeted approach to gene expression assays with the power of high-throughput sequencing. In brief, transcripts of interest (containing a known SNP) are PCR enriched and barcoded to enable large-scale multiplexing. Using this approach, we sequence only regions of interest and allelespecific read counts are used to estimate ASE for large numbers of samples using a single lane of a Solexa flowcell ( Figure 1 and 2). To demonstrate its application, we investigated variation in gene expression in a set of five Drosophila simulans genes. Using a common tester line, we ASE using Solexa Figure 1 ASE using Solexa. Summary of sample preparation: (a) The forward primer is designed to anneal 1 bp upstream of the known SNP. The 5' tail contains a unique barcode and adapter sequences necessary for hybridization to the Solexa sequencing platform. (b) Model of target enrichment and allele-specific expression represented as sequencing coverage per allele. measured ASE in an equal mix of homozygotes (parental mix), a heterozygote, and an introgression ( Figure 3). By analyzing changes in ASE under these different regulatory conditions, we elucidate the respective contributions of cis, trans, and cis-by-trans interactions on variation in gene expression. Furthermore, we tested for within-species variation in cis and trans by comparing trends in ASE between six highly inbred lines collected from a single population. Results and discussion Digital ASE assay In this study, we introduce an accurate approach to allelespecific expression assays that relies on read counts generated from Solexa sequencing. For each gene of interest, a single nucleotide polymorphism (SNP) within the mRNA transcript was identified that differs between the lines of interest and our tester line. We then designed a PCR primer that annealed immediately flanking the 5' end of the SNP and another primer that annealed 200-300 base pairs downstream (Figure 1). In the primer design, we included adapter sequences, provided by Illumina, as 5' tails to allow these PCR products to be sequenced on the Solexa platform without additional steps. When we planned on analyzing more than one sample per gene, a unique forward primer was ordered for each sample that contained the common elements described above, plus a unique one to three base pair barcode sequence in the 5' tail that will allow for individual sample identification ( Figure 1a). We used a touchdown PCR cycling program to enrich each sample for our target region and then purified the amplicons using gel extraction. We then pooled the purified samples in large numbers (in our case, 300) and sequenced them in parallel on a single lane of a Solexa flow cell. The resulting reads were analyzed by assigning each read to a specific gene based on homology and sample based on the unique barcode (see methods). We can then compare the number of reads containing each SNP to have a digital representation of ASE (Figure 1b and 2). In all comparisons, both alleles were amplified in the same reaction and thus utilized the same reagents. As a Count-based ASE assays Figure 2 Count-based ASE assays. Allele-specific expression represented as sequencing reads per W-allele (black) and tester allele (white). The sequencing coverage per allele is shown on each bar and the corrected RNA (*) is represented as a proportion of each allele. The y-axis is the proportion of the difference (allele1-allele2)/(allele1 + allele2). result, each allele should theoretically maintain the same relative abundance throughout amplification. However, this may not be the case if small differences in primer hybridization or polymerase efficiency exist between alleles. We can control for this error in amplification by analyzing heterozygous DNA samples, where the actual allele frequency is 50:50, and then correcting each RNA sample by the difference observed in the heterozygote ( [15] and Additional file 1). DNA samples from each mixed sample were also analyzed in order to correct for both allele-specific amplification error and differences in cell number between the individuals extracted from each parental line [15]. We report allelic imbalance as: the proportion of reads that are differentially expressed ((allele1 -allele2)/ (allele1 + allele2)). This approach allows us to easily estimate the binomial sampling error. If there is no difference between alleles, the bias is zero, while the bias is positive if the first allele is favored and negative if the second allele is favored. To verify the accuracy of Solexa and the normalization procedure, we created three replicate dilution series using genomic DNA from the tester line and an experimental line (W line 84). Then we estimated the allelic bias at each step of the dilution (in multiplex) using a separate sequencing lane. All four genes demonstrated a strong correlation (r >0.9 p < 0.001) between the expected and observed allelic bias ( Figure 4). The gene dsx had a relatively low correlation, because there was very limited sequencing coverage for all samples within this gene. Thus, the Solexa sequencing output can be used to accurately measure the relative abundance of alleles in a given sample. By analyzing technical variation and correlation coefficients in the verification experiment, it appears that coverage on the order of a few hundred reads is sufficient to yield reproducible results ( Figure 4 and Additional file 1). Thus, increased biological replication should be favored over sequencing coverage beyond a few hundred reads (Additional file 1: Table S3). In this study, coverage varied widely between genes (Additional file 1: Table S1), while coverage of individual assays within each gene was similar (Additional file 1: Table S3). This is most likely due to variation in initial transcript abundance, variation in amplification efficiency between genes and the resulting uneven pooling of DNA between genes. Therefore, we suggest that if the pooling of DNA is done carefully based on the concentration of each purified sample or PCR amplicon band intensity, the sequencing coverage can be more evenly distributed. This will also increase the number of high-quality ASE assays that can be performed on a single lane of Solexa. The cost per base of sequence using Solexa is very low, so for large-scale projects, the preparation costs such as barcoded primers become the limiting economic factor. To address these concerns, we suggest one of the following Nucleic acid samples approaches depending on the type of project: For studies where many assays are performed with a few select genes, barcode costs can be significantly reduced if paired-end sequencing reads are combined with multiplicative barcoding. For example, instead of ordering 900 barcoded adapters for a given gene, we can create 900 unique barcode combinations using only 30 barcoded forward primers and 30 barcoded reverse primers. For studies involving large gene sets, barcoded adapter sequences can be added to typical gene-specific annealing primers before PCR using single-strand ligation. Using this approach, barcoded adapter sequences can be used in multiple experiments. We have shown that Solexa can effectively estimate ASE using this targeted approach and we mention these additional modifications to allow easier adaptation for future researchers. Error analysis To understand the error in quantifying ASE, we looked at sources of variation at multiple levels. First, we estimated sequencing error at the SNP position by identifying the proportion of reads with an incorrect base-call. Sequencing error was well below 2% for most of the genes. Furthermore, this error rate did not seem to change when the position of the SNP within the read shifted due to barcode lengths changing from one to three base pairs (Additional Verification of ASE assays using Solexa Figure 4 Verification of ASE assays using Solexa. Each data point represents one of three replicate dilutions analyzed at each step of the dilution series (9:1, 8:2, 7:3, 5:5, 3:7, 2:8, 1:9). The distribution of sequencing reads within each gene is demonstrated as follows: mean ± SE (n = 21). DSX = 11.3 ± 3.8, CG2604 = 2,478.3 ± 288.8, CG10824 = 2,756.4 ± 333.5, CG11459 = 11,578.6 ± 2515.1. file 1: Table S2). Second, we estimated the binomial sampling error and found that this error quickly becomes negligible with the high coverage obtained with this method (Additional file 1: Table S3). Third, we assessed the error introduced by the method, such as polymerase and reverse transcription error by comparing allelic imbalance between technical replicates (cDNA templates created separately from the same RNA pool). This technical variation was considerably more than binomial sampling once coverage was over a few hundred reads (Additional file 1: Table S3). And finally, to analyze overall variation in expression estimates, including dynamic gene expression in vivo, we compared ASE estimates between biological replicates (separately collected material from the same genetic cross). The biological variation was greater than technical variation in this study, but an accurate assessment of the relative magnitude of technical and biological variance is beyond the scope of this study and thus, both should be considered when designing future experiments (Additional file 1: Table S3). ¡ ¡ ¢ ¡ £ ¡ ¤ ¡ ¥ ¦ ¡ § § § © § § § § ! " # " $ % & ' ( ) 0 1 2 3 4 '& 3 0 ¡ ¡ ¢ ¡ £ ¡ ¤ ¡ ¥ ¦ ¡ § § § © § § § § ! " # " $ % & ' ( ) 0 1 2 3 4 '& 3 0 ¡ ¡ ¢ ¡ £ ¡ ¤ ¡ ¥ ¦ ¡ § § § © § § § § ! " # " $ % & ' ( ) 0 1 2 3 4 '& 3 0 ¡ ¡ ¢ ¡ £ ¡ ¤ ¡ ¥ ¦ ¡ § § § © § § § § ! " # " $ % & '( ) 0 1 Cis-and trans-regulatory variation within species We used six highly inbred lines of D. simulans from Winters, CA (W Lines) and a scarlet ebony (st e) tester line in this study. For each W line, we compared expression to the tester line in a parental mix (i.e. an equal mix of homozygous tester and W line flies), the related F1 heterozygote, and a corresponding introgression (see methods for details) (Figure 3). This experimental design allows us to assess intraspecific regulatory variation in cis, trans and cis-by-trans. To do this, we estimated the overall expression differences in four genes in the aforementioned parental mix. Then, we determined cis-regulatory variation from the allelic imbalance present in the related F1 heterozygote, where trans-factors affect each allele equally. We can then infer trans from the overall expression difference that is not explained by cis. In the four genes analyzed, cis-regulatory variation explains the majority of the overall divergence in gene expression between the tester line and the W lines ( Figure 5, regression coefficient = 0.91 ± 0.13, [12]). It should be noted that this is a small gene set and most of these genes have been shown to be variable within-species (see methods). Thus, we do not consider this result to be a reflection of the genome as a whole. One explanation for the small contribution of trans in these genes is that trans-variation has an increased potential for pleiotropic effects, some of which may be deleterious and removed by purifying selection in the W lines tested. Gene expression is a result of cis-regulatory elements interacting with trans-regulatory proteins. If there is variation in both cis-elements and trans-proteins, there is the potential for non-additive interactions (cis-by-trans) [29]. To test for cis-by-trans, we compared allelic imbalance in the heterozygote and the introgression within each W line ( Figure 3). If all genes act additively, allelic imbalance in the heterozygote and introgression should be equal because the cis-regulatory elements for each allele and the trans-factors within each genotype are identical. If allelic imbalance between these genotypes is not equal, that difference is due to cis-by-trans. We lacked the statistical power to individually detect these cis-by-trans interactions, but we were able to test for a systematic shift in expression between these genotypes across all genes and W lines. We hypothesized that if the cis-elements and trans-factors within the tester line and the W lines coevolved, non-additive interactions would be relatively common and therefore we might detect significant cis-bytrans effects across all genes and lines. To test for this, we analyzed the relationship between heterozygous and introgression ASE values for the 6 W lines and 4 genes using a linear model. The regression coefficient is not significantly different from one (p = 0.5) and thus, there is no systematic cis-by-trans interactions detected within our sample population and selected genes. This result, together with recent findings seems to be indicating that at least within Drosophila, epistasis may be rare or is small in magnitude [17]. This may also be due to the lack of Intraspecific regulatory variation due to cis and trans Figure 5 Intraspecific regulatory variation due to cis and trans. Expression bias is the proportion of the difference between alleles: (allele1-allele2)/(allele1 + allele2). The parental mix is plotted on the x-axis and the F1 heterozygote is plotted on the y-axis. population structure in Drosophila, which would hinder the co-evolution of divergent cis-elements and trans-factors. To compliment our previous estimates of cis and trans between the tester line and the W lines, we measured variation in cis between W lines in order to give additional insight into the type and magnitude of regulatory variation that may be segregating in natural populations. To identify this variation we tested for a significant effect of a given W line on the overall estimate of cis and trans. Using this approach we were unable to detect significant variation between lines (p = 0.31). We did however find evidence for variation between the W lines and the tester line suggesting that genetic variation may be more common between populations (Figure 2). Conclusion We have presented a new method that uses Solexa to accurately measure ASE for hundreds of samples using as little as one lane of a Solexa flowcell. This will be a valuable technique for analyzing a few genes for many individuals or under many conditions, measuring a large selection of genes for a few individuals, and verifying ASE estimates from genome-wide expression assays. (Figure 3). To create the parental mix, homozygous male flies from a given W line were homogenized together with homozygous male flies from the tester line. The heterozygous genotype was the F1 male progeny from a cross between a given W line male and two virgin tester line females. And finally, to construct introgression lines, F1 heterozygous females were created from an initial cross between a given W line female and st e male. Then they were backcrossed to the st e line. After each generation, wild-type females were selected (heterozygous genotype at both markers) for subsequent backcrossing. This process was repeated for a minimum of 20 generations to create an introgression line. Thus the introgression line is heterozygous for approximately 12 Mb between the genetic markers st and e on the third chromosome, while the remainder of the genome is homozygous for the st e line (Figure 3). Two replicate introgression lines were created in parallel for each W line (introgression A and B) to control for expression differences due to different introgression cutoff points flanking the visible markers. Reciprocal crosses were not analyzed, but the effects of genomic imprinting, the only type of parent-oforigin effects that would impact ASE, are likely rare or do not exist in adult Drosophila [31]. All flies were reared at 25°C with a 12 h:12 h light cycle. Methods Fly strains and genotypes Gene selection We chose a set of genes within the introgressed region on chromosome three for further analysis (Figure 3b). Genes were chosen based on current interest in the lab (dsx) and previous analyses showing them to vary within species (Cyp21c, CG10824, CG2604) [30] or between species (CG11459) [15]. We resequenced most of the coding region of each gene and then designed our gene-specific primers based on the location of a single nucleotide polymorphism (SNP) between the W lines and the st e tester line. The Cyp21c primers non-specifically amplified a homologous gene (Cyp4ac3), thus it was not analyzed further. Extraction of nucleic acids and cDNA synthesis We extracted DNA and RNA in parallel from 14 wholebody adult male flies for each sample using a modified protocol for Promega's SV Total RNA Extraction Kit [15]. Experimental samples included a parental mix and a heterozygous DNA sample, along with cDNA from the parental mix, F1, and introgression A and B (see above) for each W line. We included replicate biological samples (separately collected material from the same genetic cross) for all experimental samples involving W line 181 and 84. Also, we included technical replicate (cDNA templates created separately from the same RNA pool) samples for all biological samples involving W lines 84 (Additional file 3). To extract nucleic acid from the parental mix, we homogenized seven homozygous male flies from a given W line together with seven homozygous male flies from the st e line. The heterozygous and the introgression genotypes were extracted from 14 male progeny. Before extraction, adult male flies (three to five days old) were snap-frozen in liquid nitrogen in the morning and stored at -70°C until extraction. Briefly, we passed the supernatant from fly homogenate through an affinity column optimized for binding DNA. Then the flow-through was run through another column optimized to bind all RNA. We then treated RNA columns with DNAse, followed by subsequent washing steps and elution. Using Applied Biosystem's (AB) High-Capacity cDNA Reverse Transcription Kit we immediately synthesized cDNA from the RNA samples using AB's standard protocols with RNAse inhibitor. Following extraction, we stored all cDNA and DNA samples at -70°C until further preparation. PCR and purification PCR primers for the ASE assay were designed such that one annealed immediately flanking the five prime (5') end of the SNP to be sequenced and the other, 200-300 base pairs downstream (3'). Then we modified each primer design with a custom 5' tail consisting of Solexa adapter sequences. Furthermore, to allow for multiplexing, a sample-specific barcode (one to three base pairs) was added to the design between the adapter and the annealing primer (Figure 1 and Additional file 2). All PCR reactions were performed using Finnzymes Phusion High-Fidelity DNA Polymerase and a touchdown cycling program stepping down 1° each cycle from 70°C to 60°C, followed by an additional 25 cycles at 60°C annealing. Samples were run on a 2% agarose gel for amplicon size confirmation and agarose gel purification. PCR products were gel purified using Qiagen's Qiaex II Gel extraction kit and standard protocols. Following purification, a portion of each of the 300 PCR enriched samples was pooled together, ethanol precipitated and then resuspended to a concentration of 10 nM. The pooled sample was sequenced at Cornell's core sequencing center using a custom primer. Data analysis Using a custom Perl script, the 6 million reads generated from a single lane of a Solexa flow cell were assigned to one of our five genes based on the first eight base pairs (allowing for one mismatch) of the annealing primer. Within each gene pool, the reads were then separated into 60 unique ASE assays using the one to three base pair sample-specific barcodes ( Figure 1). We then checked for a known sequence surrounding the SNP, including five base pairs 5' of the SNP and eight base pairs 3' of the SNP. If these 13 base pairs (5 bp pre-SNP + 8 bp post-SNP) could not be identified (allowing for a single mismatch) the reads were discarded. We then scored each read for the nucleotide in the SNP position to determine the allelespecific read count (Additional file 3). To determine the respective contributions of cis-and transregulatory variation, we compared ASE in the parental mix flies and the heterozygous flies. ASE differences in the parental mix reflect total variation in gene expression, including cis and trans. In contrast, trans is shared between alleles in the heterozygote, thus only cis contributes to ASE differences in this genotype. As a result, trans can be inferred from the percent of the total variation estimated in the parental mix (cis+trans) that is not explained by the variation estimated in the heterozygote (cis). To perform this test this we used the model: Y ij = μ+BX ij +e ij where for the i th W line and the j th gene, Y is the estimated difference between alleles in the heterozygote (cis) and X is the total difference in expression estimated from the parental mix. Then, we estimated trans-regulatory variation from the percent of the overall expression differences found in the parental mix that are not explained by this model ( Figure 5). Missing data points are due to lack of a SNP within a given gene and W line. To identify cis-by-trans interactions (i.e. non-additive effects), we tested whether the allelic bias in heterozygotes was significantly different from the allelic bias in introgressions. To examine this effect, we fit the model: Y ij = μ+BZ ij +e ij where for the i th W line and for the j th gene, Y is the estimated difference between alleles for the heterozygote and Z is the estimated difference between alleles for the introgression line. The cis-regulatory elements for each allele in the heterozygote are identical to the cis-regulatory elements in the introgression, hence the allelic bias cannot vary due to cis between these genotypes. Furthermore, although the trans-factors change between genotypes (heterozygous W/st e to homozygous st e) the trans-factors are shared equally between alleles within each genotype and thus, differences in trans cannot contribute to allelic bias. As a result, any change in allelic bias between these genotypes must be due to cis-by-trans interactions. To test for natural variation (i.e. variation between W lines) in the relative contribution of cis and trans, we fit the model Y ij = μ+B 1 X ij +B 2 L i +e ij where for the i th line and the j th gene, Y is the average bias between alleles for the parental mix, and X is the average bias between alleles for the heterozygote. We then tested for the effect of line. To verify the accuracy of ASE assays using Solexa, we used a separate lane of a flow cell to analyze three replicate dilution series (1:9, 2:8, 3:7, 5:5, 7:3, 8:2, 9:1). These dilutions were created using genomic DNA extracted separately from homozygous W line 84 and the homozygous st e tester line ( Figure 4). Each of the four genes analyzed in this study were tested for the ability to accurately detect known allele frequencies. Heterozygous DNA from a cross between these lines was also analyzed in parallel to correct for allele-specific amplification bias ( [15] and Additional file 1). To correct for differences in starting concentration of the homozygous DNA used for the dilution series, we corrected the expected values by the average allelic bias found in the 1:1 mix [15]. Correlations between the known dilution and the measured ASE using Solexa were estimated separately for each gene using Pearson's correlation coefficient [32]. Figure 3 3Nucleic acid samples. The three nucleic acid sample types used in this study: the parental mix, F1 heterozygote, and introgression lines. (a) Representation of all chromosomes for each genetic background assayed. Red represents the W line and black represents the tester line. (b) Zoom-in of chromosome 3 for each genotype. All genes analyzed are between the genetic markers scarlet (st) and ebony (e). A 1:1 relationship would indicate 100% cis and a slope of zero would indicate 100% trans. A 1:1 line is shown for reference. The vast majority of the expression differences between the W lines and the tester line are attributed to cis,for the four genes and six lines tested (regression coefficient = 0.91 ± 0.13). Color code: line 84 -blue, line 181 -red, line 147 -purple, line 192 -orange, and line 68 -green. (page number not for citation purposes) AcknowledgementsWe would like to thank Hyo-sik Jang for fly work. Also, we thank Joe Dunham and Johanna Main for discussions and comments on the method and paper, respectively. This work was supported by NIH grant RGM076643 (SVN) and 1R01GM077618 (LMM, SVN).Authors' contributionsBJM conceived the method and performed the experiments.SVN Quantitative genetic analyses of complex behaviours in Drosophila. R Anholt, T Mackay, Nat Rev Genet. 5Anholt R, Mackay T: Quantitative genetic analyses of complex behaviours in Drosophila. Nat Rev Genet 2004, 5:838-49. Genetic basis of fitness differences in natural populations. 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