Influenza virus infection is an important cause of under-five mortality. Maternal vaccination protects children younger than 3 months of age from influenza infection. However, it is unknown to what extent paediatric influenza-related mortality may be prevented by a maternal vaccine since global age-stratified mortality data are lacking.We invited clinicians and researchers to share clinical and demographic characteristics from children younger than 5 years who died with laboratory-confirmed influenza infection between January 1, 1995 and March 31, 2020. We evaluated the potential impact of maternal vaccination by estimating the number of children younger than 3 months with in-hospital influenza-related death using published global mortality estimates.We included 314 children from 31 countries. Comorbidities were present in 166 (53%) children and 41 (13%) children were born prematurely. Median age at death was 8·6 (IQR 4·5–16·6), 11·5 (IQR 4·3–24·0), and 15·5 (IQR 7·4–27·0) months for children from low- and lower-middle-income countries (LMICs), upper-middle-income countries (UMICs), and high-income countries (HICs), respectively. The proportion of children younger than 3 months at time of death was 17% in LMICs, 12% in UMICs, and 7% in HICs. We estimated that 3339 annual influenza-related in-hospital deaths occur in the first 3 months of life globally.In our study, less than 20% of children is younger than 3 months at time of influenza-related death. Although maternal influenza vaccination may impact maternal and infant influenza disease burden, additional immunisation strategies are needed to prevent global influenza-related childhood mortality. The missing data, global coverage, and data quality in this study should be taken into consideration for further interpretation of the results.Bill & Melinda Gates Foundation. Influenza infection was associated with an estimated 15,300  in-hospital acute lower respiratory infection (ALRI) deaths in children younger than 5 years in 2018, of which 36% occurred in infants under 6 months. The potential impact of maternal vaccination on paediatric mortality is unclear as age-stratified data under 6 months at time of death are lacking and maternally-derived influenza antibodies provide protection up to the first 3 months of life. We searched for articles published in English from January 1, 2014 to February 2, 2020 using PubMed and search terms concerning influenza infection and childhood mortality. Publications originated from high-income countries (HICs) only and there were no multi-country case series.To our knowledge, this is the first global case series of children who died with laboratory-confirmed influenza infection reporting age-stratified data in young children. We provide clinical and demographic characteristics of 314 paediatric in-hospital deaths with laboratory-confirmed influenza infection from 31 countries. In our study, 91 children (29%) were from LMICs. Less than 20% of children dying in-hospital with influenza were younger than 3 months at time of death and children from LMICs were younger (8·6 months) than children from HICs (15·5 months) at time of death.Our data show that maternal vaccination may prevent a higher proportion of influenza-related in-hospital deaths in children in LMICs than in other World Bank income regions. However, even in LMICs, more than 80% of children are 3 months or older at time of influenza-related in-hospital death. This implies that a maternal vaccine could maximally prevent a small proportion of global paediatric influenza-related in-hospital mortality and other strategies are expected to have greater impact. By applying the proportions of children younger than 3 months at time of death to global mortality estimates, we calculated that maternal vaccination can potentially prevent up to 3339 (UR 1287–9886) influenza-related in-hospital deaths in children younger than 5 years annually. The missing data, global coverage, and data quality in this study should be taken into consideration for further interpretation of the results.Alt-text: Unlabelled box1Influenza virus is an important cause of mortality due to acute lower respiratory infection (ALRI) in young children. In 2018, influenza was associated with an estimated 15,300  in-hospital deaths in children younger than 5 years of age globally Paediatric influenza vaccines are licensed for children aged 6 months and older In the secondary analysis of a maternal influenza vaccine trial duration of protection by maternal vaccination was shown to be limited to the first 3 months of life due to the decline of maternally-derived antibodies To gain insight into the age distribution and clinical characteristics of influenza-related mortality in young children worldwide, we initiated the FLU Global Online Mortality Database (FLU GOLD) study. Using retrospective data, we determined the characteristics and age distribution of children younger than 5 years who died in-hospital with laboratory-confirmed influenza infection worldwide. Applying the proportions of children younger than 3 months at time of death to global mortality estimates, we estimated the potential impact of maternal influenza vaccination on in-hospital paediatric-related death.22.1n = 8) and children with influenza-related mortality after stem cell transplantation. Additionally, we searched the literature using PubMed for “influenza” combined with “death”, “deaths”, “died”, “mortality”, “fatality”, or “case fatality ratio (CFR)” and “pediatric”, “pediatrics”, “child”, or “children” and invited authors to share additional (unpublished) cases. Collaborators were invited to share data between October 13, 2017, and March 31, 2020 through a link to a questionnaire  in Research Online, an electronic data capture platform The FLU GOLD study was initiated in October 2017. We invited our existing global respiratory syncytial virus (RSV) GOLD network 2.2We collected demographic and clinical characteristics and compared these between children from different income groups. Countries of origin were categorised as LMIC (LIC and LMIC combined), upper-middle-income countries (UMIC), and HIC according to the World Bank classifications for 2020 2.3We calculated age at influenza infection by subtracting the number of days between onset of influenza-related symptoms and influenza-related death from age at influenza-related death. We then determined the proportion of children under 3 months of age at time of influenza infection.2.4We differentiated between children with community-acquired and hospital-acquired influenza infection. In case the setting where influenza had been acquired was not provided, and if there were no strong indications of nosocomial infection based on timeframe and clinical disease course, we assumed the infection had been community-acquired. We assumed that deaths occurred within the hospital if data on location of death were missing.2.5To evaluate the minimum expected impact of maternal vaccination on influenza-related deaths assuming 100% vaccine efficacy and complete vaccination coverage, we multiplied the proportion of children younger than 3 months at time of community-acquired in-hospital death by the estimated total number of global influenza-associated ALRI in-hospital deaths under 5 years of age for each World Bank income group 2.6n = 145) by excluding these countries from our analyses.We compared demographic and clinical characteristics between different income groups, excluding cases with missing data for comorbidities or prematurity. We performed subgroup analyses and compared characteristics for children with hospital-acquired and community-acquired influenza-related death. Furthermore, we differentiated between seasonal and pandemic influenza-related deaths by excluding children with influenza A(H1N1)pdm09 who died within the timeframe of the WHO-declared pandemic (June 2009 - August 2010) from the analyses. Lastly, we analysed to what extent our results were sensitive to the contribution of a large number of cases from Ecuador, United Kingdom, Kenya, Turkey and South Africa .We report descriptive statistics for all variables. Continuous variables are presented as median with interquartile ranges (IQR). Categorical variables are presented as frequencies and proportions. We used the χ2.9The funder had no role in study design, data collection, data analysis, data interpretation, writing of the report, or the decision to submit the paper for publication. YNL, NIM, FSV, and LJB had full access to all the data in the study and NIM had final responsibility for the decision to submit for publication.3We obtained data from both published and unpublished cases and included 314 influenza-related deaths from 6 WHO regions and 31 countries  across the world  had mothers who had been vaccinated against influenza during pregnancy. Age at death for these children was 15 and 16 months, respectively. Type of diagnostic influenza tests used and additional characteristics for children from different income countries are included in Supplemental Tables 2 and 3, respectively. Co-pathogens were identified in respiratory samples of 114 children. RSV was the most common co-infection .Comorbidities were present in at least 166 (52·9%) children and 41 (13·1%) children were born prematurely . Paediat3.2p<0·001). The age distribution for preterm children without comorbidities, term children with comorbidities, and healthy term children is shown in Supplemental Figure 1.Globally, more than 80% of paediatric influenza-related deaths occurred after the first 3 months of life. The proportion of children younger than 3 months at time of death was 16·5% in LMICs, 12·2% in UMICs and 7·4% in HICs A. The pr3.3p = 0·09 and p = 0·14, respectively). More children with hospital-acquired influenza-related death were born preterm as compared to children with community-acquired influenza-related death . Although timing of intensive care unit (ICU) admission in relation to influenza infection was unknown, all children with hospital-acquired influenza-related death had been admitted to the ICU. This differed from children with community-acquired influenza-related death, of which 70·1% had been admitted to the ICU (p<0·001).We separately described variables for 38 (12·1%) children with hospital-acquired influenza-related death and compared these to children with community-acquired influenza-related death . Age at death, and time between onset of influenza-related symptoms and death did not differ significantly between both groups  influenza-related in-hospital deaths occurred annually in children younger than 3 months .Table 2G3.5n = 38) from the analyses, differences in characteristics between income groups remained unchanged, except for the length of hospital and ICU stay, and the presence of comorbidities between children from LMICs and UMICs .When excluding children with hospital-acquired influenza-related mortality (n = 150). Results for age distribution remained unchanged, including a higher age at death for children from HICs (14·5 months [IQR 6·1–28·6]) than from UMICs (12·0 months [IQR 4·0–28·9]) and LMICs (9·2 months [IQR 4·0–23·2]) , although the difference in age at death for children from HICs compared to children from LMICs was no longer significant. Furthermore, when excluding 26 children with influenza A(H1N1)pdm09 virus infection who died within the timeframe of the WHO-declared pandemic (June 2009 - August 2010), conclusions did not change .We also analyzed our data after excluding children with missing data for prematurity and comorbidities (n = 145), as these accounted for almost half of all influenza-related deaths (n = 89), median age at death for children from UMICs increased to 23·5 months (IQR 9·9–39·0) and there were more children with comorbidities from HICs (62·8%) as compared to children from LMICs  .Lastly, we analysed our data after excluding children from Ecuador, Kenya, United Kingdom, Turkey, and South Africa pdm09 virus were proportionately higher in HICs than LMICs. This is likely caused by publication bias since there were more studies on influenza A(H1N1)pdm09 virus available from HICs than from LMICs. Furthermore, information on influenza subtype in LMICs was not always available, which also may have contributed to this difference.Beyond the scope of this paper, we compared our data to 358 previously reported cases from the RSV GOLD I study of children under 5 years of age with RSV-related in-hospital mortality For the first time, we report global age-stratified data for children dying with influenza. This enabled us to assess the potential impact of maternal vaccination on paediatric influenza-related mortality. Furthermore, we verified each case through direct communication with the local collaborators.There are also limitations to this study. First, our results reflect only a small proportion of paediatric influenza-related mortality occurring worldwide, since limited data are available due to lack of diagnostic testing, in particular from children who died outside of the hospital. Our database included only 18 (5·7%) children from LICs, and this proportion should be much higher based on global mortality burden estimates. Since children dying in the community may be younger and are mainly from LMICs, our results are an underestimation of the potential impact of maternal vaccination on paediatric influenza-related mortality in LMICs. Furthermore, since we only included individual-level patient data, it was not possible to use larger published case series from the US for which only aggregated data could be provided . The perThere are important factors to support maternal influenza vaccination: it has proved to be safe *FLU GOLD collaborators are:1, Marta Nunes2, Richard Chawana2, Shabir A. Madhi2, Anna C. Vittuci3, Quique Bassat4,5,6,7,8, Azucena Bardají4,5,8, Edward Goka9, Srđan Roglić10"?>, Bosco Paes11, LouAnn Elliott11, Hitoshi Oshitani12, Socorro Lupisan13, Angela Gentile14, María Florencia Lucion14, Michael C. Spaeder15, Warwick Butt16, Jenny Thompson16, Asuncion Mejias17, Octavio Ramilo17, Rodrigo A. Fasce18, Marta Werner19, Diego R. Hijano20, Kim J. Allison20, Márcia Rosane Pires21, Fernanda de-Paris21, Giorgi Chakhunashvili22, Irakli Karseladze23, Grieven P. Otieno24, D. James Nokes24, Oded Scheuerman25, Dario Prais25, Mohammed Al Amad26, Abdul Wahed Al Serouri26, Asad Ali27, Mohammad Tahir Yousafzai27, Alfredo Bruno28,29, Domenica de Mora28, Jenny Ojeda30, Ghassan Dbaibo31, Rima Hanna-Wakim31, Vassiliki Papaevangelou32, Elpiniki Kartisouni32, Cheryl Cohen33, Sibongile Walaza33, Rosalie S. Linssen34, Hsin Chi35, Aykut Eşki36, Esen Demir37, Senjuti Saha38, Samir K Saha38, Anthony A. Sochet39, Beatriz E. Teppa-Sanchez39, Thyyar M. Ravindranath40, J. Scott Baird40, Shaun K. Morris41, Waison Wong41, Robert F. Breiman42, Emily S. Gurley43, Shams El Arifeen44, Nega Assefa45, J. Anthony G. Scott46, Dickens Onyango47, Karen L. Kotloff48, Samba O. Sow49, Inacio Mandomando50,51, Ikechukwu U. Ogbuanu52, Amara Jambai53, Tanil Kendirli54, Edin Botan54, Franco Díaz Rubio55, Alberto Serra56, Luis Martinez57, Luis Pedroso58, Soledad Menta59, Rosalba Pardo60, Alejandro Donoso61, Syed Faisal Mahmood62, Naveera Khan631Alder Hey Children's Hospital, and The University of Liverpool, Liverpool, UK2South African Medical Research Council: Vaccines and Infectious Diseases Analytics Research Unit, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand; and Department of Science and Technology/National Research Foundation: Vaccine Preventable Diseases Unit, University of the Witwatersrand, Johannesburg, South Africa3Bambino Gesù Hospital, Rome, Italy4ISGlobal, Hospital Clínic - Universitat de Barcelona, Barcelona, Spain5Centro de Investigação em Saúde de Manhiça (CISM), Maputo, Mozambique6ICREA, Pg. Lluís Companys 23, 08,010 Barcelona, Spain7Paediatrics Department, Hospital Sant Joan de Déu, Universitat de Barcelona, Esplugues, Barcelona, Spain8Consorcio de Investigación Biomédica en Red de Epidemiología y Salud Pública (CIBERESP), Madrid, Spain9Institute of Inflammation and Repair, The University of Manchester, Manchester, UK10University Hospital for Infectious Diseases, Zagreb, Croatia11Department of Pediatrics , McMaster University, Hamilton, Canada12Department of Virology, Tohoku University Graduate School of Medicine, Sendai, Japan13Research Institute for Tropical Medicine, Muntinlupa City, Philippines14Department of Epidemiology, Ricardo Gutiérrez Children's Hospital, Buenos Aires, Argentina15University of Virginia School of Medicine, Charlottesville, USA16Department of Paediatric Intensive Care, Royal Children's Hospital, Melbourne, Australia17Department of Paediatrics, Division of Infectious Diseases, and Center for Vaccines and Immunity, Nationwide Children's Hospital, Ohio State University, Columbus, USA18Department of Viral Diseases, Public Health Institute of Chile, Santiago, Chile19Epidemiology Unit Hospital Guillermo Grant Benavente, Concepcion, Chile20Paediatric Infectious Diseases, St. Jude Children's Research Hospital, Memphis, USA21Hospital de Clínicas de Porto Alegre, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil22One Health Division, National Center for Disease Control and Public Health, Tbilisi, Georgia23Vaccine Preventable and Respiratory Disease Division, National Center for Disease Control and Public Health, Tbilisi, Georgia24Kenya Medical Research Institute, Wellcome Trust Research Programme, Centre for Geographic Medicine Research - Coast, Kilifi, Kenya25Schneider Children's Medical Center of Israel, Petah Tikva, Sackler school of medicine, Tel Aviv university, Tel Aviv, Israel26Yemen Field Epidemiology Training Program (Yemen-FETP), Sana'a, Yemen27Department of Paediatrics and Child Health, Aga Khan University Hospital, Karachi, Pakistan28Instituto Nacional de Investigación en Salud Publica, Guayaquil, Ecuador29Universidad Agraria del Ecuador, Guayaquil, Ecuador30Ministerio de Salud Pública del Ecuador, Quito, Ecuador31American University of Beirut Medical Center, Beirut, Lebanon32Third Department of Paediatrics, National and Kapodistrian University of Athens, Athens, Greece33National Institute for Communicable Diseases (NICD), Johannesburg, South Africa34Paediatric Intensive Care Unit, Emma Children's Hospital Amsterdam UMC, Amsterdam, the Netherlands35Department of Paediatric Infectious Disease, MacKay Children's Hospital, Taipei, Taiwan36University of Health Sciences, Department of Pediatric Pulmonology, Dr. Gazi Yaşargil Women's and Children's Health, Education and Research Hospital, Diyarbakır, Turkey37Department of Paediatric Pulmonology, Ege University Medical Faculty, Ege University Children's Hospital, Izmir, Turkey38Child Health Research Foundation (CHRF), Dhaka, Bangladesh39Division of Paediatric Critical Care Medicine, Johns Hopkins All Children's Hospital, St Petersburg, USA40Division of Paediatric Critical Care Medicine, Morgan Stanley Children's Hospital of NewYork-Presbyterian, New York, New York, USA41Division of Infectious Diseases, The Hospital for Sick Children, Toronto, Canada42Emory University, Global Health Institute, Child Health and Mortality Prevention Surveillance (CHAMPS) network, Atlanta, USA43Johns Hopkins Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA and International Centre for Diarrhoeal Disease Research, Dhaka, Bangladesh44International Centre for Diarrhoeal Disease Research, Dhaka, Bangladesh45College of Health and Medical Sciences, Haramaya University, Harar, Ethiopia46London School of Hygiene & Tropical Medicine, London, United Kingdom47Kisumu County Department of Health, Kisumu, Kenya48Department of Pediatrics, Center for Vaccine Development and Global Health and Division of Infectious Disease and Tropical Pediatrics, University of Maryland School of Medicine, Baltimore, Maryland, USA49Center for Vaccine Development, Bamako, Mali50Centro de Investigação em Saúde de Manhiça (CISM), Maputo, Mozambique51Instituto Nacional de Saúde (INS), Ministério da Saúde, Maputo, Mozambique52Crown Agents Sierra Leone, Freetown, Sierra Leone53Ministry of Health and Sanitation, Freetown, Sierra Leone54Division of Paediatric Critical Care, Ankara University School of Medicine, Ankara, Turkey55Instituto de Ciencias e Innovación en Medicina, Universidad del Desarrollo, Santiago, Chile56Casa de Galicia, Montevideo, Uruguay57Hospital Paysandu, Paysandu, Uruguay58Hospital Regional Salto, ASSE, Salto, Uruguay59Hospital Tacuarembo, Tacuarembo, Uruguay60Clínicia Infantil de Colsubsidio, Bogotá, Colombia61Hospital La Florida, Santiago, Chile62Section of Infectious Diseases, Department of Medicine, Aga Khan University, Karachi, Pakistan63Research Facilitation Office, Medical College, Aga Khan University.Kentigern Thorburn5The FLU GOLD team believes data sharing is important for the global scientific community. Our data are available upon request to the corresponding author, except for personally identifiable data due to privacy reasons.6NIM, IR, LJB, HN and MN conceptualized the study. IR and FSV collected the data. YNL and FSV Analyzed the data. NIM, YNL and LJB interpreted the results. YNL wrote the initial draft. All authors critically reviewed and revised the manuscript. YNL, FSV, NIM and LJB had full access to all the data in the study and the corresponding author had final responsibility for the decision to submit for publication.7This publication is based on research funded in part by the Bill & Melinda Gates Foundation. The findings and conclusions contained within are those of the authors and do not necessarily reflect positions or policies of the Bill & Melinda Gates Foundation.LJB reports grants from Bill and Melinda Gates Foundation, during the conduct of the study. LJB has regular interaction with pharmaceutical and other industrial partners. He has not received personal fees or other personal benefits. The University Medical Centre Utrecht (UMCU) has received major funding obtained by LJB  for investigator initiated studies from AbbVie, MedImmune, Janssen, the Bill and Melinda Gates Foundation, Nutricia (Danone) and MeMed Diagnostics. UMCU has received major cash or in kind funding as part of the public private partnership IMI-funded RESCEU project from GSK, Novavax, Janssen, AstraZeneca, Pfizer and Sanofi. UMCU has received major funding by Julius Clinical for participating in the INFORM study sponsored by MedImmune. UMCU has received minor funding for participation in trials by Regeneron and Janssen from 2015 to 2017 . UMCU received minor funding for consultation and invited lectures by AbbVie, MedImmune, Ablynx, Bavaria Nordic, MabXience, Novavax, Pfizer, Janssen . LJB is the founding chairman of the ReSViNET Foundation. HN reports grants and personal fees from the World Health Organisation, personal fees from the Bill & Melinda Gates Foundation, personal fees from Sanofi, outside the submitted work. MCN reports grants from the Bill & Melinda Gates Foundation, personal fees from Sanofi Pasteur, personal fees from Pfizer, outside the submitted work. NIM has regular interaction with pharmaceutical and other industrial partners. She has not received personal fees or other personal benefits. UMCU received minor funding for NIMs consultation and invited lectures by Abbvie and Merck. All other authors have nothing to declare.


Despite the intricate involvement of the endocannabinoid system in various physiological processes, it remains one of the most under-studied biological systems of the human body. The scope of endocannabinoid signalling is widespread, ranging from modulation of immune responses in innate and adaptive immunity to gestational processes in female physiology. Cannabinoid receptors are ubiquitously distributed in reproductive tissues and are thought to play a role in regulating the immune–reproductive interactions required for successful pregnancy, specifically among uterine natural killer cells and placental extravillous trophoblasts. The use of cannabis during pregnancy, however, can perturb endocannabinoid homeostasis through effects mediated by its major constituents,  In human pregnancy, the maternal endometrium undergoes a process of decidualization wherein differentiation of endothelial, epithelial and stromal cells, paired with recruitment of immune cells, primes the decidua for blastocyst implantation . This moOf the 26–37% of leukocytes localized in the decidua, up to approximately 80% are identified as uterine natural killer (uNK) cells ,5. The pN-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD) and fatty acid amide hydrolase (FAAH), respectively [The endocannabinoid system (ECS) plays a necessary role in a variety of physiological processes in the human body, including modulation of immune responses and reproductive events . Unfortuectively ,15,16,17ectively . 2-AG isectively ,20,21,22ectively ,23. CB1Rectively ,25. In cectively ,27. Otheectively ,30,31,32i)-mediated actions [Both CB1R and CB2R are classified as GPCRs that regulate an array of signal transduction cascades, particularly the adenylyl cyclase pathway . Cannabi actions ,35. Cycl actions ,37,38. T actions ,40,41,42The presence of ECS components at the maternal-fetal interface dates to 1999 when CB1R and CB2R localization was demonstrated in the human placenta and the BeWo cell line, which is representative of cytotrophoblasts . Product9-THC) and cannabidiol (CBD) [Furthermore, alterations in ECS signalling during early pregnancy are associated with impaired decidualization due to inhibited differentiation of endometrial stromal cells, and decreased secretion of cytokines and angiogenic growth factors by uNK cells ,49. Thesol (CBD) ,51. In f+), CD2+, CD3− and CD19− [dim while the remaining, including uNK cells, are CD56bright [bright NK cells, uNK cells possess lytic granules that contain perforin and granzyme [+ NK cells [uNK cells are a phenotypically distinct subset of human natural killer (NK) cells as they are cluster differentiation 56 positive  ,9,10. Th (TGF-β) . Moreove (TGF-β) .+ NK cells [bright uNK cells or CD56dim/CD56bright peripheral NK cells are implicated [bright NK cell numbers in pregnancies complicated with preeclampsia and fetal growth restriction [uNK cells have also been implicated in gestational complications. Endometrial biopsies obtained from women suffering from recurrent miscarriage show an increase in CD56NK cells ,61. Howeplicated . Conversplicated . Placenttriction ,64. ThisIn addition to decidualization, successful pregnancy depends on proper trophoblast differentiation and functionality ,66. In bAround 3 weeks post-fertilization, CTBs differentiate into another type of trophoblast, the EVTs ,66. EVTsPeripheral NK cells display cytotoxic activity against infected and foreign cells, particularly those that have reduced expression of human leukocyte antigen class I (HLA-I) molecules . This redim NK cells to uNK cells, and migration of CD56bright peripheral NK cells [bright peripheral NK show increased expression of CXCR3 and CXCR4 compared to CD56dim peripheral NK cells [bright peripheral NK cells to the decidua through CXCR4-CXCL12 interactions in early pregnancy [The increase in number of uNK cells in the late secretory phase of the menstrual cycle and early pregnancy can be attributed to increased proliferation, differentiation of peripheral CD56NK cells ,76,77. ECommunication between uNK cells and EVTs is also important for the regulation of trophoblast invasion . uNK celIn addition, matrix metalloproteinases (MMPs) are necessary for the breakdown of the decidual extracellular matrix, which is an event required for invasion and angiogenesis . The actAlongside facilitating trophoblast invasion, first trimester uNK cells possess important roles in vascular remodelling as they secrete VEGF, placental growth factor (PlGF), Ang-1, and Ang-2 ,78,83. EThe immune system functions to defend the body from foreign substances and infection through two lines of defense: innate and adaptive responses . The expᴋB (NF-ᴋB) via the myeloid differentiation factor 88 (MyD88) pathway [Cannabinoid receptor activation in cell types of the innate immune system is associated with apoptosis and recruitment of dendritic cells, inhibited migration of neutrophils, inhibited phagocytic activity and Th1-type cytokine secretion of macrophages, and inhibited cytotoxicity of NK cells ,97,98,99 pathway . In part pathway . The phy pathway .+) and Boy/J (Ly5.1+) mice, which is linked to reduced maturation [+ T cells demonstrate that AEA (2.5 µM) suppressed the proliferation and secretion of TNF-+ T cells and CD4+ Th17 cells, respectively [9-THC (5 µM) is reported to balance CD3+ Th-cell responses by decreasing the ratio of Th1/Th2 cytokines, therefore providing this phytocannabinoid with an immunomodulatory role [ECS signalling modulates events in B cells and T cells of the adaptive immune system as well. The majority of experimental investigations, however, have examined B cell functions in mouse models. In BALB/c mice, 2-AG (1 µM) stimulated naïve B cell chemotaxis through a CB2R-dependent mechanism . CB2R alturation . Moreoveturation . On the and IL-2 . AEA  arrested blastocyst development in a dose-dependent manner, indicating that elevated 2-AG is detrimental during the pre-implantation period . While o9-THC (20 µM), AEA (10 µM), and 2-AG (10 µM) have been shown to impair the biochemical and morphological differentiation of CTBs, leading to poor trophoblast cell-to-cell fusion [9-THC (20 µM) exposure also decreased the secretion of human chorionic gonadotropin, placental lactogen, and insulin-like growth factors in BeWo cells, all of which are important for fetal development and pregnancy success [9-THC (10 mM) has been associated with decreased trophoblast invasion in the human HTR-8/SVneo cell line that models EVTs [Another important role for endocannabinoid signalling during pregnancy is the modulation of trophoblast functions . In the feration . 2-AG  is associated with cell cycle arrest at the Ged cells . AEA exped cells . Since Sed cells . Furthered cells . This oued cells . The ECSed cells .In human uNK cells isolated and purified from first trimester decidual tissues, alterations in the balance between AEA synthesis and degradation have been linked to impaired decidualization . RelativThe process of EVT invasion is dependent on successful decidualization as trophoblasts come into close contact with decidual leukocytes for the remodelling of maternal vasculature . Complical cells . Primaryal cells . Similarcells [α . Other gcells [α ,135.Moreover, IL-25 (IL-17E) is involved in mediating Th2-type immune responses . TreatmeTo conclude, alterations in cannabinoid receptor-mediated signalling can impair decidualization, which can subsequently interfere with EVT-uNK cell crosstalk as well as lead to compromised trophoblast invasion and vasculature remodelling . ComplicCannabis is one of the most commonly used drugs during pregnancy, with a self-reported incidence of 1.4–7.1% ,138,139.Maternal cannabis use is also associated with various complications for both the mother and fetus, including decreased fetal birth weight (<2500 g), increased admissions to the neonatal intensive care unit (NICU), preterm birth (<37 weeks of gestation), small-for-gestational-age (SGA) status, miscarriage, and placental abruption ,144. Mor9-THC and CBD, can readily cross the placenta due to their lipophilic nature [9-THC transfer across the placental barrier have not been evaluated, however animal models indicate that 10–28% of its maternal plasma concentrations are found in the fetal circulation [9-THC is dependent on route of administration, placental permeability, and individual variations in frequency of use [In addition, the primary bioactive phytocannabinoids, ∆c nature . Human iculation ,147. Whiy of use ,148. Unf9-THC is a partial agonist for CB1R (Ki = 5.05 nM) and CB2R (Ki = 3.13 nM)   [149,1509-THC (40 µM) in chorionic villous explant cultures has been associated with increased local AEA levels after a 72-h exposure period [9-THC can mediate its actions through CB1R and has been shown to alter ECS homeostasis in the placenta, it may interfere with placental-decidual communication. Specifically, ∆9-THC exposure may compromise the crosstalk between uNK cells and EVTs by altering their production of cytokines and growth factors, which are required for successful decidualization, invasion, and angiogenesis.Another point of contrast is that endocannabinoids are associated with local effects whereas phytocannabinoids act systemically and can influence interactions across systems, including those between uNK cells and EVTs in pregnancy . Treatmee period . Since ∆Due to its multi-faceted biological roles, the ECS holds therapeutic promise for conditions associated with altered endocannabinoid homeostasis . HoweverAs of early 2021, the FDA has approved 4 cannabinoid-based drugs: Epidiolex, Cesamet, Marinol, Syndros ,163. Ces9-THC with the trade name Cesamet [9-THC varies depending on route of administration, although it has been shown to reach peak plasma concentrations within minutes of inhalation, and has a half-life of 21.5 h [Nabilone is a synthetic analogue of ∆ Cesamet . The app Cesamet . While t Cesamet ,168. The Cesamet . In term Cesamet ,170. It  Cesamet . In contf 21.5 h .9-THC [9-THC, can stimulate appetite through activation of CB1R in areas of the CNS that regulate energy balance, specifically the hypothalamus and brainstem [9-THC, it is similarly an agonist for both CB1R and CB2R [Dronabinol is another cannabinoid that contains synthetically manufactured ∆9-THC . This co9-THC ,168. Dro9-THC . It is a9-THC . Cannabirainstem ,175,176.and CB2R ,150. Droand CB2R .Epidiolex is an oral solution of CBD that is used for treating seizures in the Lennox-Gestaut and Dravet syndromes . Althoug9-THC and CBD in equimolar amounts [Finally, nabiximols is marketed under the trade name Sativex as an oromucosal spray containing ∆ amounts . It is u amounts . Similar amounts . These e amounts ,184. Whi amounts .Human pregnancy presents a unique dilemma wherein the maternal immune system must accept the fetus, which is comparable to a semi-allogenic graft, and create a microenvironment conducive to its growth. The interactions between uNK cells and EVTs are necessary for this process, particularly since these cell types work together for successful decidual invasion and remodelling of the maternal vasculature. Furthermore, components of the ECS are involved in regulating diverse biological processes, including immune and reproductive events in pregnancy. In cases of maternal cannabis use, however, endocannabinoid homeostasis and signalling may become dysregulated. An important consideration is that phytocannabinoids lack the retrograde feedback mechanism utilized by endocannabinoids and can thus continuously activate the cannabinoid receptors. In particular, activation of CB1R by phytocannabinoids is associated with inhibited decidualization through a cAMP-dependent mechanism. As a consequence of cannabis exposure, the crosstalk between uNK cells and EVTs can become compromised, resulting in impaired invasion, angiogenesis, and potentially, later pregnancy events that promote fetal development.In addition, while some synthetic cannabinoids are approved by the U.S. FDA and Health Canada, other formulations are unregulated and lack data pertaining to drug interactions and long-term outcomes. If consumed by pregnant women, synthetic cannabinoids may have consequences for pregnancy establishment, maintenance, and success. Therefore, further molecular and clinical research is necessary to ascertain how endocannabinoids, phytocannabinoids, and synthetic cannabinoids may impact immune–reproductive interactions during pregnancy.


Rodent protoparvovirus 1 species, which is currently under validation as an anti-cancer agent), we generated tools with which to monitor infections by human cutavirus (CuV) and B19 virus (B19V) . As well as mAbs able to detect NS1 from a broad range of parvoviruses, we obtained entities specific for either (distinct) members of the Rodent protoparvovirus 1 species, human CuV, or human B19V.For many applications it is necessary to detect target proteins in living cells. This is particularly the case when monitoring viral infections, in which the presence (or absence) of distinct target polypeptides potentially provides vital information about the pathology caused by the agent. To obtain suitable tools with which to monitor parvoviral infections, we thus generated monoclonal antibodies (mAbs) in order to detect the major non-structural protein NS1 in the intracellular environment and tested them for sensitivity and specificity, as well as for cross-reactivity towards related species. Using different immunogens and screening approaches based on indirect immunofluorescence, we describe here a panel of mAbs suitable for monitoring active infections with various parvovirus species by targeting the major non-structural protein NS1. In addition to mAbs detecting the NS1 of parvovirus H-1 (H-1PV) (belonging to the  Autonomous, vertebrate parvoviruses (PVs) are icosahedral, non-enveloped particles of approximately 24 nm in diameter, with a 5 kb linear single-stranded DNA as their genome. This small nucleic acid encodes the VP proteins composing the capsid shell and a small number of nonstructural (NS) proteins, necessary to reprogram the host cell in order to support viral DNA amplification, packaging, and spreading. In an exposed organism, tissue tropism can be restricted through a limiting viral receptor at the cell surface, required for virus attachment and entry as well as the delivery into the nucleus and decapsidation of the single-stranded DNA . Moreove(Parvoviridae) have been recognized as  pathogens that infect mammals  and humans , respectively [While rodent PVs, including H-1PV, were initially discovered to be opportunistic infectants of human-cancer-derived cell lines, and are currently validated as therapeutics for cancer treatments, other members of this virus family ectively ,4,5,6,7.ectively , as wellectively , potentiectively . Althougtrans activates the P38 promoter to ensure capsid protein production and packaging [Besides the two capsid proteins VP1 and VP2, which together compose the capsid shell, parvoviruses are able to produce a number of non-structural proteins (NS), which are involved in multiple aspects of the viral life cycle, thereby ensuring progeny particle production and spreading. For rodent protoparvoviruses, there are four polypeptides produced from the early P4 promoter (NS1 and three forms of NS2) and one regulatory protein (SAT) generated from the P38 promoter . Among tackaging  and the ackaging , and facackaging . At lateackaging . To fulfackaging . In accoackaging , which iackaging , NS1 appSimilar to rodent protoparvoviruses, the newly identified primate protoparvoviruses, bufavirus (BuV), tusavirus (TuV), and cutavirus (CuV), encode, besides VPs, a large non-structural protein, NS1, and are recognized by a middle open reading frame, which could encode for an additional regulatory protein . B19V waIndeed, monoclonal antibodies recognizing NS1 from B19V and the minute virus of mice (MVM), respectively, have been generated in the past and have been proven to be excellent tools not only to monitor the viral polypeptide in its cellular environment but also to capture and purify the polypeptide for functional analyses ,21. TherThus, we generated a number of mAbs using antigens for H-1PV, CuV, and B19V NS1, respectively, and characterized them for their specificity/cross-reactivity towards different parvovirus species and, consequently, their suitability to detect viral activity in human tissues through immunological assays.2+ binding sites, and a second polypeptide (aa 547–672) corresponding to the variable C-terminus comprising the trans activation domain  reagents able to detect NS1 from a broad spectrum of PVs and (ii) mAbs with a distinct species specificity, in order to selectively identify oncolytic PVs in the host without cross-reactivity through the detection of human (proto)parvoviruses and vice versa. Thus, we decided to choose a combination of (a) conserved domain(s) and (a) variable region(s) for immunization. For the prototype strain of the minute virus of mice (MVMp), the functional domain structure of NS1 together with target phosphorylation sites together with sequence alignments between different rodent PV species have been determined , and wern domain A. It is blotting ,24. To pH, respectively (framed brown).To identify mAbs recognizing NS1, we set up large-scale experiments to screen supernatants from hybridoma cells by indirect immunofluorescence. This approach has the advantage of directly identifying reactive antibodies and assessing their sensitivity and specificity for native proteins. Different strategies for the detection of NS1-specific Abs are illustrated in Although rodent protoparvovirus NS1 proteins are rather conserved among the genus, there are also significant variations, particularly in regions that potentially interact with host cell proteins . In ordeThe screening for mAbs to detect CuV NS1, expressed by means of recombinant baculoviruses in Sf-9 insect cells, proved to be significantly less reliable than expected with the use of established reagents B. Thus, To identify mAbs for recognizing B19-NS1, we used recVV to express the viral protein in HeLa cells. As shown in H [H and NS1-MK/SP8, as well as Western blot experiments with selected candidate mAbs, which on the one hand detected all PV-NS1  while others were restricted to detecting NS1 species-specifically .Depending on the research question, it is not only important to unambiguously identify the presence of parvovirus activity in infected cells and tissues but also to eventually discriminate between potential human pathogen(s) and therapeutic rodent PVs used in cancer gene therapy. To determine the host range specificity versus cross-reactivity with the NS1 of other PV species, we took advantage of the vaccinia virus expression system. Indeed, recVV allow the production of post-translationally modified and functionally active NS1 polypeptides that closely resembled the virus-produced polypeptides . This prH  and MK3/H , the mAbH ,26, and We next examined those mAbs which were able to detect H-1PV NS1, assessing whether they had the capability to detect the different rodent protoparvovirus strains, i.e., possessed cross-reactivity with only closely related or more distant PV-NS1. Thus, HeLa and NBK cells, according to the respective host range, were infected (or not) with H-1PV, KRV, LuIII, TVX, and MVM, respectively, and subjected to immunofluorescence experiments using hybridoma supernatants obtained from either H-1PV or CuV NS1 immunizations. No specific signals were obtained with B19V-NS1-derived hybridoma. As shown in Interestingly, a significant number (6/19) of H-1PV-NS1-specific mAbs, including the clone m#236/10, that showed cross-reactivity with NS1 from different rodent PV species  completely failed to detect TVX NS1 by indirect immunofluorescence. This is of particular interest since TVX was recognized by an in-frame deletion in the C-terminal region of NS1/middle exon of NS2, similar to an H-1PV variant described by Faisst et al. ,38, provSome laboratory research and clinical applications require immunohistochemical analyses of paraffin-embedded tissues. In order to test whether some of our H-1PV NS1 mAbs were able to specifically detect NS1 in paraffin-embedded sections, we used the cell block approach for sample preparation. The latter mimics the histological processing of tissues and allows cell cultures to be used as controls for tissue immunohistochemical analyses. As illustrated in To determine and analyze viral infections in vivo, appropriate sensitive and specific tools are required. Apart from molecular biological methods, such as quantitative PCR to monitor the presence of viral DNA in tissues, blood, and/or body fluids, as well as RT-PCR to assess the presence of RNA transcripts determining viral activity in susceptible cells, it is valuable to detect viral proteins with defined functions in the target cell by immunohistochemistry. For instance, co-localization studies with cellular target proteins were able to provide important information regarding the replication process , virus pParvovirinae NS1 proteins, since B19V NS1 plays a similar key role during infection, also significantly differing in its primary sequence and ability to interact with host cell proteins and processes [Due to the small coding capacity of PVs, the amount of viral target proteins required to monitor infections is very limiting. Indeed, most viral activities are driven by a single multifunctional protein, NS1, which is regulated through the infection process by differential phosphorylations, enabling this polypeptide to exert distinct enzymatic activities and interact with multiple different proteins ,25,33. Trocesses ,19.Ideally, it is desirable to establish tools that are not only able to specifically recognize a distinct viral protein of a single species but can be used for related viruses as well. This is particularly the case for a class of emerging viruses, such as the human protoparvoviruses, for which three new viruses have been identified in the last five years ,43,44. OTo obtain optimal tools for distinct applications it is necessary to choose a screening system that provides the target protein in a suitable set-up. For our objective, obtaining mAbs that interact with the native, potentially post-translationally modified protein, we decided to use indirect immunofluorescence rather than Western blot. Indeed, our extensive comparison of seed clones by IF and WB  clearly To screen for mAbs with optimal reactivity, it is desirable to analyze the detection of native target proteins after genuine virus infections, and, as demonstrated with baculovirus expression of NS1 in insect cells, alternative expression systems might not deliver the desired results. However, particularly regarding emerging viruses, suitable virus/host cell systems are not always easily accessible. In addition, it might be useful to assess the cross-reactivity of viral proteins from different origins using the same conditions, i.e., the same host cells. In this regard, high-level expression of the polypeptides with recombinant vaccinia viruses in human cell lines appeared to be justified, since it was shown to generate functionally active and post-translationally properly modified NS1 ,30,31. Iin-frame deletions in the C-terminus of NS1 and the middle exon of NS2 [in-frame deletions. Thus, we concluded that this region of NS1 is exposed in the native protein and offers excellent epitopes not only for the binding of specific antibodies during the selection procedure but also for potential interactions with cellular targets, which are essential to drive the virus through the replication cycle [Interestingly, recent investigations with potential oncolytic parvoviruses have identified n of NS2 . TVX, whn of NS2 , and an n of NS2 . Moreoven of NS2  were fouon cycle . Indeed,on cycle ,45, and on cycle ,45. AltoIn conclusion, the presented approach generated a panel of mAbs with different specificities towards PV species to detect and monitor parvovirus infections in tissue, including paraffin sections. This will allow us to study persistent and/or chronic infections of human parvoviruses as a potential disease-causing agent. We also generated tools to monitor the effects of a virotherapy of cancer, not only for H-1PV currently under investigation  but addiExperiments on animals were conducted according to institutional and legal regulations for animal experimentation, as approved by the Animal Welfare Committee of the German Cancer Research Center and by the Land Baden-Württemberg Az.35-9185.82/A8-17.Synthetic DNA: Cuta- (Accession KT868814.1 AMS35105.1) and Bufa-NS1 JQ918261.1 DNAs were obtained through DNA synthesis, cloned as PmeI to NcoI fragments into pFastBac1 from BioCat.Immunogens: To obtain constructs to express H-1PV, B19V, and CuV immunogens in bacteria, the required PV sequences were generated by PCR using primer pairs containing appropriate restriction enzyme sequences to clone these sequences as in-frame insertions into pQE-32 (Qiagen). PCR was performed using a LongAmp PCR-KIT (NEB), using the primer pairs listed in Heli, H1-NS1TA, B19-NS1Heli, B19-NS1TA, and CutaNS1Im were cloned separately as BamHI/NotI fragments in a pQE-32 plasmid derivative, containing a NotI cleavage site downstream of BamHI.Plasmids for recombinant baculo- and vaccinia viruses: Full-length NS1, Flag-, YDGASS- or His-tagged NS1 sequences were obtained by PCR using a LongAmp PCR-KIT (NEB), using the primer pairs listed in H [Y was generated with a KLH-coupled YDGASS peptide [Primary antibodies and antisera: Antisera NS1H  and NS1-H , as wellH ,26, were peptide . MonocloSecondary antibodies: Horseradish-peroxidase-conjugated anti-rabbit and anti-mouse IgGs were from Promega. All fluorescent-dye-labeled IgGs were from Invitrogen.Cell lines: HeLa, NBK, A9, CV-1, and BSC40 cells were maintained as monolayers in Dulbecco’s Modified Eagle’s Medium , while Sf9 cells were grown in suspension using TNM-FH insect medium (SIGMA) containing 10% fetal calf serum (FCS).Viruses: Parvoviruses: MVMp (pMVMp) [TM-KIT  and grown in Sf-9 cells. Expression of the respective recombinant proteins was ensured by indirect immunofluorescence using appropriate antibodies   was prop (pMVMp) , hgH1#1, (pMVMp) , LuIII [ (pMVMp)  KRV68 (I (pMVMp)  in NBK,  (pMVMp)  was prop variant ; FL-Cuta variant . In brie variant , followeBacterial expression of peptides used for immunization was performed in E. coli SURE by induction with 1 mM IPTG for 24 h. Bacteria were then harvested by centrifugation at 2500 rpm, suspended in 400 ul Buffer 1  containing 10 mg/mL lysozyme, incubated for 20 min at room temperature, and treated with three cycles of freezing and thawing, before adding 1.6 mL TET  and 2 mL 2xRIPA  buffer. The suspension was then sonicated for 60 s and centrifuged at 8000 rpm for 20 min at 4 °C, and the pellet containing bacterial inclusion bodies was suspended in TE (10 mM Tris/1 mM EDTA pH 7.5). Proteins were then separated by SDS-PAGE, stained with Coomassie blue, and the protein bands corresponding to the immunogens were cut from the gel, eluted in 500 mM ammonium acetate, 10 mM MgAc, and 0.1% SDS overnight at 4 °C, before being dialyzed against PBS containing 20% sucrose and 10% glycerol.For the generation of monoclonal antibodies, mice  from Jackson Immuno Research Laboratories, West Grove, PA, USA, were immunized via five subcutaneous injections into the hind leg with 20 μg recombinant bacterially expressed protein. To enhance the immune response, 100 μL of Freund’s Complete Adjuvant  was added. Booster injections were conducted with Freund’s Incomplete Adjuvant, followed by injection with buffer only. Injections were given 3 to 8 days apart over up to a 30-day time period. Sero-positivity of peripheral blood samples of the animals was verified by Western blot. Lymph nodes of the popliteal fossa were isolated and lymphocytes were fused with Sp2/0 cells as described previously . Seed clCells were grown in 96-well plates (Greiner), infected or not with the indicated viruses, fixed after 24 h with 3% formaldehyde, and permeabilized with 0.2% Triton X-100. The specimens were pre-adsorbed, incubated with primary antibodies, and stained with Alexa-Fluor488-conjugated anti-species antibodies. Analyses were performed with a Keyence microscope. Quantification of fluorescence intensity was performed using Fiji ImageJ software. The total fluorescent area was analyzed, integrated densities were measured, and fluorescence intensity  was determined as follows: negative, <20,000 RFU; (+), 20,000–30,000 RFU; (++), 30,000–40,000 RFU; and (+++), >40,000 RFU.Cell extracts were produced by incubating cell pellets in an extraction buffer containing 20 mM Hepes-KOH pH 7.5, 300 mM NaCl, 1 mM EDTA, and 0.2% NP-40 on ice, and were clarified by centrifugation. Proteins were separated by SDS-PAGE, blotted onto nitrocellulose membranes, and identified with appropriate primary antibodies in 10% dry milk/PBS stained with horseradish-peroxidase-conjugated secondary antibodies for 1 h, and were detected by chemiluminescence . Protein bands were visualized in an ECL Chemocam .® Histofix 4% . The resulting cell blocks were further processed by paraffin embedding and sectioning using Leica HistoCore Arcadia H  and a Microm HM 355S , respectively. Next, 10-µm-thick sections were stained with the purified mAbs, m#236/10, H1#1420/5, and H1#1539/2, specific for the H-1PV NS1 protein. Signals were visualized by secondary staining with a cyanine-3-conjugated anti-mouse antibody . Fluorescence was assessed using a Zeiss Cell Observer Z1 and documented with ZEN blue edition software.NBK cells were seeded in 150 mm × 25 mm cell culture dishes (Nunc). After subconfluent monolayer formation, cells were either infected with five plaque-forming units (PFU) of H-1PV or mock-treated with an equal volume of a virus-free cell culture medium. Then, 24 h after treatment, cells were scraped into the medium and centrifuged for 5 min/1000 rpm at room temperature. The pellet was washed with PBS, immobilized in agarose, and fixed overnight at room temperature in ROTI


Plants are constantly exposed to environmental stresses during their growth and development. Owing to their immobility, plants possess stress-sensing abilities and adaptive responses to cope with the abiotic and biotic stresses caused by extreme temperatures, drought, flooding, salinity, heavy metals and pathogens. Acyl-CoA-binding proteins (ACBPs), a family of conserved proteins among prokaryotes and eukaryotes, bind to a variety of acyl-CoA esters with different affinities and play a role in the transport and maintenance of subcellular acyl-CoA pools. In plants, studies have revealed ACBP functions in development and stress responses through their interactions with lipids and protein partners. This review summarises the roles of plant ACBPs and their lipid and protein interactors in abiotic and biotic stress responses. At. At88]. on in PA ,88. PLDαVvACBP was upregulated in leaves upon cold and heat shock stresses in comparison to the nontreated control . At. At90]. atacbp3, atacbp4 and atacbp6 mutants each had an abnormal and more permeable cuticle in comparison to the wild type, resulting in water loss after drought stress . Tr. Tr25]. olerance . Data min et al.  revealed+) and chloride (Cl+) ions by plant roots, which eventually accumulate in leaves . In. InChACBCBP1-OEs . Four sated rice . On top ted rice .ZmACBP1 and Class II ZmACBP3 gene expression was induced after NaCl or mannitol treatment. Transgenic Arabidopsis overexpressing ZmACBP1 and ZmACBP3 exhibited better growth and longer roots in comparison to the vector control , cadmium [Cd(II)] and zinc [Zn(II)] are major pollutants threatening the environment and living organisms. Therefore, several studies have been performed to investigate the role of AtACBPs in response to heavy metal stresses ,135. Usio Pb(II) . Accumulnd roots . When trediation .AtACBP2-overexpressing plants, the overexpression of AtACBP2 enhanced tolerance to Cd(II) and oxidative stress  in transgenic Arabidopsis (AtFP6 expression was induced after Cd(II) treatment . In. InAtACBreatment . LYSOPL2xidation  76]. Po. PoAtACBIn plants, wounding results following biotic attack , mechanical damage or weather-induced damage, which may culminate in the entry of pathogens and nutrient loss. Mechanical injury triggers the transduction of mobile signals in the plants, leading to localised responses at the wound sites  and distal responses in the undamaged tissues (systemic response) . Cell waAtACBP6 and AtACBP3 were induced in Arabidopsis  . Fathogens . The expnfection .AtACBP4 and its protein interactor AtEBP were elevated following B. cinerea infection, as well as the ethylene precursor ACC and MeJA treatments  to fungal (B. cinerea and C. higginsianum) and bacterial (P. syringae) pathogens [B. cinerea [The expression of Class IV eatments  74]. Th. ThAtACBathogens . Further cinerea .Rhizoctonia solani and Cercospora oryzae), hemibiotrophic (Magnaporthe oryzae and Fusarium graminearum) and biotrophic (Xanthomonas oryzae) phytopathogens  identified, EMSAs showed that two of them bound nuclear proteins from wild-type rice infected with R. solani, C. oryzae, M. oryzae and X. oryzae [OsACBP5 5′-flanking region containing both these W-boxes fused to the gene encoding β-glucuronidase (GUS) exhibited higher GUS activity upon SA, MeJA or R. solani treatment compared to the promoter deletion lacking both W-boxes [OsACBP5.Apart from AtACBPs, recent studies have depicted that Class III OsACBP5, the homologue of AtACBP3, protects rice plants against representative necrotrophic , LEUCINE-RICH REPEAT EXTENSIN-LIKE PROTEINS (LRX4 and LRX5), XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE PROTEIN4 (XTH4) and PECTINESTERASE INHIBITOR18 (PME18), proteins involved in glucosinolate (GSL) degradation including GDSL-LIKE LIPASE23 (GLL23), EPITHIOSPECIFIER MODIFIER1 (ESM1), MYROSINASE1, MYROSINASE2, NITRILASE1 (NIT1), and a protein involved in JA synthesis, ALLENE OXIDE CYCLASE2 (AOC2), suggesting their potential in protection against R. solani [Both Lipidex assays and ITC showed that rOsACBP5 binds to 18:3-CoA esters, suggesting that 18:3-FA, a precursor for JA biosynthesis, plays a role in basal defence against fungal pathogens ,93. Furt. solani .Phakopsora pachyrhizi fungal infection of soybean, seven GmACBPs comprising all four classes were detected in microarray data analysis [GmACBP2, Class II GmACBP4, Class III GmACBP5 and GmACBP6, and Class IV GmACBP11 declined after 6 h post inoculation (hpi) of soybean with avirulent Hawaii 94-1 and virulent Taiwan 80-2 strains [Phytophthora sojae infection caused an induction of only Class IV GmACBP9 at 72 hpi, but a reduction of other GmACBPs such as Class I GmACBP2, Class III GmACBP5, GmACBP6 and GmACBP7 as well as Class IV GmACBP11 after 48 dpi [GmACBPs compared to other Class III ACBPs such as AtACBP3, VvACBP and OsACBP5 in pathogen response, suggested differential roles of GmACBPs in plant-pathogen interactions.Upon analysis . The exp strains . In addir 48 dpi . Such diStudies on ACBPs in the past two decades have strongly implicated their roles in the regulatory mechanisms of development and stress responses. All classes of ACBPs are known to play a role in abiotic and biotic stress signalling, although the detailed mechanistic events remain to be further elucidated. Through phospholipid and acyl-CoA ester binding, AtACBPs and OsACBPs are involved in stresses arising from drought ,81, adveOsACBPs [GmACBP expression in soybean upon salt and drought treatment suggests that soybean stress responses may differ from Arabidopsis [Taken together, dicot AtACBPs have been widely studied in relation to their acyl-CoA and phospholipid binding properties, protein-protein interaction and the resultant signalling cascades of abiotic and biotic stress responses. However, the roles of the monocot OsACBPs and ZmACBPs, as well as the emerging leguminous GmACBPs, remain to be further investigated given that preliminary studies have shown stress-induced changes in ACBP expression ,103,104.OsACBPs , it woulbidopsis ,93,94 anbidopsis ,118 and bidopsis . The ovebidopsis ,131,180.It is known that ACBPs play significant roles in plant development and stress responses ,119,132,


Gas chromatography–mass spectrometry (GC–MS) analyses revealed enhanced attenuation (>90%) of the total polyaromatic hydrocarbons (PAHs); the manure amendments significantly enhancing (up to 30%) the degradation of high molecular weight (HMW) PAHs. Microbial community analysis showed the dominance  of sequences affiliated to phyla Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. The core genera enriched were related to hydrocarbon metabolism . However, manure-treated samples exhibited high number and heterogeneity of unique operational taxonomic units (OTUs) with enrichment of additional hydrocarbon-degrading bacterial taxa . Thirty-three culturable hydrocarbon-degrading microbes were isolated from the co-composting microcosms and mainly classified into Burkholderia, Sanguibacter, Pseudomonas, Bacillus, Rhodococcus, Lysinibacillus, Microbacterium, Brevibacterium, Geobacillus, Micrococcus, Arthrobacter, Cellulimicrobacterium, Streptomyces Dietzia,etc,. that was additionally affirmed with the presence of catechol 2,3-dioxygenase gene. Finally, enhanced in situ degradation of total (49%), LMW (>75%) and HMW PAHs (>35%) was achieved with an enriched bacterial consortium of these microbes. Overall, these findings suggests that co-composting treatment of crude oil sludge with animal manures selects for intrinsically diverse bacterial community, that could be a driving force behind accelerated bioremediation, and can be exploited for engineered remediation processes.The present study aimed to characterize the bacterial community and functional diversity in co-composting microcosms of crude oil waste sludge amended with different animal manures, and to evaluate the scope for biostimulation based  Animal manure, Bioremediation, Bacterial diversity, Co-compost, Catechol 2,3-dioxygenase. However, it is associated with generation of large amounts of waste from its extraction and processing such as crude oil waste sludge (COWS). Chemically, COWS is composed of low and high molecular weights polycyclic aromatic hydrocarbons (LMW/HMW-PAHs) with characteristic strong molecular bonds and hydrophobicity. These properties make them less amenable to biodegradation and are thus recalcitrant in the environment under normal conditions. Additionally, COWS and its components are substantially cytotoxic, mutagenic and carcinogenic, especially polycyclic aromatic hydrocarbons (PAHs) , 2. Envi2 . The PCR conditions included: 1 cycle at 98 °C for 10s, then 34 cycles at 98 °C for 1s, 55 °C for 1 min and 72 °C for 15s. Then, a final elongation stage at 72 °C for 1 min. The resultant PCR fragment size (912 bp) spanning the open reading frame (ORF) of the 23O gene , was vis2.5A consortia consisting of 34 bacterial isolates exhibiting crude oil waste degrading ability was prepared by initially culturing each colony on nutrient broth overnight at 37 °C. After 24 h, 200 μL of each enriched were added simultaneously to 100 mL MSM broth supplemented with 5% crude oil waste sludge and the media incubated at 28 °C for 24 h at 120 rpm for 30 days. The experiment was performed in duplicate, and uninoculated flasks considered as controls. After 30 days, 5 mL of the bacterial consortia was resubcultured on fresh 100 mL MSM containing 5% crude oil waste sludge for 24 h at 28 °C while shaking at 120 rpm for 30 days. Similar experiments were performed using pyrene and anthracene as the sole carbon source. Using GC-MSD, the residual concentrations of the crude oil sludge and its constituent PAHs was determined. The total DNA of the enriched bacterial consortium at the end of the experiments was also extracted and subjected to Illumina Miseq sequencing to establish the bacterial community diversity and composition as described in 2.6https://www.ncbi.nlm.nih.gov/) sequence archive (SRA) as BioProject ID PRJNA794053. The 16S rDNA sequences were also deposited at the NCBI GenBank database under accession numbers MK854826 - MK854993. The data analysis results obtained during this study are included in the manuscript.All the raw datasets from Illumina sequencing have been deposited at the NCBI database (33.12 evolution (respiration rate) and pH are presented in 2 evolution (∼18 μg/dry weight/day) after 5 months of composting, with other manure compost piles  recording moderate CO2 evolution (∼10 μg/dry weight/day) during the same period  to high molecular weight (HMW) compounds. The average reduction levels of each PAH in the samples after composting with different manure amendment is presented in Overall, 36.5–99.9% PAH reduction levels were achieved under the composting conditions with reduction efficiency depending on the compost treatment and the molecular weight of the PAHs. Compost treatments, irrespective of the manure type, resulted in comparatively higher reduction of total PAHs (ΣPAHs), LMW- and HMW-PAHs than in control samples. Manure microcosms  had >300 mg/kg soil ΣPAHs reduction compared to control (CT) 175.8 mg/kg soil. HMW-PAHs such as perylene, dibenzoanthacene, benzo[ghi]perylene and benzo[e]acephenathrylene exhibited lower reduction (39.7–81.7%) b, wherea3.3Chao-1) were observed for PM and CM samples than HM and SM treatments  was observed for HM followed by PM, with SM and CM exhibiting comparable values to CT samples.Summary of the sequencing outputs and diversity indices for bacterial communities in co-composting experiments is presented in eatments . Additio3.4Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes were dominant taxa, accounting for >99% of sequences across the co-composting treatment (Proteobacteria were highly enriched in HM (99%), PM (90%), CT (85%), SM (50%). CM was dominated by Actinobacteria (35%) and Firmicutes (45%) with members of phyla Proteobacteria accounting for 20% of the detected sequences (Bacteroidetes were only detected in SM (20%) and PM (5%). Other minor phyla detected included Verrucomicrobia, TM6_(Dependetiae), Acidobacteria, Saccharibacteria, Tenericutes, Spirochaetae, Cyanobacteria, BRC1 and Chloroflexi. At class level, γ-proteobacteria, α-proteobacteria, Actinobacteria, β-proteobacteria, Clostridia, Bacilli, Bacteroidiia and Sphingobacteriia were the dominant groups in all samples.In total, 17 phyla, 42 classes, 83 orders, 162 families and 359 genera were detected in the study. OTUs assigned to the phylum reatment a. Howeveequences a. In conPropionibacteriales and Micrococcales was highly enriched in most co-composting treatments  accounting for >45 and >19% relative abundance, respectively, of Actinobacteria detected. Interestingly, CM was enriched with Corynebacteriales (67.4%) and Streptosporangiales (23.1%), whereas members of the orders Propionibacteriales and Micrococcales were detected at 0.7 and 4.0% relative abundance, respectively  in PM treatment only. On the other hand, members of the order Acidithiobacillales were highly enriched in CM (80%). In contrast, order Pseudomonadales was relatively more abundant in most co-composting treatments including control samples, but very low (3% relative abundance) in CM. Other important orders detected included Rhizobiales, Burkholderiales, Xanthomonadales, Caulobacterales, and Enterobacteriales (only detected in SM and CT)  were detected in CT sample.The relative distribution of the bacterial orders belonging to the top three phyla is illustrated in ectively a. Anothe and CT) c. FigurePseudomonas, Rhodanobacter, Proteiniphilum, Enterobacter, Achromabacter, Delftia, Bacillus, and Methylobacterium were dominant groups (Pseudomonas (49.0%), Rhodanobacter (22.6%), Achromobacter (10.7%), unclassified Rhizobiales (4.6%), Marinilabiaceae_ge (2.2%) and Propionbacterium (1.4%) were highly enriched in PM, whereas Pseudomonas (70.2%), Achromobacter (12.0%), Stenotrophomonas (4.5%), Sphingobium (3.2%), Pseudoxanthomonas (2.3%), Delftia (1.2%) Massilia (1.2%) and Methylobacterium (1.1%) were the main genera in HM. By contrast, genera Bacillus (17.4%), KCM-B-112_ge (11.3%), Nocardiopsis (7.6%), Gordonia (4.0%), Clostridium sensu stricto_1 (3.0%), Mycobacterium (2.4%), Corynebacterium (2.2%) and Rhodococcus (1.8%) were enriched in CM treatment.At genus level, t groups a. Overal3.5Pseudomonas, Delftia, Methylobacterium, Dietzia, Bacillus, Propionibacterium, Bradyrhizobium, Streptomyces, Achromobacter, Microbacterium and Sphingomonas. However, manure-treated had the highest unique OTUs with 233 (84.4%), while CT had 9 (3.3%). The major unique genera in manure-treated samples included Proteiniphilum, unclassified Micrococcales, unclassified Lachnospiraceae, Sphingobium and Stenotrophomonas, whereas Afpia, Klebsiella and Burkholderia-Paraburkholderia were dominant unique genera in CT. The Venn diagram of the shared and unique microbiome in manure-treated samples is presented in According to Venn diagrams consistent overlap patterns of genus clusters among different manure-treated co-composting and control treatment of crude oil waste sludge were obtained b. The sh3.63 CFU/g, with PM and CT yielding the highest  and lowest counts , respectively. A total of 211 bacterial colonies were picked from MSA plates supplemented with crude oil waste sludge as a sole C-source, and were identified based on partial 16S rDNA sequence to putatively belong to 31 phylotypes based on OTUs. A heatmap showing the relative abundance of the bacterial phylotypes recovered from the different manure and control samples after 10 months composting of crude oil waste sludge is presented in To augment metagenomic-based studies, axenic culture studies were performed. The average number of culturable bacteria for manure-treated compost was 4.1 × 10Bacillus, Lysinibacillus, Microbacterium, Burkholderia, Dietzia, Rhodococcus, Pseudomonas and Paeniclostridium were recovered in high frequency (>4% relative abundance) across all samples. However, only five isolates belonging to 4 genera: Bacillus; Microbacterium; Rhodococcus; and Paenibacillus were recovered in CM. In contrast, members of genera Burkholderia, Bacillus, Gordonia, Enterococcus, Geobacillus, Sanguibacter and Bhargavaea were isolated from control (CT) samples. Overall, higher species diversity and number of isolates (n = 119) were recovered in PM samples, with phylotypes belonging to genera Dietzia, Burkholderia, Arthrobacter, Lysinibacillus, Sporosarcina, Staphylococcus, Sanguibacter, Bhargavaea Rhodococcus and Streptomyces were unique to PM samples only . The ability of each axenic culture to degrade the crude oil waste sludge was considered to be proportional to the decolourization of DCPIP incorporated into the growth media.4 CFU/g culturable bacteria. Furthermore, 29 out of 33 isolates were positive for catechol 2,3-dioxygenase gene (C23O). Cultures found to be efficient degraders of the crude oil sludge could be phylogenetically grouped into 4 clades  belonging to the phylum Actinobacteria. These isolates were recovered from all treatments, including the control (CT) samples, with exception of swine/pig manure (SM). In clade II, six isolates belonging to orders Streptomycetales (genus Streptomyces) and Corynebacteriales  within the phylum Actinobacteria. In contrast, Clade III were mostly members of th phylum Firmicutes. The bacterial genus Bacillus and Lysinibacillus were the most recovered group in this study. Others included Clostridium, Enterococcus, Staphylococcus, Sporosarcina and Bhargavaea. In clade IV, included isolates members of class Betaproteobacteria (genus Burkholderia) and Alphaproteobacteria (genus Sphingomonas and Ochrobactarum). Interestingly, one novel PAH-degrading bacterial isolate that exhibited the highest similarity of ∼76.7% to members of genus Bacillus was also recovered from cow manure (CM) sample. These bacterial isolates were the most efficient degraders of the crude oil sludge. The evolutionary relationship of these isolates and their GenBank relatives are displayed in Axenic cultures of thirty-three isolates were qualitative positive for crude oil waste sludge degradation, with 6, 7, 6, 1 and 13 recovered from CM, CT, HM, SM and PM microcosms, respectively . These i4 clades . Clade I3.8In this study, an enriched consortia of the 33 efficient PAH-degraders were added to crude oil sludge (BC1)-, pyrene (BC2)- and anthracene-contaminated (BC3) media to test their bioaugmentation capacity. The PAHs were degraded gradually in media; total PAH (ΣPAHs), pyrene and anthracene contents dropped from 359.7 to 106.7, 14.0 to 3.36 and 42.0 to 0.084 mg/kg, respectively, after 30 days of treatment a. The reSphingomonas, Pseudomonas, Microbacterium, Rhodococcocus, Burkholderia-Paraburkholderia, unclassified Bradyrhizobiaceae, Bacillus, Delftia, Cellulomonas, Enterobacter and Aflpia were relatively viable and dominant across all samples. However, subtle variation in the enrichment of the bacterial groups was observed. Hierarchical clustering based on weighted UniFrac distance showed a clear separation of pyrene treated samples (BC2) from other two treatments. Overall, BC1 had higher species richness (Chao-1 = 89.1) than other two treatments . BC2 also had a higher species diversity and relative abundance of top 40 OTUs recovered at day 30. The most abundant genus observed in BC2 included Microbacterium, Sphingomonas, Rhodococcus, Burkholderia-Paraburkholderia and unclassified Bradyrhizobiaceae. Interestingly, OTUs assigned to genera such as Sporosarcina, Pseudoarthrobacter, Cellulomicrobium and Ochrobactarum associated with bacterial consortium were only observed in BC2. In contrast, only BC3 exhibited enriched abundance of genus Enterobacter.To further gain insight on the viability and antagonistic effects of bacterial isolates during PAHs degradation, a cultivation-independent detection method using 16S rDNA amplicon sequencing was carried out for three microcosms  at day 30. Heatmap of the normalized abundance at the genus level for bacteria in the three microcosms on day 30 is illustrated in 4in situ and ex situ physicochemical and environmental conditions. To improve the natural ability of microorganisms to degrade contaminants in oil associated environments, several successful engineered bioremediation approaches utilizing bioaugmentation (addition of known degraders of the contaminant) and or biostimulation (addition of nutrients in the form of fertilizers) have been reported [Bioremediation has been established to be a reliable cost-effective technology for oil spill and crude oil waste remediation , 41. Howreported , 43. In 4.12 evolution  showed a baseline attenuation of 52% for total PAH.In engineered bioremediation for oil-contaminated soils and refinery wastes, addition of N and P containing fertilisers to alleviate nutrient limitation is key in enhancing microbial activity and concomitant PAHs biodegradation , 44. In volution a and higvolution b during Assessment of the nature of the hydrocarbons within the co-composting microcosms was also done to gain information on possible role of manure amendment in the biostimulation of indigenous microorganisms capacity to efficiently degrade different PAH compounds in the crude oil sludge . GC/MS a4.2in situ and ex situ bacterial community for the improved crude oil sludge remediation. Metagenomic analysis showed that Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes were the predominant phyla present in all the microcosms at day 300. The shared genera between manure-treated and control microcosm included Pseudomonas, Delftia, Methylobacterium, Dietzia, Bacillus, Propionibacterium, Bradyrhizobium, Streptomyces, Achromobacter, Microbacterium and Sphingomonas, some of which has been previously reported in oil-contaminated soils [Pseudomonas and Sphingomonas have been previously associated with degradation of LMW PAHs [2-assimilating and methanogenic microorganisms  are known to be key players in HMW PAH degradation [Bacillus, Dietzia and Achromobacter are potent biosurfactant producers that promotes efficient emulsification and eventual biodegradation of HMW PAHs [Delftia, Bradyrhizobium, Microbacterium and Streptomyces can also decompose various aromatic compounds during denitrification [Proteiniphilum, unclassified Micrococcales, unclassified Lachnospiraceae, Sphingobium and Stenotrophomonas [Based on Baas-Becking hypothesis that “everything is everywhere, but the environment selects” , we envied soils , 43, 45,ed soils , 52, 53.LMW PAHs , 55, ferHMW PAHs , 56. Bacfication . Persistphomonas . The obsγ-proteobacteria , β-proteobacteria , α-proteobacteria , were prominent in PM and HM, while Firmicutes  and Actinobacteria  were found to predominate CM sample. As Proteobacteria are known for their catabolic versatility, genetic plasticity and metabolic diversity leading to broad substrate specificities for several classes of hydrocarbons in environments such as natural oil deposits, asphalt, crude oil, oil sand, oil contaminated water, soil and sludge [Proteobacteria, imply that the amendment with manures rich in utilizable C, N and other nutrients could be responsible for the increase in the relative abundance of the phylum Proteobacteria in PM and HM systems. In contrast, the genetic plasticity, metabolic versatility and production of extracellular and cellular biosurfactants by members of Firmicutes and Actinobacteria, have been reported to enhance the uptake and biodegradation of hydrophobic pollutants [As shown in d sludge , their ellutants . This ma4.3C23O)   were idel sludge . The detRhodococcus, Paenibacillus and Bacillus; 3 species from HM belonging genus Rhodococcus, Sphingomonas and Pseudomonas; and Lysinibacillus, Bhargavaea and Burkholderia. The elevated activities of these isolates in early stages of PAH degradation, indicate that they are fast PAH degraders, mainly associated with degradation of LMW PAHs. In support of these findings, Obi et al. [Pseudomonas and Bacillus species from crude oil sludge that had characteristic fast PAH degradation . To complement 2,6-DCPIP test, cell viability test of the isolates after 30 days incubation in MSM media supplemented with crude oil sludge as sole carbon and energy source was undertaken. All isolates yielded between 0.1-3.12 × 104 CFU/g culturable bacteria, indicating their ability to grow and utilize crude oil sludge PAHs. Further, PCR amplification with degenerate primers was also performed to screen for the presence of the cathecol-2,3-dioxygenase (C23O) genes in the isolates. The results showed that all the isolates possessed the catabolic C23O genes, providing clues on the their potential capability to degrade the crude oil sludge PAHs.Redox indicator 2,6-DCPIP (an electron acceptor) undergoes a colour change from blue (oxidised form) to colourless (reduced form). This color change can be used to determine the capability of microorganisms to utilise and to estimate the PAH biodegradation capacities of axenic bacterial cultures , 61. In The functional profile of the selected bacterial isolates related to their ability to utilize crude oil PAHs suggests that crude oil sludge-soil-woodchips-manure co-composting microcosms is a reservoir for the recovery of important bacterial species that can be exploited for engineered remediation processes. In this study, an enriched consortia of the identified 33 PAH-degraders was used for laboratory scale bioaugmentation study of crude oil sludge in absence of manure amendments. The treatment resulted in accelerated degradation of total PAHs, anthracene and pyrene within 30 days a, with v52 evolution) and temperature increase during co-composting indicated that manure treatments enhanced microbial activities which could be linked to overall improvement of PAH degradation. Culture independent analysis revealed that higher bacterial diversity was observed for HM followed by PM, with SM and CM exhibiting comparable values to CT samples. Members of Proteobacteria were highly enriched across all samples , with exception of CM which was dominated by Actinobacteria (35%) and Firmicutes (45%) members. Notably, the members of order Streptomycetales (11.7%) were only identified in the PM treatment. The major unique genera across all samples were Pseudomonas, Delftia, Methylobacterium, Dietzia, Bacillus, Propionibacterium, Bradyrhizobium, Streptomyces, Achromobacter, Microbacterium, Sphingomonas etc., that could be recovered from the microcosm samples by culturing. However, most OTUs were unclassified, which further warrants a comprehensive taxonomic approach to identify their identity and novelty. Many isolated strains were able to grow on crude oil sludge as carbon source, albeit with significant number of strains exhibiting both tolerance and high catabolic activity potential against crude oil sludge PAHs. Therefore, our findings have demonstrated the potential utility of the readily available animal manures as economically feasible co-composting agent for improved bioremediation crude oil sludge waste. Further, this study provides theoretical insights on co-composting with different animal manures as an interesting approach for the exploitation of novel PAH-degrading bacteria, which can further be formulated for engineered bioaugmentation of crude oil pollution remediation.In conclusion, the current study attempted static co-composting technique with different manures to understand the bacterial diversity and its catabolic potential against hydrocarbon degradation using both culture-dependent and culture-independent analysis. Acceleration of respiration rates .This work was supported by South Africa https://www.ncbi.nlm.nih.gov/) sequence archive (SRA) as BioProject ID PRJNA794053 under the accession number MK854826 to MK854993.Data associated with this study has been deposited at NCBI database (The authors declare no conflict of interest.No additional information is available for this paper.


To resolve whether the lack of AR is the driving factor for the emergence of the NE phenotype, molecular, cell, and tumor biology analyses were performed on 23 xenografts derived from patients with PC, recapitulating the full spectrum of genetic alterations proposed to drive NE differentiation. Additionally, phenotypic response to CRISPR/Cas9-mediated AR KO in AR+ CRPC cells was evaluated. These analyses document that (a) ARSi-resistant NEPC developed without androgen deprivation treatment; (b) ARS in ARSi-resistant AR+/NE+ double-positive “amphicrine” mCRPCs did not suppress NE differentiation; (c) the lack of AR expression did not necessitate acquiring a NE phenotype, despite concomitant mutations/deletions in PTEN and TP53, and the loss of RB1 but occurred via emergence of an AR–/NE– double-negative PC (DNPC); (d) despite DNPC cells having homogeneous genetic driver mutations, they were phenotypically heterogeneous, expressing basal lineage markers alone or in combination with luminal lineage markers; and (e) AR loss was associated with AR promoter hypermethylation in NEPCs but not in DNPCs.Resistance to AR signaling inhibitors (ARSis) in a subset of metastatic castration-resistant prostate cancers (mCRPCs) occurs with the emergence of AR S-transferase π [GSTP1]), but not AR , and hK2 (KLK2) with no expression of NE markers  is subverted from a growth suppressor of c-Myc expression to a cell autonomous oncogenic stimulator of c-Myc expression and thus malignant growth –26. Due – (+ adenocarcinoma (ARPC) “losing” its luminal cell differentiation via the loss of AR activity , such as abiraterone acetate (Abi), enzalutamide (Enza), and apalutamide, were developed and clinically documented to increase the survival of men with mCRPC, progressing after first-line ADT and when given in combination with first-line ADT . Despite– . The proactivity –34. It hhenotype –35.n = 23) of previously characterized patient-derived xenografts (PDXs) in addition to several newly established PDXs, which collectively recapitulate the full spectrum of clinically important genetic alterations in mCRPC. Second, hypermethylation of the AR promoter as a putative mechanism for the loss of AR expression was evaluated. Third, the in vitro and in vivo growth characteristics versus marker expression of the ARPC LNCaP-95 (LN-95) mCRPC cells initially exhibiting AR activity were determined following CRISPR/Cas9 dependent elimination of total AR protein expression.Therefore, to interrogate the relationship between ARSi resistance and NE differentiation, 3 complementary approaches were taken. First, the growth characteristics and expression of basal versus luminal versus NE lineage markers were evaluated in a large series  and hemizygous deleterious mutation (R130Q) in PTEN with a loss of PTEN protein expression . They also expressed REST and YAP1  metastasis at rapid autopsy , which are consistent with the PDX. Despite the patient never being exposed to ARSi treatment, the in vivo growth of BCaP-1 PDX is ARSi resistant because it grows equally well in intact or castrated hosts, with a doubling time of 20 ± 5 days  allele and loss of the other WT RB1 allele  mRNA  PC (DNPC) heterogeneously composed of cells expressing both basal and luminal cell characteristics, suggestive that their cancer-initiating cell was an AR– progenitor cell whose malignant transformation did not require exposure to ARSis.The BCaP-1 PDX was derived from a soft tissue metastasis adjacent to the right tibia obtained at rapid autopsy from a 63-year-old African American patient, who, at the time of initial presentation, had bone and lymph node metastases and an initial diagnostic prostate biopsy that was positive for carcinoma with a Gleason sum score of 9. Over the next year, the patient was treated with ADT followed by palliative external beam radiation of the bone before death but never received treatment with next-generation ARSis . At auto± 5 days , which warcinoma . Based u3 R175H;  coupled 1 allele . These cdeletion , resultideletion . They alB1 S45F; , which wB1 S45F; , presumaand Ki67 , with a and Ki67 . This isGR) mRNA  and no eal cells . Howevernd KRT18 , while h18  who underwent resection for a noninvasive urothelial carcinoma of the bladder a year before having a prostate biopsy that was positive for Gleason 9 PC with perineural invasion. Over the next 3 years, the patient had a radical prostatectomy for locally advanced disease , followed by ADT, external beam radiation, taxane chemotherapy, and olaparib before undergoing a rapid autopsy upon his death  (TP53 (R282W), and no expression of PTEN due to homozygous deletion. In addition, they minimally expressed RB1 mRNA with no detectable nuclear expression of RB1 protein (KLK3 (PSA), FOLH1 (PSMA), or NKX3.1   or mutat protein . None of protein  and Ki67 protein . They dir NKX3.1 . Again, essed GR . All 3 hessed GR . Again, B13 mRNA  and protB13 mRNA  than ARPn factor .KRT8 , FOLH1, KLK2, and KLK3 , and mutated PTEN (V317fs), but with no expression of NE markers like CHGA, CHGB, and SYP  and then a PC vaccine (GVAX), followed by ADT, docetaxel plus strontium-89, external beam radiation, Abi, etoposide, and cisplatin ; ref. 46ted TP53 . The mosted TP53 . Similarlized PC . Essentilized PC . A high and Ki67 . LvCaP-1and KLK3 , but witand KLK3 . This is3 R248Q; , SOX2, m and SYP . LvCaP-1 and SYP , p75 , mutated PTEN (V317fs), mutated SPOP (F133L), and mutated ATRX  media containing a low level of androgen . ARSi reSYP, CHGA, or CHGB  than the parental AR-expressing LN-95 cells in castrated male hosts  as the parental LN-95 cells despite the loss of AR expression  showed differential methylation consistent with the suppression of the NE phenotype in the DN versus NE PDXs (CHRNB2) was the promoter methylation pattern in the LvCaP-2/-2R amphicrine PDXs, consistent with that expected based on the NE phenotype in LuCaP 93  and growth arrest ; or (b) an AR– prostate progenitor cell is the cancer-initiating cell and gives rise to malignant progeny heterogeneously expressing various combinations of basal and luminal markers . In this second scenario, no promoter hypermethylation-dependent silencing of AR would be necessary for selective outgrowth of these DN cells under extreme androgen deprivation. Presently, these PDX models are being utilized to resolve whether adaption versus selection is the mechanism for the emergence of ARSi-resistant DNPC. Earlier studies demonstrated that PC with a luminal phenotype could be derived from genetically manipulated primary human benign prostate basal cells, suggesting that histology does not necessarily correlate with cell of origin  an initially ARf origin .– PCs are increasing . WNB, YZ, IMC, SLD, LA, RAP, BH, RC, AKM, SLZ, JEH, JL, AMDM, EC, JX, SY, MCH, PSN, WGN, WBI, and JTI analyzed and interpreted the data . WNB, YZ, IMC, SLD, LA, RAP, BH, RC, AKM, SLZ, JEH, JL, AMDM, EC, JZ, SY, MCH, PSN, WGN, WBI, and JTI wrote, reviewed, and/or revised the manuscript. YZ, IMC, JEH, EC, and JL provided administrative, technical, or material support . WNB and JTI supervised the study.


The underlying mechanism of fibroblast growth factor receptor 1 (FGFR1) mediated carcinogenesis is still not fully understood. For instance, FGFR1 upregulation leads to endocrine therapy resistance in breast cancer patients. The current study aimed to identify FGFR1-linked genes to devise improved therapeutic strategies. RNA-seq and microarray expression data of 1,425 breast cancer patients from two independent cohorts were downloaded for the analysis. Gene Set Enrichment Analysis (GSEA) was performed to identify differentially expressed pathways associated with FGFR1 expression. Validation was done using 150 fresh tumor biopsy samples of breast cancer patients. The clinical relevance of mRNA and protein expression of FGFR1 and its associated genes were also evaluated in mouse embryonic fibroblasts (MEFs) and breast cancer cell line (MDA-MB-231). Furthermore, MDA-MB-231 cell line was treated with AZD4547 and GANT61 to identify the probable role of FGFR1 and its associated genes on cells motility and invasion. According to GSEA results, SHH pathway genes were significantly upregulated in FGFR1 patients in both discovery cohorts of breast cancer. Statistical analyses using both discovery cohorts and 150 fresh biopsy samples revealed strong association of FGFR1 and GLI1, a member of SHH pathway. The increase in the expression of these molecules was associated with poor prognosis, lymph node involvement, late stage, and metastasis. Combined exposures to AZD4547 (FGFR1 inhibitor) and GANT61 (GLI1 inhibitor) significantly reduced cell proliferation, cell motility, and invasion, suggesting molecular crosstalk in breast cancer progression and metastasis. A strong positive feedback mechanism between FGFR1–GLI1 axis was observed, which significantly increased cell proliferation and metastasis. Targeting FGFR1–GLI1 simultaneously will significantly improve the prognosis of breast cancer in patients. The fibroblast growth factor receptor (FGFR) signaling pathway plays an important role in a variety of biological processes including angiogenesis, cell growth, differentiation, and survival . The gen1. The mRNA expression levels of FGFR1 gene was estimated using z-score >2.0. The clinicopathological relevance of FGFR1 expression was assessed against multiple features including age, stage, grade, node stage, and metastasis. The details of clinical information are available in The study design of the current study is described in n = 150) along with matched control tissues were collected for validation of discovery cohort findings after receiving informed consents of participants. Clinicopathological information of these patients was collected through subsequent follow-up from pathology reports. Information regarding clinicopathological features of validation cohort is available in The present study was conducted after obtaining approval from institutional ethical review committees at our university and concerned hospital. Tumor biopsies . cDNA was generated using RevertAid First-Strand cDNA Synthesis Kit  as per manufacturer’s instructions. Conventional PCR was performed with β-actin primers to confirm the cDNA synthesis. Amplified products were electrophoresed on 2% agarose gel and stained with ethidium bromide for further use.Total RNA was extracted from tumors and matched normal samples using TRIzol–ΔΔCt method.Primers for FGFR1 and GLI1 were designed using Integrated DNA Technology (IDT) software and synthesized for Macrogen, Korea . Target 2, and MDA-MB-231 cells were transfected with them for assessing the differential expression of FGFR1, SHH, and GLI1.MDA-MB-231 was maintained as per American Type Culture Collection (ATCC). Expression of FGFR1, SHH, and GLI1 were assessed both at RNA and protein level. Similarly, the effect of these aforementioned molecules was evaluated on CRISPR/Cas9-mediated SHH knockout MDA-MB-231 cells . Moreove5 cells were seeded in six-well plates until confluency and treated with variable concentrations of GANT61. Similarly, AZD4547 (Selleckchem3) was added to the medium at a concentration of 100 nM. Both RNA and protein were quantified from treated and untreated wells for functional assays performed after 48 h of treatment with both inhibitors.A stock of GANT61  was dissolved in dimethyl sulfoxide (DMSO) maintaining a stock concentration of 1 mM. Briefly, 3 × 10Total protein content from respective cell lines were extracted and quantified using the Pierce BCA protein assay kit . Extracted proteins were separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membrane, and blocked by 5% non-fat milk at room temperature. Membranes were incubated with primary antibodies for FGFR1 , SHH , and GLI1  . After o5 cancer cells from respective cancer cell lines were seeded in six-well plates. The plate was left at 37°C incubation till it reaches confluent monolayer. Once attained, the medium was aspirated, and N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) medium was introduced in each well. Wounding measurements were recorded using the previously mentioned protocol  placed in 24-well plate. These inserts were precoated with 50 μg/ml Matrigel  prior to cell seeding. A total of 5 × 10 earlier .U test and Kruskal–Wallis ANOVA were applied to explore any probable association of these genes with clinicopathological features. Furthermore, correlation between molecules was observed using Spearmen test. All p-values of 0.05 were considered statistically significant.IBM SPSS 21 software  was used for all the statistical analyses. Data were represented as mean ± SD, and Wilcoxon signed-rank test was performed to evaluate difference between tumor and control. Mann–Whitney p = 0.05) and node-positive patients (p = 0.04)  .Next, GSEA was performed to identify common differentially expressed pathways in both breast cancer cohorts. A total of 20 pathways were found to be upregulated along with FGFR1 overexpression in TCGA dataset . Similarp = 0.0001), node positive (p = 0.044), and metastasis (p = 0.022) (p = 0.047). Therefore, based on all the expression and statistical analyses in discovery cohorts, we further established the potential prognostic association of GLI1 and FGFR1 genes using normal and tumor pairs of 150 breast cancer patients.Interestingly, 5 out of 16 SHH pathway genes showed core enrichment in FGFR1-expressed breast cancer patients using leading-edge subset method . Of note= 0.022) . Similarp = 0.0001) and GLI1 (p = 0.0001) were significantly overexpressed in tumor samples compared to their respective controls. Interestingly, FGFR1 and GLI1 expression showed strong positive correlation . Of note, consistent with discovery cohort findings, overexpression of GL1 and FGFR1 genes was significantly associated with high grade (p = 0.05), late stage , and metastasis , respectively  and AZD4547 (FGFR1 inhibitor) on both GLI1 and FGFR1 was studied in MDA-MB-231 cell line. To perform the analysis, multiple genes including SHH, AKT, and MAPK were also selected to compare the effect. According to the results, a significant decrease in the expression of GLI1, SHH, and FGFR1 was observed after treatment with GANT61 at 24 and 48 h. MAPK expression was reduced after 48 h of treatment. However, no change in the expression of AKT gene was observed . InteresNext, using Western blot analysis, the impact of GANT61, AZD4547, SiGLI1, and SiSHH was assessed in MDA-MB-231 cells on SHH, GLI1, and FGFR1 expression. First, GANT61 treatment was administered until 48 h with 12-h intervals. A significant decrease in protein expression was observed after 12 h for all three genes including SHH, GLI1, and FGFR1 . In addiFurthermore, the effect of FGFR1–SHH pathway signaling on the motility of breast cancer cells was evaluated using wound-healing assay. MDA-MB-231 cells were treated with AZD4547 only, GANT61 only, and AZD + GANT61 combined . Breast Fibroblast Growth Factor Receptor 1 DNA amplification is the most frequently reported FGF-pathway alteration in breast cancer  between FGFR1 and GLI1 and significant association with late stage, high grade, and metastasis, further emphasizing on the FGFR1–GLI1 nexus in breast carcinogenesis. Next, the prognostic impact of FGFR1–GLI1 axis inhibition was evaluated. Cyclin-dependent kinase 6 (CDK6) inhibitors are the most widely used treatment options in breast cancer patients (in vitro functional assays strongly suggested that targeting FGFR1–GLI1 crosstalk can significantly improve the prognosis of breast cancer. Interestingly, FGFR1 and GLI1 also activate many downstream oncogenic genes, which lead to cancer cell migration, proliferation, and survival (These observations were further validated using qPCR method in 150 paired tumor-normal cases. The results showed strong correlation (patients . Howeverpatients . Recentlpatients . For thapatients . Moreovepatients . Furthersurvival . Hence, The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the ERB Shaheed Zulfiqar Ali Bhutto. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Ethics Review Board of the Texas A&M University.SKR, WK, FW, AM, TK, ER, JK, ShH, AH, SA, NA, StH, FH, and MFM: conceptualization, visualization, and writing—original draft. SKR, WK, FW, TK, AM, ER, and JK: data curation. SKR, WK, FW, TK, AM, ER, JK, FH, and MFM: formal analysis, methodology, and validation. FW, FH, and MFM: funding acquisition. SKR, ER, TK, JK, ShH, AH, SA, NA, StH, FH, and MFM: investigation. ShH, AH, SA, NA, StH, FH, and MFM: project administration. ShH, AH, StH, FH, and MFM: resources and supervision. FH and MFM: software. ShH, AH, StH, FH, and MFM: writing—review and editing. All authors contributed to the article and approved the submitted version.The 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.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.