Myeloproliferative neoplasms (MPNs) comprise polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The relationship between JAK2 p.(V617F) mutation and MPNs was first described in 2005. The purpose of this study was to determine the prevalence of JAK2 p.(V617F) mutation in Tunisian patients assessed for MPNs and try to set a genotype-phenotype correlation. A retrospective study was conducted between January 2015 and April 2019. We collected the clinical data of all patients with MPNs suspicion or atypical splanchnic vein thrombosis (SVT). JAK2 p.(V617F) mutation was detected by allele specific real-time quantitative fluorescence PCR (AS-qPCR). We gathered 974 patients who underwent molecular analysis, 55.5% of them were male and 44.5% were female. The median age of all studied patients was 56 years. JAK2 p.(V617F) was found in 349 (35.8%) of total enrolled cases. It was reported in 44%, 37%, 29% and 25% of all patients diagnosed as having respectively ET, PV, PMF and atypical SVT. JAK2 p.(V617F) was negative in 62.2% of patients addressed for suspicion of PV. There was a significant positive correlation between the JAK2 p.(V617F) mutation status, age, gender, white blood cell counts and platelet counts. To our best knowledge, this is the first vast investigation of JAK2 p.(V617F) variant in Tunisia and North Africa with the lowest mutation rate in entire cohort and MPNs subgroups, underlying a specific presentation of this mutation. It is considered as an essential marker of MPNs’ diagnosis and prognosis and is associated with differences in the phenotype of these disorders, helpful for the follow-up of these patients. Myeloproliferative neoplasms (MPNs) are a heterogeneous group of disorders which arise from genetically altered myeloid stem or progenitor cells. MPNs are characterized by an over production of mature myeloid cells. Classical MPNs have been classified into 3 entities: polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) . It has JAK2) gene in a significant proportion of patients with MPNs [JAK2 gene, resulting in the substitution of Valine to Phenylalanine at codon 617 p.(V617F) [JAK2 protein, leading to constitutive activation of JAK2 and JAK/STAT signaling pathway in the absence of ligands [JAK2p.(V617F) is detected in 50-60% of ET and PMF patients and up to 97% of PV patients [JAK2p.(V617F) mutation as a major criterion for the diagnosis of MPNs [JAK2p.(V617F) mutations in a cohort of 45 patients [JAK2p.(V617F) mutation in MPNs Tunisian patients and try to set a genotype-phenotype correlation.The year 2005 was a watershed in understanding of the molecular pathogenesis of MPNs with the identification of an acquired recurrent somatic mutation in the Jak homology domain 2(JH2) of Janus kinase 2 .V617F) . This do ligands . JAK2p. was prescribed by a clinical hematologist. We gathered clinical information in the prescription of all screened patients: age, gender and the applied classification and diagnostic criteria proposed by the World Health Organization (WHO) in 2008 and the updated version 2016 relative to MPNs [Samples´ collection: blood samples were obtained after written informed consents from all included patients. Blood routine examination involved hemoglobin, white blood cell (WBC) counts and platelet counts.JAK2 p.(V617F) mutation detection: DNA extraction was carried out from lymphocytes by QIAMP DNA mini-Kit  according to the manufacturer´s protocol. DNA quality was assessed with the Nanodrop 2000 spectrophotometer  with an approved DNA concentration ranging between 100-200 ng/µL and A260/A280 ratio between 1.8-2.JAK2 MutaSearch Kit, designed for the specific and quantitative determination of DNA copy number of JAK2p.(V617F) mutation and JAK2 wild type with respective primers [JAK2), negative control (NC-V617F JAK2), cut-off sample (COS-V617F JAK2) were provided by the kit. A 96-well plate qPCR device were used and all measures were analysed in duplicate. For one reaction, the mix contained 12.5 μL of TaqMan Universal PCR Master Mix, 1μL of specific primers and probe and 6.5 μL of H2O. Five μL of DNA samples were added to 20 μL of the corresponding qPCR premix well (V617F or WT). Five μL of each control and COS-V617F JAK2 were added in each corresponding 20 μL qPCR premix well. The following cycling conditions were run in the 7500 Sequence Detection System  to amplify in real time the increase in fluorescence generated: One cycle at 50°C for 2 minutes of incubation, followed by a 10-minute incubation at 95°C, then 50 cycles at 95°C (15 seconds), and an extension step at 63°C (1 minute). The point at which the fluorescent signal rises above the background called crossing threshold (CT), is directly proportional to the quantity of starting DNA. The average CT value obtained (CT V617F and CT WT) for each sample  were calculated. The load limit verified that the patient DNA sample used for the test has been correctly manipulated to ensure the reliability of the final results for JAK2 V617F status. Then we calculated the ΔCT value for all valid samples (ΔCT Sample) and controls  in order to obtain the ΔΔCT value for each patient sample (ΔΔCT Sample) and each control (ΔΔCT PC- V617F and ΔΔCT NC- V617F). The JAK2 V617F mutation was detected if ΔΔCT Sample > + ΔCT COS x 0.07 and was absent if ΔΔCT Sample < - ΔCT COS x 0.07.We performed an Allele specific real-time quantitative fluorescence PCR (AS-qPCR) using ipsogen  primers . p.. The tests used for comparison of distributions of quantitative variables were Student's T test and Mann-Whitney U test. The degree of significance “p” retained for all the tests was 0.05.Ethics in publishing: this study was approved by the regional committee for medical and health research ethics of Pasteur Institute of Tunis.Population basic characteristics: we tested 974 Tunisian patients for JAK2p.(V617F) mutation with clinical suspicion of MPN or a typical splanchnic vein thrombosis. The median age of all studied patients was 56 years with extremes ranging from 1 to 90 years and M/F sex-ratio was 541/433. From the total studied population, the MPNs were divided into: 29.4% (286/974) PV, 28.2% (275/974) ET and 9.2% (89/974) PMF. Unclassified MPNs were found in 25.5 % (249/974) of all cases and 7,7% (75/974) patients were addressed for SVT. We did not report any secondary polycythemia. The clinical and biological characteristics in all tested Tunisian patients are summarized in Genotype-phenotype correlation:JAK2p.(V617F) mutation was detected in 349 of all 974 patients (35.8 %). JAK2 p.(V617F) was found in 44% (121/275), 37.8% (108/286), 30.1% (75/249), 29.2% (26/89) and 25.3% (19/75) of patients diagnosed with ET, PV, Unclassified MPNs, PMF and thrombosis respectively. The SVT subgroup were carriers of JAK2p.(V617F) mutation despite the absence of overts signs of MPNs. We compared the subgroup with mutant variant to wild type subgroup and found a statistically significant difference in the distribution of age and sex ratio (p= 0.000) (JAK2p.(V617F) mutation in total cohort, three subgroups of MPNs and unclassified MPNs (p=0.000-0.032) (JAK2 in the different subgroups is represented on = 0.000) . Mean ag0-0.032) , Table 2JAK2 wild type subgroup, the sex presentation was significant (p<0.001) where PV phenotype (63.2% vs 14.9%) was more common in men than women whereas ET (66.9% vs 22.3%) and PMF (18.2% vs 14.5%) phenotypes were more reported in women than men (JAK2p.(V617F), the sex repartition was not significant : women were more common to have ET  and PMF (11.5% vs 8.8%) phenotypes while men tend to have more PV phenotype (50.4% vs 34.6%) (JAK2p.(V617F) was associated with a meaningfully higher level in WBC and platelet counts (p= 0.000). There was no significant correlation between the JAK2 mutational status and hemoglobin levels (p=0.150) (Men were more concerned with PV (p<0.001) than women who were more prevalent with PMF clinical phenotype  and ET phenotype  . In JAK2than men . In patis 34.6%) . JAK2p.(p=0.150) .JAK2p.(V617F) had significantly higher mean age (p=0.000) and higher platelet counts (p=0.000) than in wild type group. Men were more concerned with JAK2 wild type (p=0.000)  . In ET, p=0.000) . The mutJAK2 p.(V617F) mutation in the first large Tunisian patients cohort examined for suspected MPNs or SVT. We also investigated the relationship between this mutation and its influence in hemogram variation in 974 patients divided into different subgroups of MPNs.In the present study, we determined the prevalence of JAK2 p.(V617F) mutation is central to the diagnosis of a clonal process and exclusion of non-neoplastic disorders [JAK2 gene, as a major criterion of MPNs´ diagnosis specifically ET, PV and PMF. It is detected in around 50-60% of ET and PMF patients and in more than 95% of patients with PV [Myeloproliferative neoplasms mainly occur in adults and only infrequently in children . Patientisorders . In 2008 with PV .JAK2 p.(V617F) mutation was detected in around 35.8 % of all enrolled patients, 44% of ET, decreasing the distribution of this mutation between PV and ET accounting for 37.8% of PV and 29.2% of PMF. It is striking that as many as 62% of those with PV were JAK2 p.(V617F) negative [JAK2 p.(V617F) in all cohorts and their different subgroups of MPNs [In this Tunisian study, negative -27 (Tablnegative . We mana of MPNs -27 (TablJAK2 p.(V617F) mutation. These results support the previous view that routine screening of this mutation appears to be indicated in the presence of SVT [JAK2p.(V617F) mutant status, age, gender, white blood cell counts and platelet counts in different MPNs groups [JAK2p.(V617F) mutation are more elder in MPN groups which is conform to recent reports [et al. (2020) in term of women predominance in ET and men in PV independantly of JAK2p.(V617F) status but contrasting in PMF where they found more men related to this phenotype (p=0.001) [JAK2 mutant subgroup, we did not established a correlation while their women present more ET and their men more PMF (p=0.001) [Nineteen patients with SVT were positive for e of SVT . Many sts groups -27 (Tabl reports -27 (Tablp=0.001) . In theip=0.001) . In wildp=0.001) .JAK2 p.(V617F) mutation rate was related with variations in hemoglobins, WBC and platelet counts compared with other populations [In MPNs subgroups, ulations -27  mutation. The mutational landscape in MPN is much more complex than initially thought [JAK2 p.(V617F) was informative. However, our study suffers from limitations and bias since we only search for JAK2 p.(V617F). We should consider these data in MPN with caution since JAK2 mutation in exon 12, MPL and CALR mutations were not investigated and further additional studies should test them in the large proportion of the JAK2 negative patients.Despite advances in understanding MPNs biology, an important fundamental question of how the same genetic event can be associated with distinct clinical phenotypes remains unresolved, as it is for  thought . A combi thought . In our JAK2p.(V617F) mutation in our population and found the lowest mutation rate of this variant in all broad cohorts. Unbalanced rates in different subgroups of MPNs were highlighted. The allele-specific quantitative real-time fluorescence PCR in JAK2p.(V617F) keeps its place in routine. The clinical and biological characteristics established in this study could be helpful for the clinicians during the diagnosis and the follow-up of MPNs patients. Furthermore, we must also refine the mutational spectrum of MPNs in Tunisia with additional studies of others driver mutations.From our first large study on Tunisian patients with MPNs and SVT, we have settled the prevalence of Molecular genetics is a cornerstone in the diagnosis and follow-up of MPN subgroups.This is the first large retrospective study from Tunisia that provides an overview on clinical and biological characteristics in JAK2 p.(V617F) carriers addressed for suspected MPNs or SVT;The lowest rate in JAK2 p.(V617F) mutation (35.8%) was reported in Tunisia in total cohort with 37.8% in PV, uncommon with previous studies.


Plasma efavirenz concentrations were quantified at 4, 8, 12, 24, and 48 weeks of cART. Genotyping for CYP2B6, CYP3A5, UGT2B7, ABCB1, and SLCO1B1 common functional variant alleles was performed. (3) Results: The efavirenz plasma concentration reached a peak at two months, declined by the 3rd month, and stabilized thereafter, with no significant difference in geometric mean over time. On average, one-fourth of the children had plasma efavirenz concentrations ≥4 µg/mL. On multivariate analysis, CYP2B6*6 and ABCB1c.3435 C > T genotypes and low pre-treatment low-density lipoprotein (LDL) were significantly associated with higher plasma efavirenz concentration regardless of treatment duration. Duration of cART, sex, age, nutritional status, weight, and SLCO1B, CYP3A5, UGT2B7, and ABCB1 rs3842 genotypes were not significant predictors of efavirenz plasma exposure. (4) Conclusion: Pre-treatment LDL cholesterol and CYP2B6*6 and ABCB1c.3435 C > T genotypes predict efavirenz plasma exposure among HIV-infected children, but treatment-duration-dependent changes in plasma efavirenz exposure due to auto-induction are not statistically significant.(1) Background: Efavirenz plasma concentration displays wide between-patient variability partly due to pharmacogenetic variation and autoinduction. Pediatric data on efavirenz pharmacokinetics and the relevance of pharmacogenetic variation are scarce, particularly from sub-Saharan Africa, where >90% of HIV-infected children live and population genetic diversity is extensive. We prospectively investigated the short- and long-term effects of efavirenz auto-induction on plasma drug exposure and the influence of pharmacogenetics among HIV-infected Ethiopian children. (2) Method: Treatment-naïve HIV-infected children aged 3–16 years old ( HIV infections remain a global public health problem, particularly in sub-Saharan Africa (SSA). According to the 2020 UNAIDS estimates, around two-thirds of the 1.7 million HIV-infected children 0–14 years) worldwide live in SSA ,4. Recen years woABCB1 genotype on plasma efavirenz concentrations in healthy volunteers [Efavirenz has served as the preferred first-line antiretroviral drug for more than a decade. Currently, the World Health Organization (WHO) recommends dolutegravir as the preferred first-line regimen for adults and children to whom dosing recommendation is available . Howeverlunteers  and HIV lunteers ,17,18.Efavirenz plasma concentration displays wide variations between patients and different populations, partly due to pharmacogenetic variations ,19. The Overall pediatric data on efavirenz pharmacokinetics and the relevance of pharmacogenetic variation is scarce, mainly from sub-Saharan Africa, where >90% of HIV-infected children globally live and population genetic diversity is extensive ,27. The This multi-centered prospective pharmacokinetic and pharmacogenetic study was conducted between April 2017 and August 2019. A total of 111 ART-naïve HIV-infected children aged from 3 to 16 years were prospectively recruited and enrolled at seven HIV/AIDS treatment centers located in two adjacent administrative regions in Ethiopia: Oromia and Southern Nations Nationalities and Peoples Region (SNNPR), which have diverse ethnic groups. In the Oromia region, the study centers were Adama Hospital, Asela Referral and Teaching Hospital, and Shashemene General Hospital. The study sites in SNNPR region were Hawassa University Comprehensive Specialized Hospital, Adare General Hospital, Hosana Referral Hospital, and Arbaminch General Hospital. These regions were selected because of high HIV prevalence and population diversity, which is generalizable to the nation.At enrollment, complete medical history, clinical examinations, and laboratory tests including complete and differential blood counts, platelet counts, CD4 count, HIV viral load, and liver enzymes were performed. Subsequently, study participants initiated a first-line pediatric cART regimen containing efavirenz (EFV) and lamivudine (3TC) with either zidovudine (AZT), abacavir (ABC), or tenofovir (TDF) following the Ethiopian national pediatric HIV treatment guideline . All chi® assay reagents for allelic discrimination (Applied Biosystems Genotyping Assay) as described previously [® genotyping assays with the following ID numbers for each SNP: C__7817765_60 for CYP2B6*6 , C__26201809_30 for CYP3A5*3 , C__30203950_10 for CYP3A5*6 (rs10264272), C__32287188_10 for CYP3A5*7 (g.27131_27132insT rs41303343), C__11711730_20 for ABCB1 (c.4036A > G rs3842), C__7586657_20 for ABCB1 (c.3435C > T rs1045642), C__30720663_20 for UGT2B7 (372G > A rs7662029), C__1901697_20 for SLCO1B1*1B (c.388A > G rs2306283), C__30633906_10 for SLCO1B1*5 (c.521T > C rs4149056), and C__1901709_20 for SLCO1B1 (g.38664C > T rs4149032). Genotyping was performed using a QuantStudio 12K Flex Real-Time PCR system . The final volume for each reaction was 10 µL, consisting of 2X TaqMan® fast advanced master mix , TaqMan® 20 X/40 X drug metabolism genotyping assay mix (Applied Biosystems), and genomic DNA. The PCR profile consisted of an initial step at 60 °C for 1 min, a hold stage at 95 °C for 20 s, and 40 cycles , and then a read stage with 60 °C for 30 s.Genomic DNA was extracted from whole blood samples using a QIAamp DNA Blood Midi Kit (Spin Protocol) . Genotyping for common functional variant alleles in five genes relevant for efavirenz disposition was conducted by real-time PCR using Taqmaneviously ,35. CommEfavirenz reference standard was purchased from Merck . Deuterated-efavirenz (rac Efavirenz-d4) was purchased from Toronto Research Chemicals . Acetone, acetonitrile, ammonium acetate, isopropanol, methanol, and acetic acid of mass spectrometry (MS) grade were purchased from Merck . Blank human plasma was obtained from the blood bank of the Karolinska University Hospital Huddinge .An LC-MS/MS system consisting of an Acquity Ultra Performance LC-system coupled to a Xevo TQ-S Micro  was used to quantify plasma efavirenz concentration. The MS/MS was operated in positive ionization mode. The data were processed using MassLynx 4.2 (Waters). In brief, plasma calibration samples were prepared by spiking blank plasma samples with efavirenz and were included in each analytical run. Quality control samples were also prepared by spiking plasma blanks to obtain low and high concentrations. The lower and upper limits of quantification were 15.8 and 15,800 ng/mL, respectively.g. An amount of 5 µL of the supernatant was injected onto the LC-MS/MS system. The chromatographic system consisted of an Acquity UPLC BEH C18 reversed-phase column 2.1 × 50 mm, 1.8 μm with 0.1% aqueous formic acid as mobile phase A and MeOH as mobile phase B. The initial composition of the mobile phase was 50% B, followed by a linear gradient to 99% in 1.5 min, with a flow rate of 0.4 mL/min. Total analysis/run time for each sample was approximately 3 min.Plasma efavirenz concentration was determined by direct injection of the supernatant after protein precipitation using acetonitrile. In brief, a 50 µL aliquot of plasma samples was added into 200 µL acetonitrile solution containing internal standard (500 ng/mL of efavirenz-d4 in methanol), vortexed for 30 s followed by centrifugation for 5 min at 2100× Efavirenz was monitored by the transition m/z 316 > 168 and the efavirenz-d4 by 320 > 172. The calibration curve was constructed by linear regression of the analyte/internal standard area ratios with an applied weighing of 1/x2. Accuracy was within ±10% throughout the quantification range. Precision was below 6 CV% except for LLOQ (below 10 CV%). The method was validated according to the European Medicines Agency Guideline on bioanalytical method validation .Study participants’ sociodemographic and baseline clinical parameters are summarized as the median and interquartile range (IQR) or as frequency and percentages. For assessment of the nutritional status of the children, anthropometric measurements were converted to height for age Z score (HAZ) and weight for age Z score (WAZ) using WHO Anthro-Plus software version 1.0.4. Body mass index (BMI) for age percentiles was calculated using the age-and-sex-specific percentile for BMI as per the Centers for Disease Control calculator for children and teens aged 2–19 years.Efavirenz plasma concentrations were transformed to log10 values before statistical analysis. The dataset was reshaped from a wide to a long format in which each individual subject had five records or less for weeks 4, 8, 12, 24, and 48. One-way analysis of variance (ANOVA) was used to identify genotype association with efavirenz plasma concentration measured at different weeks of ART. Change in efavirenz concentration over time was analyzed using a generalized linear mixed-effect model (LME). Univariate followed by multivariate analyses using LME models were conducted to determine predictors of efavirenz concentrations and to quantify the unexplained between- and within-subject variations in efavirenz exposure at different weeks of treatment. The LME modeling estimated fixed effect (predictor coefficients) and random effect parameters (between and within-subject variabilities in efavirenz concentrations). The LME models were therefore formulated as follows.p-values ≤ 0.2 were included in the final LME models.p values < 0.05 were considered statistically significant.Descriptive statistics analysis for baseline characteristics was performed using Statistical Package for Social Sciences (SPSS) software, version 20.0 . The R software package  and GraphPad Prism version 7  were used for the analysis and presentation of the results. The study received ethical approval from the SNNPR Regional Health Bureau Institutional Review Board (IRB), Addis Ababa University College of Health Sciences (Protocol No: 053/16/Pharma), and the National Research Ethics Review Committee, Ministry of Science and Higher Education-Ethiopia (Ref NO 3.10/166/2016). Prior to giving consent, study participants and their parents/guardians received written and oral information in their local language about the study. Written informed consent from parents or legal guardians was obtained. For participants ≤12 years, written informed consent was obtained from their parent or guardian. For participants >12 years of age, informed consent was obtained from the parent or guardian, and assent was obtained from the study participant.A total of 111 newly diagnosed and ART-naïve HIV-infected children were enrolled prospectively to participate in this study, of whom 63 (56.8%) were boys. Thirty-nine (35.1%) children were orphans (having lost one parent or both). Eleven (9.9%) children did not complete the study due to lost to follow-up or transfer out from the study area.The sociodemographic, clinical, and biochemical characteristics of the study participants are presented in CYP2B6, CYP3A5, UGT2B7, ABCB1, and SLCO1B1 are presented in CYP3A5*7 (27131_27132insT) was absent. Haplotype analysis indicated no linkage disequilibrium between CYP3A4*1B (−392A > G), CYP3A5*3 (c.6986A > G), and CYP3A5*6 (c.14690G>A) [CYP3A5*1 alleles .The variant alleles and respective genotype frequency distribution of 4690G>A) ,38. HencA total of 396 plasma samples were collected during the 48-week follow-up period. There was wide between-patient variability in plasma concentrations of efavirenz, and the coefficients of variation at the 4th, 8th, 12th, 24th, and 48th weeks of ART were 143%, 128%, 161%, 157%, and 142%, respectively. The proportions of children with sub-therapeutic (<1 µg/mL) plasma efavirenz concentration at the 4th, 8th, 12th, 24th, and 48th weeks of ART were 12.3%, 13%, 17.9%, 17.7%, and 12.3%, respectively. The proportions of children with supra-therapeutic (>4 µg/mL) plasma efavirenz concentration at the 4th, 8th, 12th, 24th, and 48th weeks of ART were 19%, 30%, 29%, 22%, and 29%, respectively.p = 0.89). Though not significant, the pattern of change in the mean log plasma efavirenz concentration over time displayed a moderate increase at the 8th week of ART, declined by the 12th week, and stabilized after that.The pattern and extent of change in plasma efavirenz concentration in each individual and the geometric mean at the 4th, 8th, 12th, 24th, and 48th weeks of ART are presented in CYP2B6*6/*6 genotype had significantly higher mean log efavirenz plasma concentration than homozygous wild-type (CYP2B6*1/*1) or heterozygous (CYP2B6*1/*6) at week 4 , week 8 , week 12 , week 24 , and week 48 . The CYP2B6*6 genotype was significantly associated with therapeutic plasma efavirenz concentration categories (p < 0.001). There was no significant impact of CYP3A5, UGT2B7, SLCO1B1, and ABCB1 c.4036A > G genotypes on efavirenz plasma concentration at all study time points, except for ABCB1 c.3435C > T. Carriers of at least one ABCB1 c.3435C > T allele had significantly higher mean log efavirenz plasma concentration than those with the ABCB1 c.3435 C/C genotype at weeks 4 and 12 with p-values of 0.004 and 0.03, respectively. Although not statistically significant, children with at least one variant allele of ABCB1 c.4036A > G had a higher mean log efavirenz plasma concentration at all treatment weeks. The pattern and extent of change in the geometric mean of plasma efavirenz concentration at the 4th, 8th, 12th, 24th, and 48th weeks of ART among the different CYP2B6 genotypes stratified by age group are presented in Association of genotype with plasma efavirenz concentration measured at the 4th, 8th 12th, 24th, and 48th weeks of efavirenz-based antiretroviral treatment among HIV-infected children using one-way analysis of variance is presented in p-values ≤ 0.2 from the univariate analysis were included in the model simultaneously, and the result is presented in CYP2B6*6 (c.516G > T), ABCB1 c. 3435 C > T genotype, and baseline LDL were significant predictors of efavirenz exposure regardless of weeks of treatment  as a categorical predictor was included in the model and found to have no significant effect on the plasma efavirenz exposure.A univariate followed by a multivariate analysis was conducted to identify predictors of plasma exposure over time using the developed linear mixed effect (LME) models. The impact of sociodemographic, plausible clinical, and biochemical characteristics of the study participants presented in reatment . The coeCYP2B6*1/*1 genotype. In a univariate analysis, the impact of sociodemographic, potential pre-treatment clinical, and biochemical characteristics of the study participants presented in p-values ≤ 0.2 from the univariate analysis were included in the LME model simultaneously to predict plasma exposure over time, and the results are presented in CYP2B6*1/*1 genotype, the unexplained between-subject and within-subject variability in efavirenz concentration was about 22% and 26%, respectively. The effect of duration of therapy on plasma efavirenz concentration was noted at week 8 of ART. While pre-treatment higher liver enzymes (ALT and ALP), total cholesterol, and triglycerides were positive predictors, LDL was a negative predictor of plasma efavirenz exposure regardless of weeks on ART among children homozygous for the functional CYP2B6*1 variant allele.A subgroup analysis was performed to identify predictors of plasma efavirenz concentration over time among study participants with the CYP2B6*6 (c.516G > T), ABCB1 3435C > T genotype, and low pre-treatment LDL concentration are significant predictors of high plasma efavirenz exposure regardless of the duration of therapy. (iii) Among the CYP2B6*1/*1 genotype, altered liver enzymes  and lipid profiles  influence plasma efavirenz exposure. (iv) There is no significant association of nutritional status (stunting and wasting), sex, age, SLCO1B1, CYP3A5, and UGT2B7 genotype on plasma efavirenz concentration or autoinduction profile. To our knowledge, this is the first study to explore the short- and long-term efavirenz plasma exposure, and autoinduction profile among HIV-infected children using efavirenz plasma level as an index and to examine any implications of pharmacogenetic variations.Previous studies reported the association of pharmacogenetic variations and plasma efavirenz concentration with antiretroviral-treatment-induced neurotoxicity and liver enzyme abnormalities among HIV-infected patients ,34. TherCYP2B6*1/*1 genotype. This contrasts with our current findings among children, where minor changes in plasma efavirenz concentrations over time were observed regardless of the CYP2B6 genotype. Such inconsistent efavirenz autoinduction profiles between adults and children could be due to differences in age-related metabolic capacity or differences in administered dose. The observed non-significant effect of treatment duration on efavirenz plasma exposure over time may indicate that maximal induction reaches a “steady state” within the first four weeks of therapy at the given weight-band efavirenz dose for children. Nonetheless, the current efavirenz pediatric dosing based on the child bodyweight category results in no significant change on long-term plasma efavirenz exposure.Efavirenz induces its own metabolism by increasing the expression of hepatic CYP2B6 enzyme via the human constitutive androstane receptor (hCAR) . CurrentCYP2B6*6 genotype was significantly associated with plasma efavirenz concentration and therapeutic range categories among children. Although CYP2B6*6 allele frequency distribution is not significantly different between Ethiopian adults (29.7) [Notably, our study shows the existence of wide between-patient variability in efavirenz plasma concentration among children. In line with our previous reports from adult patients ,19, the s (29.7) ,34, incrs (29.7) , and poos (29.7) .CYP2B6*6 (c.516G > T) and ABCB1 c3435C > T genotypes were significant predictors of higher efavirenz plasma concentration. CYP2B6 is the main enzyme responsible for the metabolism of efavirenz. ABCB1 encodes P-glycoprotein, an ATP-dependent drug efflux pump, which is responsible for drug transport across extra- and intra-cellular membranes. Our findings reveal that sex, stunting, wasting, weight, and age had no significant role in predicting plasma efavirenz among children. The current pediatric HIV treatment guideline is only weight-based and does not consider enzyme maturation and genetic variations [CYP2B6 defective variant allele occurs at a higher frequency. Consideration of a pharmacogenetics algorithm might be more useful for determining the optimal dose of efavirenz for pediatric patients [This study used linear mixed effect (LME) modeling to identify variables that predict plasma efavirenz concentration in children . In lineriations . Hence, patients ,47.Both HIV infection and efavirenz-based ART alter lipid profiles ,49. ReceThis study has some limitations. We could not utilize changes in intrinsic hepatic clearance of efavirenz to study the impact of efavirenz induction on its plasma exposures, as was done in previous studies among healthy adult subjects. Intensive pharmacokinetics sampling, which requires multiple blood withdrawals at each study time point over a one-year period from vulnerable patient populations (HIV-infected children) just for study purposes, was not possible due to ethical concerns. Likewise, we could not perform fasting lipid profile analysis for ethical reasons as children did not fast for the study purpose. Instead, we characterized the time course of induction and extent of changes in efavirenz plasma concentration profile in the same individual over time, whereby each child was effectively functioning as their own control, lowering the level of unexplained variance.CYP2B6 and ABCB1 pharmacogenetic variations. In addition, pre-treatment plasma LDL is a significant predictor of plasma efavirenz exposure. Duration of therapy-dependent changes in plasma efavirenz concentration due to autoinduction (as observed in adult patients) is not significant with the current weight-band efavirenz dosing among children. About one-fourth of the children had plasma concentrations exceeding supratherapeutic plasma efavirenz concentration (4 µg/mL), indicating that the current weight-band efavirenz dosing maybe unnecessarily high. Weight and age, which are the basis for the current pediatric efavirenz dosing, had no significant effect on plasma efavirenz concentration. For estimating the optimum pediatric efavirenz dose, CYP2B6 genotype-based dose modifications may have more importance. Future pharmacokinetic and pharmacogenetic association studies with treatment outcome (efficacy and safety) measures are required to support a low dose efavirenz regimen among the pediatric population as confirmed in adult HIV patients.We report the presence of wide between-patient variability in plasma efavirenz exposure among HIV-infected children, partly due to


Background: Vitamin D is a well-established regulator of calcium and phosphate metabolism that has neurotrophic and neuroprotective properties. Deficiency of vitamin D has been proposed to promote cognitive dysfunction and brain atrophy. However, existing studies provide inconsistent results. Here we aimed to investigate the association between vitamin D metabolites, cognitive function and brain atrophy in a cohort of well-characterized community-dwelling elderly individuals with normal neurological status and without history of stroke and dementia.3, 25(OH)D2 and 24,25(OH)2D3 were measured by liquid-chromatography tandem mass-spectrometry in serum samples from 390 community-dwelling elderly individuals. All participants underwent thorough neuropsychiatric tests capturing memory, executive function and visuopractical skills. In 139 of these individuals, MRI of the brain was performed in order to capture neurodegenerative and vascular changes.Methods: 25(OH)D2D3  and vitamin D metabolite ratio (VMR)  were significantly related to memory function. Adjustment for multiple testing weakened these relationships, but trends (p≤0.10) remained. 24,25(OH)2D3 and VMR showed similar trends also for visuopractical skills and global cognitive function. No significant relationships existed between vitamin D metabolites and MRI derived indices of neurodegeneration and vascular changes. Sub-group analyses of individuals with low concentrations of 25(OH)D and 24,25(OH)2D3 showed significantly worse memory function compared to individuals with normal or high concentrations.Results: Total 25(OH)D , 24,25(OH)Conclusions: Vitamin D deficient individuals appear to have a modest reduction of memory function without structural brain atrophy. Future studies should explore if vitamin D supplementation can improve cognitive function. Dementia, and in particular Alzheimer’s disease (AD), is a major cause of disability and dependency among older people worldwide . It has At present, curative therapies are lacking and preventive measures that potentially delay onset and progression of the disease are of particular importance. Factors known to lower the incidence of Alzheimerʹs disease are educational attainment, social integration, regular physical activity, and treatment of vascular risk factors at midlife . An adeq2D). Besides the intracerebral synthesis of 1,25(OH)2D, vitamin D metabolites can enter the brain by crossing the blood-brain barrier [Several lines of evidence indicate that vitamin D has neurotrophic and neuroprotective properties, and is involved in brain development . The vit barrier . In the  barrier  and exer barrier , 13. Bas barrier . While s barrier , 16, oth barrier –19. So f barrier , 21.2D is the catabolite of 25(OH)D that is only formed through the action of 24-hydroxylase (CYP24A1) when sufficient amounts of 25(OH)D are available. The simultaneous analysis of 24,25(OH)2D3 may be helpful in identifying individuals with functional vitamin D deficiency or defects of 24-hydroxylase [2D3 might indicate CYP24A1 deficiency or functional vitamin D deficiency where all 25(OH)D is needed to maintain an adequate cellular supply with active 1,25(OH)2D. Another limitation of previous studies is the measurement of 25(OH)D with different immunoassays, which have shown variable accuracy in comparison to validated LC-MS/MS reference methods [In line with current recommendations, all existing studies assessed vitamin D status by measuring serum 25(OH)D. 24,25(OH)roxylase , 23. Low2D3, 25(OH)D3 and 25(OH)D2 simultaneously with a validated in-house LC-MS/MS method, calculating the vitamin D metabolite ratio (VMR) [The present study aimed to expand existing knowledge by analysing 24,25(OH)io (VMR) , and ass3, 25(OH)D2 and 24,25(OH)2D3 were performed in 390 stored serum samples at -80° C from community-dwelling elderly individuals with normal neurological status and without history of stroke and dementia. Neuropsychiatric tests capturing memory, executive function and visuopractical skills were used for analysing cognitive function of all participants. In 139 individuals MRI of the brain was performed in order to capture neurodegenerative and vascular changes. Only subjects with a complete set of biochemical and cognitive test results and without vitamin D supplementation were included in this study. Results were used to explore a potential association between vitamin D status, cognitive function and structural markers of neurodegeneration. The selection of participants for the final study cohort is shown in Measurements of the vitamin D metabolites, 25(OH)DThe study population was assembled by community-dwelling elderly individuals with normal neurological status and without history of stroke and dementia from the Austrian Stroke Prevention Study (ASPS) and the Austrian Stroke Prevention Family Study (ASPS-Fam). The ASPS is a prospective single-centre study examining the effects of vascular risk factors on brain structure and function , 27. RanInclusion criteria were absence of stroke and dementia, and a normal neurologic examination. ASPS-Fam is an extension of ASPS with a similar study protocol consisting of ASPS participants and their first-grade relatives , 27. SinThe risk factors hypertension, diabetes mellitus and atrial fibrillation were examined in all participants. Hypertension was defined as history of hypertension or systolic blood pressure over 140 mmHg or a diastolic blood pressure over 90 mmHg  and curr3, 25(OH)D2 and 24,25(OH)2D3. Details of this method have been reported previously [3, d3-25(OH)D2, and d6-24,25(OH)2D3 were used as internal standards. In the next step, samples were separated on an Agilent HPLC 1260 system using a Kinetex® 5 μm F5 100Å LC Column  and a water/acetonitrile gradient. A Sciex 4500 MS/MS instrument was employed for the detection of vitamin D metabolites with a run time of 17 min. Quantitation of the metabolites was performed by direct determination of peak area ratios of 25(OH)D3  = 8.35 min), 25(OH)D2  and 24,25(OH)2D3  = 6.24 min) [2D3/25(OH)D3/ x 100  [Vitamin D metabolites were determined in serum samples (50 μl) using a validated in-house liquid-chromatography tandem mass-spectrometry (LC-MS/MS) method for the simultaneous measurement of 25(OH)Deviously . Briefly.24 min) . VMR was .Cognitive function was assessed with dedicated test batteries. A description of these test batteries has been published previously , 31–35. Additionally, principal components analysis was used to calculate a measure of global cognitive ability (g-factor) combining the results from all individual tests .ASPS-Fam participants underwent MRI on a 3T whole-body MR system . MRI scans from ASPS participants could not be considered in the present study as they were obtained in the 1990s, when 3D T1 and FLAIR sequences were not yet available.Total, cortical and subcortical gray matter volume, hippocampus volume and lobar cortical volume, thickness and surface area were computed from the T1 weighted MPRAGE images using FreeSurfer 5.3 , 38. BasStatistical analysis was performed using the R software version 3.6.1 . We asseViolin plots showing the distribution of the laboratory parameters were generated using the R package ggplot2 . Normall2D3 and the VMR on the basis of existing literature [2D3 and < 3% for the VMR. All analyses were adjusted for age, sex, hypertension, diabetes and atrial fibrillation, and the season of blood draw. Cognition analyses were additionally adjusted for education, and, as we pooled ASPS and ASPS-Fam data for these analyses, we also used the study as covariate to adjust for any undetected differences between the two studies. ASPS-Fam is a family study and therefore we calculated linear mixed models with the family structure as a random effect as implemented in the lmekin function of the R package coxme [We first determined the linear association between vitamin D metabolites and cognition as well as MRI by including the vitamin D metabolites as continuous predictors. Subsequently, we compared the measures of cognition between individuals with and without deficient vitamin D metabolite concentrations. Based on the recommendation of the IOM , 25(OH)Dterature  and our ge coxme . A kinshge coxme . The resge coxme  to compe3 and 24,25(OH)2D3 as well as the VMR were not significantly different between both study cohorts. Only the 25(OH)D2 concentration was significantly lower in ASPS than in ASPS-Fam participants (p = < 0.001)  and the two sub-cohorts, ASPS (n = 242) and ASPS-Fam (n = 148) are listed in < 0.001) .2D3 and VMR in ASPS and ASPS-Fam. 25(OH)D, 24,25(OH)2D3 and the VMR were lower in spring compared to all other seasons (p<0.05)  .2D3 and VMR were significantly related to the test performance on memory. However, after adjustment for multiple testing, none of these associations remained significant. In addition, there was a non-significant association between 24,25(OH)2D3, VMR and visuopractical skills as well as general cognitive functioning D and 24,25(OH)abolites . Correctabolites .2D3, as well as a low VMR. When considering only 24,25(OH)2D3 and VMR, similar trends are also detectable for visuopractical skills and global cognitive function. In contrast, none of the vitamin D metabolites is linked to any of the structural indices of neurodegeneration and vascular brain changes.The present results suggest a reduced memory function in individuals with low serum concentrations of 25(OH)D and 24,25(OH)2D3 and VMR, similar relationships were seen for visuopractical skills and global cognitive function as assessed by the g-factor.Several studies have investigated a potential relationship between serum 25(OH)D and cognitive function with mixed results. For example, amongst 2,777 well-functioning, community-dwelling elderly individuals of the Health ABC cohort, serum 25(OH)D was associated with global cognitive function and cognitive decline . Miller 2D3 as it was done in our study allows to improve the identification of individuals with vitamin D deficiency, CYP24A1 lack of function mutations, vitamin D intoxication or uncontrolled 1,25(OH)2D production in granulomatous diseases [2D are available. Consequently, in the absence of genetic enzyme defects, detectable amounts of 24,25(OH)2D3 imply a sufficient supply with vitamin D that allows the maintenance of an adequate vitamin D metabolism. In contrast, undetectable 24,25(OH)2D3 concentrations suggest functional vitamin D deficiency. This concept is supported by a recent study from Cavalier et al., were in a large cohort of children, adolescents and young adults more than 80 % of individuals with 25(OH)D concentrations between 30 and 50 nmol/L had detectable levels of 24,25(OH)2D3, which suggests vitamin D sufficiency from a biochemical point of view [2D3 and cognitive function. In the linear model 24,25(OH)2D3 was positively associated with memory, visuopractical skills and global cognitive function. Correction for multiple testing attenuated these effects slightly, but did not abolish the respective trends (p ≤ 0.1). Sub-group analyses comparing individuals with and without low 25(OH)D, 24,25(OH)2D3 and VMR confirmed the results of the linear model. When considering all results together, there appears to be a consistent pattern that supports a link between serum vitamin D metabolite concentrations, VMR and cognitive function. This approach of interpreting statistical results is in line with a recent publication of Amrhein et al. that question the simple binary interpretation of p-values without considering confidence intervals and patterns of results [It is important to note that previous studies assessed 25(OH)D mainly by immunoassays. These assays are known for their variable analytical performance, which may represent a source of substantial bias –54. Nonediseases , 55. Recdiseases . Accordi of view . In line results .2D3 are related to MRI derived indices of neurodegeneration and vascular changes. In contrast, previous studies reported reduced gray matter volume, lower volumes of hippocampal subfields and connection deficits in 25(OH)D deficient people [Another key finding of the present study is that neither 25(OH)D nor 24,25(OH)t people , 12. In t people .2D3 and MRI derived indices of brain atrophy, suggests a functional rather than a structural cause of cognitive impairment in subjects with low vitamin D concentrations. This assumption is supported by studies demonstrating expression of the VDR and 1α-hydroxylase in the hippocampus, hypothalamus, thalamus, cortex, subcortex and substantia nigra, which are related to cognitive function [The absence of a significant relationship between serum 25(OH)D, and 24,25(OH)function , 9. Obsefunction , 13, 58.function , which afunction , 60.2D are very stable compounds that change minimally when stored at -80° C frozen [2D3 in ASPS and ASPS family. The samples of both cohorts have been collected several years apart and despite this fact their mean concentrations were almost identical.Our study has limitations. The percentage of vitamin D deficient participants (25(OH)D <50 nmol/L) is small. This might have reduced the chance of identifying potential associations with cognitive impairment and brain abnormalities in our cohort of community-dwelling persons. Furthermore, the detailed assessment of cognitive function and brain structure resulted in a substantial number of statistical tests requiring correction for multiple testing. In conjunction with the limited number of participants this correction weakened several significances leaving only trends. Recently, scientists and statisticians have proposed to change the traditional approach of interpreting statistical results in a binary fashion on the basis of p-values and to consider confidence intervals and patterns as well . When coC frozen . This poIn conclusion, vitamin D deficient individuals appear to have a modest reduction of memory function without structural brain atrophy. As vitamin D deficiency is highly prevalent and effective therapies for cognitive dysfunction and dementia are lacking, future studies should explore if vitamin D supplementation can improve cognitive function.


Spodoptera exigua, an important global pest of many crops. Unfortunately, field resistance to avermectins recently evolved in the beet armyworm, threatening the sustainable use of this class of insecticides. Here, we report a high-quality chromosome-level assembly of the beet armyworm genome and use bulked segregant analysis (BSA) to identify the locus of avermectin resistance, which mapped on 15–16 Mbp of chromosome 17. Knockout of the CYP9A186 gene that maps within this region by CRISPR/Cas9 gene editing fully restored EB susceptibility, implicating this gene in avermectin resistance. Heterologous expression and in vitro functional assays further confirm that a natural substitution (F116V) found in the substrate recognition site 1 (SRS1) of the CYP9A186 protein results in enhanced metabolism of EB and abamectin. Hence, the combined approach of coupling gene editing with BSA allows for the rapid identification of metabolic resistance genes responsible for insecticide resistance, which is critical for effective monitoring and adaptive management of insecticide resistance.The evolution of insecticide resistance represents a global constraint to agricultural production. Because of the extreme genetic diversity found in insects and the large numbers of genes involved in insecticide detoxification, better tools are needed to quickly identify and validate the involvement of putative resistance genes for improved monitoring, management, and countering of field-evolved insecticide resistance. The avermectins, emamectin benzoate (EB) and abamectin are relatively new pesticides with reduced environmental risk that target a wide number of insect pests, including the beet armyworm,  CYP9A186 gene that maps within this region by CRISPR/Cas9 gene editing fully restored avermectin susceptibility. Heterologous expression and in vitro functional assays further confirm that a natural substitution (F116V) found in the substrate recognition site 1 (SRS1) of the CYP9A186 protein results in enhanced metabolism of avermectin. Hence, the combined approach of coupling gene editing with BSA allows for the rapid identification of metabolic resistance genes responsible for insecticide resistance, which is critical for field monitoring of such mutations, for making improved decisions on appropriate use of effective chemistries, as well for improvements in the design of future compounds that target S. exigua.Insecticide resistance is a global constraint to agricultural production, and rapid identification of resistance genes is critical for better monitoring and management of resistant insect pests. Identification of metabolic resistance genes has always been a challenging task due to the high diversity of insect detoxification enzyme genes. Here, we report a high-quality chromosome-level assembly of the beet armyworm genome and use bulked segregant analysis (BSA) to identify the locus of avermectin resistance, which mapped on 15–16 Mbp of chromosome 17. Knockout of the  Avermectins are a group of 16-membered macrocyclic lactones possessing potent anthelmintic and insecticidal activities . SimilarDrosophila melanogaster  and B: acetonitrile, with a constant flow rate of 0.3 mL min-1. The gradient elution conditions were as follows: 0 min A: B 50: 50; 0.3 min A: B 50: 50; 2 min A: B 5:95; 2.5 min A: B 5: 95; 2.6 min A: B 0:100; 3 min A: B 0: 100; 3.1 min acetonitrile: Water 50: 50; 5 min acetonitrile: Water 50: 50. Samples separated by UPLC were directly analyzed using a tandem triple quadrupole mass-spectrometer  and run in positive ESI mode with multireaction monitoring (MRM) (S7 Table).The degradation of EB and abamectin was monitored and quantified by UPLC-MS/MS. Samples (0.5 μL each) were separated by Acquity BEH C8 column  using a Waters Acquity UPLC system (Waters ACQUITY UPLC I-Class) and eluted with a gradient of mobile phase consisting of A: 10 mM ammonium acetate [+0.1%  and the same mobile phases and gradient as outlined above. The partitioned samples were then analyzed by high-resolution mass spectroscopy  under +ESI mode with collision energy at 50V.S1 FigA) Schematic diagram of the sgRNA-targeting sites. The gray line indicates the genome locus of CYP9A186 and the boxes represent the exons of CYP9A186. The sgRNA-targeting site was located on the sense strand of exon 1. The sgRNA-targeting sequence is shown in purple, and the protospacer adjacent motif (PAM) sequence is in red. (B) Representative chromatograms of PCR-product sequencing in G1 individuals and the CYP9A186 knockout strain (dA186) showing presence of 4-bp indel mutation. (C) Diagram detailing crossing scheme used to obtain the homozygous CYP9A186 knockout strain .((TIFF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S3 FigA, samples with CYP9A186-F116V; B, samples with CYP9A186wt; C, samples with non-insertion control (CK). Metabolism of abamectin: D, samples with CYP9A186-F116V; E, samples with CYP9A186wt; F, samples with non-insertion control (CK). MRM spectra of parent compound (emamectin benzoate and abamectin), hydroxy-metabolite (-OH) and O-desmethyl-metabolite (-O-CH3) were shown in the top, middle, and bottom respectively within each block. Red arrows indicate metabolites.Metabolism of emamectin benzoate: (TIFF)Click here for additional data file.S4 FigA) Mass spectra of the metabolite hydroxyl-emamectin benzoate (HO-groups correspond to either 24’ or 26’ position). (B) Mass spectra of the metabolite O-desmethyl-emamectin benzoate. (C) Mass spectra of the metabolite hydroxyl-abamectin (HO-groups correspond to either 24’ or 26’ position). (D) Mass spectra of the metabolite O-desmethyl-abamectin.((TIFF)Click here for additional data file.S5 FigA) sgRNA targeting site of CYP9A40 and CYP9A98 genes and the two primer pairs for allele-specific PCR detection. Target sequences and protospacer adjacent motifs (PAMs) are shown in light blue and in red, respectively. The positions of the two sgRNAs (sgRNA-A40 and sgRNA-A98) and a representative chromatogram of direct sequencing of PCR products of individuals from the dA40-A98 strain with the primer pair A40F/A98R are shown. (B) Genotyping of individual S. exigua for deletion of the CYP9A cluster according to banding patterns of the PCR products amplified with a set of five primer pairs. M, 2000 bp MW Marker; Lane 1, A40F/A98R; Lane 2, A40F/A40R; Lane 3, A9F/A9R; Lane 4, A186F1/A186R1; and Lane 5, A98F/A98R.((TIFF)Click here for additional data file.S6 FigA) sgRNA targeting site of CYP9A40 and CYP9A107 genes and the two primer pairs for allele-specific PCR detection. Target sequences and protospacer adjacent motifs (PAMs) are shown in light blue and in red, respectively. The positions of the two sgRNAs (sgRNA-A40 and sgRNA-A107) and a representative chromatogram of direct sequencing of PCR products of individuals from the dA40-A107 strain with the primer pair A40F/A107R are shown. (B) Genotyping of individual S. exigua for deletion from CYP9A40 to CYP9A107 according to banding patterns of the PCR products amplified with a set of five primer pairs. M, 2000 bp MW Marker; Lane 1, A40F/A107R; Lane 2, A40F/A40R; Lane 3, A107F/A107R; Lane 4, A9F/A9R; and Lane 5, A186F2/A186R2.((TIFF)Click here for additional data file.S7 FigA) sgRNA targeting site of CYP9A107 and CYP9A98 genes and the two primer pairs for allele-specific PCR detection. Target sequences and protospacer adjacent motifs (PAMs) are shown in light blue and in red, respectively. The positions of the two sgRNAs (sgRNA-A107 and sgRNA-A98) and a representative chromatogram of direct sequencing of PCR products of individuals from the dA107-A98 strain with the primer pair A107F/A98R are shown. (B) Genotyping of individual S. exigua for deletion from CYP9A107 to CYP9A98 according to banding patterns of the PCR products amplified with a set of five primer pairs. M, 2000 bp MW Marker; Lane 1, A107F/A98R; Lane 2, A107F/A107R; Lane 3, A186F2/A186R2; and Lane 4, A98F/A98R.((TIFF)Click here for additional data file.S8 Fig(TIFF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.


Our study characterizes genetic mutations, as well as pathogenesis in both in vitro and in vivo model of a new Korea H7N3 viruses in 2019, carrying multiple potential mutations to become highly pathogenic and develop an ability to infect humans; thus, emphasizing the need for routine surveillance of avian influenza viruses in wild birds.Low-pathogenicity avian influenza viruses (LPAIV) introduced by migratory birds circulate in wild birds and can be transmitted to poultry. These viruses can mutate to become highly pathogenic avian influenza viruses causing severe disease and death in poultry. In March 2019, an H7N3 avian influenza virus—A/Spot-billed duck/South Korea/WKU2019-1/2019 (H7N3)—was isolated from spot-billed ducks in South Korea. This study aimed to evaluate the phylogenetic and mutational analysis of this isolate. Molecular analysis revealed that the genes for HA (hemagglutinin) and NA (neuraminidase) of this strain belonged to the Central Asian lineage, whereas genes for other internal proteins such as polymerase basic protein 1 (PB1), PB2, nucleoprotein, polymerase acidic protein, matrix protein, and non-structural protein belonged to that of the Korean lineage. In addition, a monobasic amino acid (PQIEPR/GLF) at the HA cleavage site, and the non-deletion of the stalk region in the NA gene indicated that this isolate was a typical LPAIV. Nucleotide sequence similarity analysis of HA revealed that the highest homology (99.51%) of this isolate is to that of A/common teal/Shanghai/CM1216/2017 (H7N7), and amino acid sequence of NA (99.48%) was closely related to that of A/teal/Egypt/MB-D-487OP/2016 (H7N3). An in vitro propagation of the A/Spot-billed duck/South Korea/WKU2019-1/2019 (H7N3) virus showed highest (7.38 Log Avian influenza is an infectious disease caused by the influenza A virus, which is widespread in migratory birds and mammals, including humans . WaterfoSpecifically, compared to the other 14 HA subtypes identified in birds, H5 and H7 have been mostly observed in wild avian species. Although the first report of a H7 subtype AIV, which was studied in northern Italy in 1878 by Perroncito, who labelled the disease caused by this virus as a “fowl plague” . ConcomiAnas platyrhynchos domesticus) in Cambodia [Between 1995 and 2002, Aamir et al. reported that poultry, specifically chickens, were infected with HPAIV H7N3 in Pakistan; their report also clarified that this virus replicated poorly in mallard ducks . In JanuCambodia  and in MCambodia . AccordiCambodia ; althougThe aim of this study is to evaluate the phylogenetic and mutational analysis of H7N3 AIVs from spot-billed ducks in South Korea, early 2019. Moreover, as there are currently no reports related to the pathogenetic analysis of H7N3 AIVs, to the best of our knowledge, we describe, for the first time in the literature, the complete pathogenesis of H7N3 AIVs by experimental infections in a mammalian model, in vitro and in vivo.A total of 1800 fresh samples of fecal droppings were collected during an annual surveillance from November 2018 to March 2019 in different regions of South Korea, stored at 2–8 °C, and shipped to the laboratory within a day for further analysis.The wild bird fecal samples were pooled in groups of up to three and re-suspended in phosphate buffered saline (PBS) containing antibiotic solution (100 mg/µL of streptomycin)  followed by centrifugation at 3000 rpm for 10 min at 4 °C. To verify that the samples contained Influenza A viruses, reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify the matrix gene, following the World Health Organization (WHO) guideline, using universal primers and probes . The 0.1c oxidase I (COI) as previously described [Host identification was confirmed by polymerase chain reaction (PCR) using a DNA barcode and a region, consisting of 751 base-pairs (bp), of the mitochondrial gene cytochrome escribed . The hosViral RNA was extracted directly from the allantoic fluid of embryonated chicken eggs (ECEs) using a NucleoSpin RNA kit  according to the manufacturer’s instructions. Briefly, virus-containing egg fluid was mixed with lysis buffer containing β-mercaptoethanol. Suspension viscosity was reduced by filtration in a Nucleospin filter, and RNA binding conditions were adjusted by adding ethanol. Solubilized RNA was bound to a Nucleospin RNA column membrane, and desalting was performed by the addition of a membrane desalting buffer. The DNA digestion step was omitted as rDNase treatment was noted to reduce RNA stability. Elution was performed directly after the desalted membrane was washed with wash buffers. The first strand cDNA was transcribed by a Superscript III first strand cDNA synthesis kit  using a universal primer for influenza A virus (Uni12: 5′-AGCRAAAGCAGG-3′) in a final volume of 20 µL as per the manufacturer’s protocol.To evaluate the growth of the influenza virus and determine the subtype, conventional real-time RT-PCR for influenza A virus was performed using total RNA following World Health Organization guidelines .NGS sequencing was conducted by GnCBIO  following the Hiseq X method, as previously reported ,18. BrieRaw sequence reads were quality trimmed using “trim galore” (q = 20) and non-influenza virus read was removed using Deconseq (iden = 60). Python script was used as a tool to adjust the amount of data, up to 600,000 reads. Meanwhile a database of only segments four (HA), six (NA), and eight (NS1) from the influenza virus from NCBI was created to provide and align to those of the reference using Gsmapper . The open reading frame (ORF) was observed using the obtained consensus and adopted a result with an ORF similar to the reference. As the ORF length differed from the reference, sequence error was corrected using Proof Read as previously described .https://www.ncbi.nlm.nih.gov/ (accessed on: 1 October 2020)) and the Global Initiative on Sharing All Influenza Data (GISAID https://www.gisaid.org/ (accessed on: 1 October 2020)). The data were merged, the duplicated sequences were deduced, and finally phylogenetic tree analyses were conducted using MEGA6.0 software . Phylogenetic trees of all eight full length gene segments  of the H7N3 (WKU2019-1) isolate were generated by applying the neighbor-joining method with Kimura’s two-parameter distance model and 1000 bootstrap replicates. The HA and NA gene tree included sequences with high homology from the NCBI database, the H7N3 subtype reported from South Korea (complete genome), and a representative H7 HPAIV strain. The tree for the other six internal segments  included influenza virus isolate with nucleotide homologies to respective segments from different geographic regions [Nucleotide blast analysis was used to identify relevant viral genes, and reference sequences were downloaded from the National Center for Biotechnology Information database (NCBI  regions .50 (50% tissue culture infectious dose) titers as previously reported [2. Twenty-four hours after plating, cells with 80% confluence were washed with 1× phosphate-buffered saline (PBS) and inoculated with serial 10-fold dilutions of virus in media containing 1 µg/mL of L-1-tosylamide-2-phenylethyl chloromethyl ketonetreated trypsin (TPCK-trypsin). Virus-infected cells were incubated at 37 °C, 5% CO2 for 3 days and the TCID50 titers were determined via the Reed and Muench method [50, the chorioallantoic membrane (CAM) cavities of 10-day-old SPF embryonated chicken eggs were inoculated with 100 μL serial 10-fold dilutions of the viruses, five eggs for each dilution. The eggs were then incubated at 37 °C for 3 days. Allantoic fluid was harvested and tested via HA assays [50 calculation of viruses was performed using the Reed and Muench method [The enzyme-linked immunosorbent assay (ELISA) was used to measure the TCIDreported ,22. Brieh method . For theA assays  and EID5h method .50) of each supernatant was determined [The growth kinetics of virus isolates were evaluated in in vitro. MDCK cells were inoculated with the viruses at a multiplicity of infection (MOI) of 0.01 in DMEM (Dulbecco’s Modified Eagle Medium) medium containing 1% antibiotic and 1 μg/mL TPCK-trypsin. Supernatants were collected at 12, 24, 36, 48, 60, 72, and 84 hpi. ELISA were performed with anti-influenza nucleoprotein (PAN antibody) to detect infected cells and the virus titer (TCIDtermined .n = 11 mice’s/group), which were intranasally inoculated by 105 EID50/mL of each viruses , and two control groups infected with A/California/04/2009 (H1N1) (CA/04/09) (pdm09) or A/mallard/Korea/KNUGPH12/2011 (H7N7) or mock infected as normal control group). Mice were anesthetized by 1% isoflurane following manufacturer’s instructions  according to the guidelines of Vertebrate Animal Research, University of Iowa [n = 3 mice/group) were collected at days 3, 6, and 15 post-infection (dpi). The lung tissue was homogenized and the TCID50 was determined to test the viral titers of homogenate supernatant [The pathogenic potential of the new isolate was then determined in mammals; six-week-old female BALB/c mice purchased from Orient  . Results were presented as the mean ± SD. A value of p < 0.05 was considered significant.The mean, standard deviation (SD), and Student’s Samples were isolated from feces of waterfowls in South Korea on 15 March 2019. RNA sample from feces were amplified for HA and NA using universal primer, and the PCR product was ligated to vector plasmid. Five different representative colonies were selected for HA/NA amplification and ligated sequence was sent for sequencing. The result showed positive for HA7 and NA3 subtype that initially confirmed H7N3 . FurtherSurface gene HA, matrix protein (M), and polymerase basic protein (PB)1 were closely related to A/common teal/Shanghai/CM1216/2017 (H7N7), which originated from China, while NA was closely related to A/teal/Egypt/MB-D-487OP/2016 (H7N3) with nucleotide identities of 99.51%, 99.90%, 99.56%, and 99.48%, respectively. PB2 was closely related to A/Duck/Hubei/HF5/2017 (H7N8), which originated from China, with a nucleotide identity of 99.17%. Similarly, the gene for polymerase acidic protein (PA) was closely related to A/wild duck/South Korea/KNU18-114/2018 (H7N7), was originated from Korea, with a nucleotide similarity of 99.54%. The gene for nucleoprotein (NP) was closely related to A/teal/Egypt/MB-D-487OP/2016 (H7N3) and A/teal/Egypt/MB-D-698OP/2016 (H7N3), which seemed to be originated from Egypt, while that for non-structural protein (NS) was closely related to A/wild goose/dongting lake/121/2018 (H6N2), from China, with nucleotide similarities of 99.66% and 99.65%, respectively.NA gene reassortment prevailed from the African region with links to Mongolian isolates in 2016. This was followed by the transmission of the PB2 gene from South Korea through the H1N1 isolate to Bangladesh in 2016; then, the same gene reassortment occurred in H3N1, which was transmitted to China and rehabilitated with H7N8, before the PB2 gene was finally reassorted to prevail in South Korea ) before being transmitted to South Korea by wild ducks. Then, the same NS gene rehabilitated into H7N1 to H7N7 isolates. At present, the H7 subtype isolates are privileged in South Korea.Similarly, the HA and M genes was due to the reassortment from Georgia isolates (H7N7) in 2016, likewise PB1 and NP genes were formerly transmitted from Mongolia (H1N1) and Egypt (H7N9), respectively. Following that, four genes—PB1, HA, M, and NP—were collectively reassorted into A/common teal/Shanghai/CM1216/2017 (H7N7), which originated from China in 2017. Finally, the same gene was transmitted into South Korea from 2018 by various isolates of H7N1 and H7N1 through certain migratory birds like wild ducks and white-fronted geese. The detailed information on the evolutionary reassortment is presented in The backbone of the PA genes from the Cambodian isolate into South Korea from 2017–2018; our isolate may have been transmitted during this period. Additionally, there might have been a major H7N3 reassortment from China during the migration season of 2017–2018.International migration of the birds may have been involved in the transmission of the The HA cleavage site of H7N3 (WKU2019-1) contained the PELPKGR↓GLF (↓denotes the cleavage site) sequence, with a monobasic amino acid arginine in the HA cleavage site, which is a marker for LPAIV H7 virus. Modification of HA receptor binding site (RBS) can lead a switch in the preference for glycans to bind preferentially to α-2,6-linked sialic acid receptors from avian to human, leading to an AIV pandemic . We comp50 assay are shown in As the molecular characterization shows reassortment via mutation, our viral isolate could increase replication efficiency; therefore, in vitro examination of the viral replication of our isolate along with other isolates was performed. To evaluate the growth kinetics of our isolate, human-origin virus, A/California/07/2009 (H1N1), and H7N7, which is infectious to mammals, were used as controls. H1N1 replicated more efficiently in MDCK cells, as compared H7N3 (WKU2019-1) and A/mallard/Korea/KNUGPH12/2011 (H7N7) (Described as H7N7) . Raw dat50 assay (10 (TCID50/mL), respectively) and these viral loads gradually decreased at 6 dpi , respectively). In contrast, no virus was detected at 15 dpi in all groups, indicating that the virus was not yet well-adapted to murine infections. All raw data of the TCID50 assay are presented in We next examined the pathogenic potential of the new isolate by intranasally inoculating the virus in six-week-old female BALB/c mice. Mice infected with positive control, H1N1 virus, displayed severe clinical signs, including ruffled fur, depression, high body temperature, and gradual decrease in body weight from 3 dpi and gradually regained weight after 12 dpi A. In add50 assay C. As shoLPAIVs of subtypes H7 and H5 have the ability to spontaneously mutate to HPAIV variants, causing high mortality. The first outbreak of HPAIV caused by subtype H7N3 appeared in Pakistan in 1995 and affected 3.2 million birds, primarily breeders and boilers. Since then, it has caused outbreak in Chile (2004) , the NetDuring our influenza surveillance, the H7N3 strain was isolated, and through further studies was subtyped as an LPAIV isolate with a HA cleavage site sequence PELPKGR↓GLF. According to phylogenetic analysis, the HA, PA, and M genes were closely related to A/wild duck/South Korea/KNU18-114/2018 (H7N7), while the genes of PB1, NS, and NP were closer to the Korean isolates—A/wild fronted Goose/South Korea/KNU18-119/2018 (H7N7) and A/wild duck/South Korea/KNU18-114/2018 (H7N7); the genes of PB2 was similar to that of china isolate, A/ duck/Hubei/HF5/2017 (H7N8); and that of NA was similar to that of Mongolian isolate A/duck/Mongolia/782/2017 (H7N3).6 EID50 virus inoculation, the highest virus titer was achieved at 3 dpi with drastic weight loss and severe diffused pneumonia. The difference in the two experiments was the concentration of virus used, which might be the reason for such a difference in the virus titer achieved and the histopathological changes in lungs. The H7N3 (WKU2019-1) isolate carries mutations in the proteins of PB2 (H447Q) [In the H7 subtype, most of the residues, including 138 and 221, were highly conserved. The single substitutions at those two sites (S138A or T221P) does not cause obvious changes in the receptor-binding properties and retained avian receptor-binding specificity unless G186V substitution was displayed. Further negligible binding to the human RBS was observed, even if the three other sites were replaced by hydrophobic residues . V186 is the key determinant of avian-specific H7N9 HA obtaining human receptor-binding capacity . However (H447Q) , PB1 (K3 (H447Q) , which l (H447Q) . A recen (H447Q) . AlthougIn conclusion, the ultimate goals and objectives of our surveillance was to detect Avian influenza rather than any other virus and microorganism. Furthermore, RNA isolated from initial feces failed to read the virus by NGS hence virus amplified from allatonic fluid was used for sequencing and subtyping which was the limitation of our study. However, this study is first to broaden the knowledge of Korean H7N3 isolates from evolution to mammalian cell expressions and in vivo characterization. Continuous monitoring and molecular characterization of the H7N3 virus will be required for the broader understanding of evolutionary dynamics of the virus; further improving control measures is recommended.


Petunia hybrida flowers, which emit high levels of phenylpropanoid/benzenoid volatile organic compounds (VOCs), we uncovered genome-wide dynamic deposition of histone H3 lysine 9 acetylation (H3K9ac) during anthesis as an underlying mechanism to coordinate primary and secondary metabolic networks. The observed epigenome reprogramming is accompanied by transcriptional activation at gene loci involved in primary metabolic pathways that provide precursor phenylalanine, as well as secondary metabolic pathways to produce volatile compounds. We also observed transcriptional repression among genes involved in alternative phenylpropanoid branches that compete for metabolic precursors. We show that GNAT family histone acetyltransferase(s) (HATs) are required for the expression of genes involved in VOC biosynthesis and emission, by using chemical inhibitors of HATs, and by knocking down a specific HAT gene, ELP3, through transient RNAi. Together, our study supports that regulatory mechanisms at chromatin level may play an essential role in activating primary and secondary metabolic pathways to regulate VOC synthesis in petunia flowers.Biosynthesis of secondary metabolites relies on primary metabolic pathways to provide precursors, energy, and cofactors, thus requiring coordinated regulation of primary and secondary metabolic networks. However, to date, it remains largely unknown how this coordination is achieved. Using  Our study shows that post-translational modification of histones is essential for regulating the biosynthesis and emission of floral scent compounds, thus providing insights into the regulation of secondary metabolism at chromatin level. Plants have a remarkable ability to synthesize a vast array of secondary metabolites that are not only vital for plant growth, development, reproduction, and defense, but also play crucial roles for mankind in food, medicine, industrial raw materials, and biofuels. A unique subset of secondary metabolites consists of volatile organic compounds (VOCs) that plants use for communication and interaction with the surrounding environment , protectde novo in the tissues from which they are emitted, and their production and emission are spatially  or day 2 (post-anthesis) stage at 15.00 h in three replicates. The corolla tissue was fixed in 37 ml of 1% formaldehyde under vacuum twice, first for 15 min and then for 10 min. The reaction was stopped with addition of 2.5 ml of 2 M glycine, placed under vacuum for 5 min, and then washed three times with ultrapure water. The fixed tissue was then dried and frozen in liquid nitrogen. Chromatin extraction and immunoprecipitation were performed as previously described . The pellet was resuspended in 1 ml of a second extraction buffer (EB2) and centrifuged again at 4 °C . The pellet was then resuspended in 300 μl of a third extraction buffer (EB3) and overlaid on 300 μl of EB3 and centrifuged at 4 °C  to obtain the chromatin pellet. The pellet was resuspended in 300 μl of nuclei lysis buffer and sonicated with a BioRuptor Pico sonicator (Diagenode) for 26–36 cycles to obtain an ~200 bp fragment distribution. Cell debris was pelleted at 4 °C  and separated from the supernatant containing chromatin. A 20 μl aliquot of the supernatant was kept as input DNA and the rest was diluted with ChIP dilution buffer for immunoprecipitation. To perform immunoprecipitation, Protein A Dynabeads (Invitrogen) were first resuspended in ChIP dilution buffer. A 50 μl aliquot of beads was used for pre-clearance of chromatin (rotating for 2 h at 4 °C) and, during this time, a second 50 μl of beads were incubated with antibody. Anti-H3K4me3  and anti-H3K9ac  antibodies were used for immunoprecipitation. Pre-cleared chromatin was then added to antibody-bound beads and incubated with rotation overnight at 4 °C. Beads were washed twice each with a series of buffers: low salt wash buffer, high salt wash buffer, LiCl wash buffer, and Tris–EDTA. Chromatin was then eluted twice with 250 μl of elution buffer at 65 °C for 15 min. The two eluates were combined. The DNA cross-linking was reversed with addition of 20 μl of 5 M NaCl and incubation at 65 °C overnight. A 1 μl aliquot of proteinase K (Invitrogen) was added to digest the proteins at 45 °C for 1 h, then an equal volume of 25:24:1 phenol/chloroform/isoamyl alcohol, pH 8.0 (Invitrogen) was added to phase separate the DNA and the proteins. The DNA in the aqueous phase was precipitated with sodium acetate and ethanol along with 1 μl of glycogen carrier (Thermo Scientific) overnight at –20 °C and then centrifuged to pellet at 4 °C . The pellet was washed with cold 70% ethanol, dried, and then resuspended in water. The concentration was measured using a Qubit dsDNA High Sensitivity assay kit (Invitrogen).Approximately 2 g of corolla tissue from Petunia axillaris (v1.6.2) and P. inflata (v1.0.1) genomes  <0.01 cut-off for significance. Significantly increased or decreased peaks at a ≥2-fold were intersected with annotated petunia gene features using bedtools  was used for visualization of ChIP-Seq data  and P. inflata (v1.0.1) genomes (RNA sequencing data for day 0 and day 2 petunia corolla tissue were obtained from the NBCI Gene Expression Omnibus (GSE70948)  was used to calculate statistical significance of enrichment, and FDR correction (https://cran.r-project.org/web/packages/pheatmap/index.html) and ‘RColorBrewer’ (https://cran.r-project.org/web/packages/RColorBrewer/index.html).In order to determine enriched Gene Ontology (GO) terms in gene sets of interest in petunia, the best rrection  was applrrection . REVIGO P. axillaris were identified based on supplementary notes to the publication of petunia genomes  genes were identified in P. axillaris based on homology to the major CAD genes contributing to lignin biosynthesis in A. thaliana  stage at 15.00 h and floated in a 3% sucrose solution with mock treatment (0.1% DMSO) or the HAT inhibitor C646 (MilliporeSigma) or MB-3 (MilliporeSigma) at a final concentration of 100 μM in 0.1% DMSO. Treatment with inhibitors did not affect anthesis, as all flowers were open by the afternoon of day 1. Corolla tissue was harvested and frozen in liquid nitrogen 1 h before the end of the light period at 19.00 h in four biological replicates of two flowers each, on day 0, day 1, and day 2. Gibberellic acid inhibitor assays were performed similarly, with uniconazole (Lkt Laboratories) or flurprimidol (bioWORLD) at a final concentration of 100 μM in 0.1% DMSO, with a single end-point harvest at day 2. RNA was extracted with the RNeasy Plant Mini Kit (Qiagen), treated with recombinant RNase-free DNase I (Roche) at 37 °C for 1 h, followed by a clean up with the RNeasy Mini Kit (Qiagen). SuperScript IV (Invitrogen) was used to generate cDNA with an oligo(dT) primer. Quantitative reverse transcription–PCR (qRT–PCR) was performed with PowerUp SYBR Green (Applied Biosystems) using primers designed to specification with Primer3  and anti-H3K14ac  antibodies used for immunoprecipitation. Primers designed to gene body acetylation peaks (ACTIN (Peaxi162Scf00583g00529) and PP2AA3 (Peaxi162Scf00382g00122).For histone acetyltransferase (HAT) inhibitor assays, on peaks  were useP. hybrida cDNA using primers including an attB recombination site. The fragment was then cloned into the pDONR/Zeo gateway vector (Life Technologies) following the manufacturer’s instructions and subcloned into the destination vector pK7GWIWG2(II) in a sense and antisense direction to facilitate hairpin formation by Invitrogen Gateway LR Clonase II Enzyme mix (Life Technologies). Binary vectors were transformed into Agrobacterium tumefaciens strain GV3101 with freeze–thaw cycles. Transient ELP3 down-regulation was achieved similarly to as previously described (Agrobacterium containing pK7GWIWG2(II)-ELP3 or pK7GWIWG2(II) empty vector control at an OD600 of 0.8. Infiltrated flowers were kept in 5% sucrose solution in the dark for an additional 48 h before floral scent was collected for 4 h from 17.00 h to 21.00 h. Following scent collection, total RNA was extracted from petunia flowers using the Spectrum Plant Total RNA Kit (Sigma-Aldrich). About 1 μg of total RNA was reverse transcribed to first-strand cDNA in a 10 μl reaction using the EasyScript cDNA synthesis kit  and used for qRT–PCR as described above.A 300 bp sequence specific for RNAi-mediated silencing of ELP3 was chosen using the Sol Genomics Network VIGS (virus-induced gene silencing) tool  and open flowers (day 2). As activation of VOC pathway genes was of particular interest, histone marks associated with transcriptional activation, H3K9ac and H3K4me3, were chosen as the focus of this study  . Furthermore, we observed increased H3K9ac at gene loci involved in the formation of VOCs including volatile benzenoid/phenylpropanoid (CNL) and volatile phenylpropene (HCT). In addition, DMGs with increased H3K9ac were enriched with GO terms related to post-transcriptional regulation, hormone signaling, and intracellular protein transport, while DMGs with increased H3K4me3 were enriched with GO terms for post-transcriptional regulation and transport, and tissue development and the cell cycle  to open flower (day 2), in the form of increased levels of H3K9ac or H3K4me3 at hundreds to thousands of gene loci. Specifically, 4207 DMGs exhibit increased H3K9ac from day 0 to day 2, and 355 DMGs displayed decreased H3K9ac . For H3Kropanoid ; CNL andlpropene ; HCT. Inll cycle . There wTo understand the effects of observed histone modification patterns on gene expression, RNA-Seq data generated from the same tissue and the same developmental stages as the ChIP-Seq were analyzed . As expeTo investigate the extent to which changes of histone modifications at a gene locus co-occur with changes in transcript level, the DMGs identified by ChIP-Seq were compared with DEGs determined by RNA-Seq. Significant overlaps were observed between DEGs with increased transcript levels and DMGs that gained either H3K9ac or H3K4me3 on day 2 versus day 0 . In contTo further investigate the regulation of biosynthesis and emission of floral VOCs, we first examined in detail the enriched metabolic GO terms among DEGs and DMGs for their relevance to floral VOC emission . InteresS-adenosylmethionine (SAM) biosynthetic process’ and the connected ‘one-carbon metabolic process’. SAM is a methyl donor in the production of volatile metabolites such as methylbenzoate and eugenol  and DEGs with increased transcript levels ; in contrast, genes with a change in H3K4me3 levels do not appear significantly enriched. Our results therefore support that flux through the shikimate and phenylalanine biosynthetic pathways is a major target of H3K9ac and transcriptional regulation during petunia flower development to potentiate VOC production.The enzymes involved in the shikimate and phenylalanine biosynthesis pathways have been well characterized in plants  Maeda e. In totaPAL and one copy of 4CL. In the case of 4CL, two of the five copies are transcriptionally increased. Interestingly, the two induced copies also display increased H3K9ac or H3K4me3. 4CL drives the synthesis of 4-coumaryl-CoA, which has multiple fates in metabolism and can serve as a precursor for flavonoids and anthocyanins, lignin, and phenylpropene VOCs. The distinct chromatin and transcriptional regulation of these five copies could possibly indicate specialized gene functions for different metabolic fates.From phenylalanine, a set of reactions catalyzed by phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumaryl-CoA ligase (4CL) generates metabolites shared by many downstream phenylpropanoid branches, including those toward synthesis of flavonoids, lignin, and VOCs . In petu6–C3) and benzenoid (C6–C1) compounds are synthesized from cinnamate produced from phenylalanine by PAL, while formation of volatile phenylpropanoid-related (C6–C2) compounds occurs directly from phenylalanine , and coumarate 3-hydroxylase (C3H) utilize 4-coumaroyl-CoA from the general phenylpropanoid pathway to produce aldehyde intermediates p-coumaraldehyde or coniferaldehyde  are targeted for H3K9ac deposition during corolla maturation  and laccases (LACs)  Fig. 4BFig. 4B.  and LAC , which iOverall, our results suggest that important control points of the monolignol/phenylpropene metabolic network are regulated at the chromatin level, possibly to direct flux through the monolignol pathway to produce VOCs. The apparent GO term enrichment for lignin biosynthesis in both up- and down-regulated genes  is due tEPSPS1, DAHP1, and CS), benzenoid VOC biosynthesis (CNL1 and KAT1), and phenylpropene VOC biosynthesis (CCoAOMT1 and CFAT). Also included were ODORANT1 (ODO1), encoding a transcription factor involved in activating shikimate pathway and VOC genes (ATP-binding cassette subfamily G member 1 (PhABCG1), encoding an active VOC transporter required for VOC emission  during corolla development  and ent-KAURENE ACID OXIDASE (KAO) are well expressed, GA20ox, which acts downstream of KAO to produce bioactive GA, is strongly down-regulated from day 0 to day 2  (REPRESSOR OF GA 1–3 (RGA1) and PHYTOCHROME INTERACTING FACTOR3-LIKE 5 (PIL5), a positive regulator of RGA1  in petunia.Dataset S1. Increased and decreased H3K9ac and H3K4me3 DMGs.Dataset S2. Transcriptionally activated and repressed DEGs.Table S1. Primers used in the study.Table S2. Enriched GO terms among DMGs with increased H3K9ac.Table S3. Enriched GO terms among DMGs with increased H3K4me3.Table S4. Enriched GO terms among up-regulated (A) or down-regulated (B) DEGs.Table S5. Genes involved in shikimate and phenylalanine synthesis, the general phenylpropanoid pathway, or volatile benzenoid and phenylpropanoid synthesis genes.Table S6. Flavonoid and anthocyanin synthesis genes.Table S7. Genes involved in the monolignol pathway, eugenol/isoeugenol synthesis, or lignin polymerization.Table S8. Histone acetyltransferase genes.Table S9. Gibberellic acid signaling and metabolism genes.erab072_suppl_Supplementary-File-1Click here for additional data file.erab072_suppl_Supplementary-File-2Click here for additional data file.erab072_suppl_Supplementary-File-3Click here for additional data file.