Recent attempts to produce DHSA in seed oil by recombinant expression of cyclopropane fatty acid synthases (CPFASes) resulted in decreased oil content and poor germination or low DHSA accumulation. Here we explored the potential for plant vegetative tissue to produce DHSA by transiently expressing CPFAS enzymes in leaf. When CPFASes from plant and bacterial origin were transiently expressed in Nicotiana benthamiana leaf, it accumulated up to 1 and 3.7% DHSA in total fatty acid methyl ester (FAME), respectively, which increased up to 4.8 and 11.8%, respectively, when the N. benthamiana endogenous oleoyl desaturase was silenced using RNA interference (RNAi). Bacterial CPFAS expression produced a novel fatty acid with a cyclopropane ring and two carbon-carbon double bonds, which was not seen with plant CPFAS expression. We also observed a small but significant additive effect on DHSA accumulation when both plant and bacterial CPFASes were co-expressed, possibly due to activity upon different oleoyl substrates within the plant cell. Lipidomics analyses found that CPFAS expression increased triacylglycerol (TAG) accumulation relative to controls and that DHSA was distributed across a range of lipid species, including diacylglycerol and galactolipids. DHSA and the novel CPA were present in phosphatidylethanolamine when bacterial CPFAS was expressed in leaf. Finally, when plant diacylglycerol acyltransferase was coexpressed with the CPFASes DHSA accumulated up to 15% in TAG. This study shows that leaves can readily produce and accumulate DHSA in leaf oil. Our findings are discussed in line with current knowledge in leaf oil production for a possible route to DHSA production in vegetative tissue.Saturated mid-chain branched fatty acids (SMCBFAs) are widely used in the petrochemical industry for their high oxidative stability and low melting temperature. Dihydrosterculic acid (DHSA) is a cyclopropane fatty acid (CPA) that can be converted to SMCBFA  Saturated mid-chain branched fatty acids (SMCBFAs) offer unique properties that combine the benefits of saturated and unsaturated fatty acids and are thus an important component of the oleochemical industry . SMCBFA via the in planta production of cyclopropane oils that can be extracted and hydrogenated in post-harvest processing to yield SMCBFA products (Escherichia coli (Ec) CPFAS is proposed to cyclopropanate C16:1 and C18:1 that are predominantly esterified to the sn-2 position of phospholipids  and cotton (GhCPFAS) were tested, which produced 3.7 ± 0.4% (n = 7) and 0.9 ± 0.1% (n = 6) DHSA in total FAME, respectively (N. benthamiana to see if increased substrate availability would assist in higher levels of DHSA production. To do this we used a hairpin construct to silence the endogenous oleoyl desaturase (NbFAD2) via RNA interference (RNAi), herein termed hpNbFAD2.1, which was previously shown to increase total oleic acid content in the leaf transient expression system by six-fold compared to the control when tomato yellow leaf curl virus suppressor protein V2 was coexpressed  = −9.9, p < 0.01) and 4.8 ± 0.4%  = −18.9, p < 0.01) in total FAME, respectively, compared to when the CPFASes were expressed without hpNbFAD2.1  was observed with the 3-pyridylcarbinol ester indicative of a cyclopropane ring on Δ9-10, the lack of mass abundance and inconsistencies with the characteristic 12 unit mass difference between Δ9 and Δ10 formed during fragmentation of the cyclopropane ring , and V2.A more extensive analysis of the fluxes of cyclopropane fatty acids in transient leaf assays was conducted using a combination of GC analysis of thin layer chromatography (TLC) fractions and liquid chromatography-tandem mass spectrometry (LC-MSMS) of total lipid extracts from leaves expressing either Gh- or EcCPFAS, N. benthamiana leaves expressing EcCPFAS were more thoroughly investigated using OzID to elucidate positions of unsaturation, as well as composite tandem mass spectrometry methods employing both collision- and ozone-induced dissociation to identify the fatty acyl chains at each sn-position . Similar to PC 37:2, PC 37:6 was found to contain C18:2CPA on either sn-position.Total lipids extracted from leaves expressing either Gh- or EcCPFAS were subjected to TLC for GC analysis of the PC fraction. Both Gh- or EcCPFAS expression resulted in 25–27% accumulation of DHSA in the PC fraction as determined by GC analysis Figure 5position . For theN. benthamiana leaf , DAG 36:6 (C18:3/C18:3), DAG 37:4 (DHSA/C18:3), DAG 36:4 (C18:3/C18:1), and DAG 36:5 (C18:2/C18:3) (Diacylglycerol (DAG) and TAG species were analyzed by LC-MSMS to investigate the acyl chain combinations containing DHSA. While many DHSA- containing DAG and TAG species were commonly shared between the two CPFAS-expressing leaf lipid extracts, extracts expressing EcCPFAS contained quantitatively more compared to those with GhCPFAS expression based on the response factors. The most abundant DAG species in 2/C18:3) Figure 8The percent distribution of TAG species shows that the most abundant species upregulated with expression of CPFASes are: TAG 53:4, TAG 55:7, TAG 56:5 followed by lower levels of TAG 53:2&3 and TAG 55:4-6. The overall distribution of acyl chains on different TAG species was assessed on triple-quadrupole LC-MS (LC-MS QQQ) by the neutral loss of each acyl chain. The most abundant DHSA- containing TAG species were TAG 53:2 (C16:0/C18:1/DHSA), TAG 53:3 (C16:0/C18:2/DHSA), TAG 53:4 (C16:0/C18:3/DHSA), TAG 55:4 (C18:0/C18:3/DHSA), TAG 55:5 (C18:1/C18:3/DHSA), TAG 55:6 (C18:2/C18:3/DHSA), TAG 55:7 (C18:3/C18:3/DHSA), and TAG 56:5 (DHSA/DHSA/C18:3) Figure 9N. benthamiana expressing EcCPFAS, with or without GhCPFAS, compared to those without EcCPFAS expression , DGDG 36:6 (C18:3/C18:3), MGDG 34:6 (C16:3/C18:3), and MGDG 36:6 (C18:3/C18:3) in leaf lipid extracts expressing EcCPFAS, but not those with GhCPFAS  36:6 (C18:3/C18:3), MGDG 34:6 (C16:3/C18:3), digalactosyldiacylglycerol (DGDG) 34:3 (C16:0/C18:3), and DGDG 36:6 (C18:3/C18:3) accounted for over 80% of each galactolipid species in Arabidopsis thaliana diacylglycerol acyltransferase (AtDGAT1)  . TAG is AtDGAT1) . TAG canAtDGAT1) . We coexThe FAME profile of TAG fractions from leaf total lipid extracts separated by thin layer chromatography showed that DHSA produced from both Gh- and EcCPFAS accumulated equally in TAG, at approximately 15.8%  into DHSA rich oils can be boosted by the addition of specific DHSA handling enzymes. In plant seed the flux of oleic acid into DHSA could be significantly boosted by the further addition of a DHSA-specific lysophosphatidic acid acyltransferase SfLPAT2, , or phos SfLPAT2 . FurtherA. thaliana  has the potential to provide various forms of cyclopropane fatty acid as substrates for production of a wide range of SMCBFAs. Another interesting observation to note is the lack of lactobacillic acid  that could potentially be produced by E. coli CPFAS expression in these transient leaf assays. OzID analysis of PC species detected C18:1Δ11 as an acyl chain, which is the endogenous substrate for CPFAS in E. coli, from which lactobacillic acid is produced  was synthesized  with two silent nucleotide substitutions G2043A and G2551A from the published sequence to remove XhoI restriction sites.E. coli and cotton CPFAS genes were cloned into pENTR-NcoI via two EcoRI restriction sites and EcoRI and XhoI restriction sites respectively, and subsequently cloned into a cauliflower mosaic virus (CaMV) 35S promoter-driven over-expression vector pXZP393  was digested with NcoI, overhangs removed with DNA polymerase I, Large (Klenow) fragment, and re-ligated to generate pENTR-NcoI.  pXZP393  using LR35S::EcCPFAS and 35S::GhCPFAS, viral suppressor 35S::V2, visual expression marker 35S::GFP, N. benthamiana microsomal oleate desaturase silencer 35S::hpNbFAD2.1, and A. thaliana diacylglycerol acyltransferase 35S::AtDGAT1 ethanesulfonic acid (MES), 5 mM MgSO4, 500 μM acetosyringone). Infiltration mixes were prepared with each AGL1 at 0.3 OD600 units and infiltrated using a syringe on the underside of 5–6-week-old N. benthamiana leaves. Biological replicates of each experiment were produced by individual agroinfiltration spots on different leaves across different plants. Infiltrated plants were left at 24°C with 10:14 light:dark cycle for 5 days. On the fifth day expression of the infiltrated region was confirmed by presence of the GFP signal, which was harvested, freeze dried overnight, weight recorded, and stored in −80°C until analysis.Recombinant AGL1 containing :AtDGAT1  were groTotal lipid was extracted from leaf tissue as previously described , with trGC-MS was performed to confirm the identity of FAMEs not present in the base control infiltration and was carried out on a Shimadzu GC-MS QP2010 Plus ion-trap fitted with on-column injection  as previously described , equippem/z 184 or neutral loss of m/z 141, respectively. Multiple reaction monitoring lists were based on the loss of observed fatty acids using a collision of 28 V for all lipid species except DAG where 14 V was used. Chromatograms were integrated using Agilent Quantitative Analysis software version 6.0 . Statistical and graphical analysis was conducted using R (www.rstudio.com).LC-MSMS analysis of total lipid extracts was conducted based on previously described methods . Briefly using R  and data using R  and the  using R  using RsN. benthamiana expressing 35S::EcCPFAS, 35S::V2, 35S::GFP, 35S::hpNbFAD2.1, and 35S::AtDGAT1 were subjected to OzID analysis  with the Yeast Transformation Kit . Exogenous feeding of yeast, FAME preparation, and GC-MS analysis followed the methods of EcCPFAS in pENTR-NcoI was used to transfer EcCPFAS into pYES-DEST52  using LR Clonase™ II enzyme mix  according to the manufacturer's instructions. The resulting plasmid was used to transform The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.SO, SS, and CW conceived the project and designed the experiments. SO, MT, X-RZ, FN, DM, and SB conducted the experiments. All authors contributed to writing the manuscript.This project was co-funded by CSIRO and Grains Research and Development Corporation. Access to the Central Analytical Research Facility, operated by the Institute for Future Environments, Queensland University of Technology, was supported by generous funding from the QUT Science and Engineering Faculty.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.


MyoD1 and Myogenin promoters in GapmeR treated C2C12 cells. Furthermore, Ppp1r1b-lncRNA was found to bind to Ezh2, and chromatin isolation by RNA purification (ChIRP) assay revealed enriched interaction of Ppp1r1b-lncRNA with Myod1 and Tbx5 promoters, suggesting that Ppp1r1b-lncRNA induces transcription of myogenic transcription factors by interacting with the polycomb repressive complex 2 (PRC2) at the chromatin interface. Correspondingly, the silencing of Ppp1r1b-lncRNA increased EZH2 binding at promoter regions of myogenic transcription factors. Therefore, our results suggest that Ppp1r1b-lncRNA promotes myogenic differentiation through competing for PRC2 binding with chromatin of myogenic master regulators during heart and skeletal muscle development.Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators and play important roles in cardiac development and congenital heart disease. In a previous study, we identified a novel lncRNA, Ppp1r1b, with expression highly correlated with myogenesis. However, the molecular mechanism that underlies Ppp1r1b-lncRNA function in myogenic regulation is unknown. By silencing Ppp1r1b-lncRNA, mouse C2C12 and human skeletal myoblasts failed to develop fully differentiated myotubes. Myogenic differentiation was also impaired in PPP1R1B-lncRNA deficient human-induced pluripotent stem cell-derived cardiomyocytes (hiPSCs-CMs). The expression of myogenic transcription factors, including MyoD, Myogenin, and Tbx5, as well as sarcomere proteins, was significantly suppressed in Ppp1r1b-lncRNA inhibited myoblast cells and neonatal mouse heart. Histone modification analysis revealed increased H3K27 tri-methylation at  Long noncoding RNAs (lncRNAs), which comprise the bulk of the noncoding genome, have been proven to play important roles in maintaining cardiovascular system homeostasis . TherefoSkeletal and cardiac muscle both arise from myogenic mesodermal lineages and share many characteristics. It was reported that the expression patterns of cardiac and skeletal muscle transcription factors and fast-skeletal myosin heavy chain (sk-fMHC) within ventricular myocardium and skeletal muscle were similar at embryonic day (ED) 20, and the expression patterns became cardiac or skeletal muscle specific only during postnatal development . Some tySeveral myogenic transcription factors have been identified. Among them, the basic helix–loop–helix (bHLH) family  were discovered to be key regulators of muscle development . LikewisEpigenetic modifications at the transcription factor binding sites play critical roles in the regulation of gene expression, including the myogenic differentiation genes . In embrLncRNAs may carry out both gene inhibition and gene activation through a range of diverse mechanisms. Targeted modulation of epigenetic modifiers, such as the Polycomb repressive complex (PRC) subunits, by lncRNAs has been suggested as a general mechanism for trans-acting lncRNA in gene regulation . PRC-medIn this report, we investigated the expression patterns of Ppp1r1b-lncRNA during myoblast differentiation, assessed the effect of down-regulation of Ppp1r1b-lncRNA on myogenesis in vitro and in vivo, and examined its impact on H3K27 methylation status at promoter regions of myogenic transcription factors, including MyoD, Myogenin, and Tbx5. We demonstrated interaction between Ppp1r1b-lncRNA, with Ezh2, and targeted promoters of myogenic transcription factors. These findings revealed that Ppp1r1b-lncRNA regulates myocyte differentiation by negatively modulating H3K27me3 of myogenic transcription factor genes via competing for PRC2 binding with chromatin.To determine the effects of Ppp1r1b-lncRNA on myogenesis, C2C12, an immortalized mouse myoblast cell line, was used. We cultured C2C12 cells in high serum conditions for rapid proliferation and low serum conditions for differentiation. The expression of Ppp1r1b-lncRNA in differentiated C2C12 cells was significantly higher than that in the nondifferentiated cells A. SubcelIn order to elucidate the functional role of Ppp1r1b-lncRNA, we treated C2C12 cells cultured in differentiation media with 50 nM antisense Ppp1r1b-lncRNA GapmeR for 48 h. The GapmeR treatment decreased Ppp1r1b-lncRNA expression by more than 70% in differentiated C2C12 cells C. MorphoSupplemental Fig. 1). Consistent with lncRNAs properties, no peptide was produced confirming the lack of a functional ORF in both mouse and human orthologs.LncRNAs are known to be poorly conserved across species. Furthermore, lncRNAs are less conserved at the primary nucleotide sequences compared with protein coding genes . It was Like in mice, the human PPP1R1B-lncRNA was also induced during differentiation A–C. To eLncRNAs are known to have tissue specificity. To examine whether the human specific PPP1R1B-lncRNA affect myogenesis during cardiac muscle development, we used a human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) cell line. By silencing the PPP1R1B-lncRNA, myogenic differentiation of cardiomyocytes was impaired. The iPSC-CMs also exhibited significantly less myosin expression and myofibrillar bundles following PPP1R1B-lncRNA siRNA treatment A,B. FurtTo further characterize the function of Ppp1r1b-lncRNA in myogenesis, we examined the impact of its loss on mRNA expression of the muscle-specific transcription factors and muscle structure genes. It was known that the differentiation of skeletal muscle cells is governed by a group of four myogenic regulatory factors (MRFs) including MyoD, Myf5, Myogenin and MRF4 . MyoD isOur data revealed that mRNA levels of Myogenin, MyoD1, Tbx5, Dystrophin and Troponin T2, were significantly lower in the GapmeR treated C2C12 cells compared with the untreated group A. LikewiMyoD1 and Myogenin promoters were decreased in the differentiated myotubes compared to the undifferentiated myoblasts. After Ppp1r1b-lncRNA GapmeR treatment, the H3K27me3 levels were increased. These findings are consistent with the suppressed expression of these transcription regulators  possesses histone methyltransferase (HMT) activity with specificity for Lys 9 (K9) and Lys 27 (K27) of histone H3 . EZH2 is, was analyzed from the pulled RNA: DNA interactome  While our results support a role for the Ppp1r1b-lncRNA during early stages of muscle and heart development, our in vivo studies are limited to temporal Ppp1r1b-lncRNA silencing during the neonatal period. A comprehensive in vivo characterization of the functional roles during development in transgenic mouse models is required. (iii) Examining the translational value of Ppp1r1b-lncRNA manipulation requires temporal and tissue specific in vivo delivery of Ppp1r1b-lncRNA in disease models of muscular dystrophy, cardiomyopathy, and congenital heart disease.Our study has revealed the crucial roles of Ppp1r1b-lncRNA in myogenesis control and potential molecular mechanisms. Heart formation encompasses an orchestrated series of molecular and cellular events, and thus even subtle alterations in this process can lead to serious cardiac disorders. On the other hand, cardiac muscle has limited proliferative capacity and regenerative therapies are highly in demand as a new treatment strategy. Our results may facilitate our understanding of the function of Ppp1r1b-lncRNA and provide us with a promising therapeutic target for congenital heart disease and skeletal myopathy. However, we should acknowledge important limitations: (i) Although several models have demonstrated the molecular mechanisms involved in the lncRNA dependent inhibition of histone methylation , furtherSupplemental Information Appendix.Extended materials and their sources are available in the C2C12 and Human skeletal muscle myoblast cell lines were obtained from ATCC, and ZenBio. Human iPSC-derived cardiomyocytes (SCVI480-CM) were obtained from Joseph C. Wu MD, PhD at the Stanford Cardiovascular Institute (CVI), Stanford; CA. Cells were cultured according to protocols established at the CVI . Human hAll animal-related experimental protocols were approved by the University of California Los Angeles Animal Care and Use Committee (ACUC). Wild-type C57BL/6 mice were purchased from Charles River laboratory. Neonatal mice were used for Ppp1r1b-lncRNA GapmeR injection in vivo. C57BL/6 neonatal mouse pups were intravenously injected with 1.2 nmol/g body weight GapmeR at P1, P3, and P5. Mice were sacrificed at P7 and heart tissues were collected. Experimental protocols were approved by the University of California Los Angeles Animal Care and Use Committee.Transient transfection of cells with GapmeR (Qiagen) and siRNA (Bioland Scientific) was performed in 12-well plates using Lipofectamine 2000 reagent or RNAiMAX (Life Technologies). Mouse Ppp1r1b-lncRNA GapmeR (50 pmol/well) was transfected into C2C12 cells cultured in differentiation media. Human PPP1R1B-lncRNA siRNA (30 pmol/well) was transfected into human skeletal muscle myoblast and human iPSC-CM cultured in differentiation media.Cytoplasmic and nuclear RNA were isolated with the Cytoplasmic and Nuclear RNA Purification Kit (Norgen Biotek) following the manufacturer's manual.Supplemental Information Appendix, Table S1). Relative expression value was calculated using the comparative threshold cycle (ΔΔCT) method.Total RNA was isolated from the cultured cells or tissue using RNeasy Mini Kit (Qiagen). The reverse transcription reaction was performed with 1 µg total RNA and the SuperScript IV First-Strand Synthesis System (Thermo Fisher Scientific), and the real-time PCR reactions were performed by using iTaq Universial SYBR Green Supermix from BioRad. Primers were prepared by Invitrogen.  were used to transfer proteins to a membrane for analyzing with antibodies. Antibodies were listed in Cells were fixed in 4% (v/v) formaldehyde in PBS for 20 min. After rinsing with PBS, the cells were incubated with 0.1% TritonX-100 in PBS for 15 min at room temperature and rinsed. After blocking in PBS containing 10% Bovine Serum Albumin for 1 h, the cells were incubated overnight with primary antibody and then appropriate AlexaFluor conjugated secondary antibodies for 1 h. Cell nuclei were eventually counterstained by DAPI. Images were recorded on a Nikon confocal microscope and an echo revolve microscope . Fusion index was defined as the number of nuclei in myosin heavy chain (DSHB) positive cells divided by the total number of nuclei. EB density was defined as the total number of nuclei in EB divided by the EB area.The full-length mouse and human lncRNA cDNA was cloned into pCMV Sport 6.1 vector, which contains a T7 or a SP6 promoter. The Transend nonradioactive system was used to incorporate biotinylated lysine into nascent proteins. After the in vitro TNT reaction, the reaction was terminated by adding SDS loading buffer and incubation at 95°C for 5 min. After SDS-PAGE and electro blotting, the biotinylated proteins were visualized by binding Streptavidin-HRP and chemiluminesent detection.Supplemental Information Appendix, Table S1). Data were analyzed using fold enrichment method.Undifferentiated and differentiated C2C12 as well as Ppp1r1b-lncRNA GapmeR treated C2C12 cells were exposed to 1% formaldehyde, and chromatin was fragmented by sonication. Immunoprecipitation was performed using IgG (negative control) or with the primary antibodies for H3K27me3 or EZH2. DNA was purified from the immunoprecipitates using the PCR purification kit (Qiagen). Subsequently, real-time PCR was performed to amplify the promoter region DNA of Myogenin and MyoD1. Primers were prepared by Invitrogen .The assay was performed with P0 C57BL/6 mouse hearts. T7 RNA polymerase kit (Promega) and Biotin RNA labeling mix (Roche) were used for preparation of biotin-labeled Ppp1r1b-lncRNA. After incubated biotinylated RNA with cell extract at 4°C, the washed Dynabeads M280 Streptavidin (Thermo Fisher Scientific) was added to the pulldown mixture. After 1 h incubation at 4°C for 1 h, the beads were collected by using the magnetic apparatus and washed with the binding buffer. Proteins that were co-precipitated were eluted in the SDS sample buffer and fractionated in 8%–16% gradient SDS-PAGE for western analysis with EZH2 antibody  was added to cell lysate and incubated overnight at 4°C, Protein A/G beads (Thermo Scientific) were added and incubated for 1 h at 4°C with gentle rotation. After washed off unbound material, RNAs bound to immunoprecipitated EZH2 were isolated and qRT–PCR was performed using Ppp1r1b-lncRNA primers .RNA immunoprecipitation experiments were performed with P1 neonatal mouse heart tissue. Tissue lysate was cross-linked by 1% formaldehyde and lysed by sonication. After EZH2 antibody  was used. Assays were performed per manufacturer's protocol. Capture probes were designed to target Ppp1r1b-lncRNA and prepared at 50 µM total oligo concentration. P1 mouse heart tissue and human fetal heart tissue were cross-linked in 1% glutaraldehyde/PBS and sonicated to shear DNA. Probes were added to cell lysate and incubated overnight for hybridization. After hybridization, Streptavidin magnetic beads were added and incubated for an additional 30 min. After washing, beads were incubated with RNase A and RNase H in DNA elution buffer. The eluted sample was then subjected to protease K treatment, DNA isolation and quantitative PCR analysis. Primers used for real-time PCR were listed in t-test; P ≤ 0.05 was considered significant unless specified otherwise.Quantified results are presented as mean ± SEM. Comparisons between groups were evaluated using analysis of variance or the Student's www.ncbi.nlm.nih.gov/geo) under Neonatal Heart Maturation SuperSeries GSE85728 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE85728). All unique materials, resources, and reagents are available on request by qualified researchers for their own use. Generated plasmid constructs will be deposited in addgene.Gene expression data will be deposited within the Gene Expression Omnibus repository (Supplemental material is available for this article.


Breast cancer is one of the leading causes of cancer-related deaths in women, and there is a demand in developing an Asian-based genetic profiling database for breast cancer in improving the treatment response. This study aimed to determine molecular alternations and identify potential therapeutic targets by analyzing the genetic profiling from a cohort of Taiwanese breast cancers using a commercialized next-generation sequencing (NGS) targeted panel.The study population comprised a broad spectrum of breast cancer patients in Taiwan, including Group 1: planned to receive first-line surgery and followed by adjuvant therapy, or early relapse within three years, Group 2: planned to receive first-line neoadjuvant therapy and followed by surgery, and Group 3: de novo stage IV, or stage IV with recurrence beyond three years. Molecular profiles were determined using Thermo Fisher™ Oncomine™ Comprehensive Assay version 3 (TMO comprehensive assay) from Formalin-Fixed Paraffin-Embedded (FFPE) tissues. Level of actionability was evaluated with the ESMO Scale of clinical actionability of molecular targets (ESCAT).ERBB2 amplification, BRCA1/2 germline mutation, PIK3CA mutation, and NTRK translocation, and Tier II: antitumor activity associated with the matched alteration-drug but lack of prospective outcome data including PTEN loss, ESR1 mutation, AKT1 mutation, and ERBB2 mutation, and Tier III: matched drug-alteration that led to clinical benefit in another tumor type including MDM2 amplification, and ERBB3 mutation. Among them, 249 (66%) showed at least one actionable alternation based on the ESCAT criteria. The most frequent impacted genes  were PIK3CA (38%), followed by ERBB2 (23%), ESR1 (10%), AKT1 (6%), and BRCA2 (5%), and the remaining rare variants (less than 5% of assayed cohort) were BRCA1 (3%), MDM2 (2.2%), and ERBB3 (1.1%).A total of 380 TMO comprehensive assays were conducted on 372 patients, and we presented targeted sequencing analyses of Tier I: alteration-drug match associated with improved outcome in clinical trials including Targeted sequencing of actionable genes is believed to provide clinical applicability and substantial benefits for Taiwanese breast cancer patients. A valid scale of clinical actionability should be adopted for precision medicine practice under multidisciplinary molecular tumor board.The online version contains supplementary material available at 10.1186/s12885-021-07931-4. In Taiwan, breast cancer is one of the leading causes of female malignancy and ranked fourth of cancer-related deaths. Breast cancer incidence rate has continuously increased throughout the past two decades, and has reached 73 per 100,000 females, or more than 12,000 incident cases during the year 2018, according to the cancer registry annual report [Breast cancer is a highly heterogeneous disease leading to diverse morphological features and different clinical outcomes. An increasing number of studies are focusing on the early detection and proper treatment to enhance diagnostic accuracy and improve the longevity and quality of life of breast cancer patients , 3. In gThese tools, however, do have limitations in the immediate detection of occult recurrence or metastasis. For mammography and breast ultrasound, detection is not always feasible synchronously when recurrence or metastasis occurs even though they are the gold-standard for breast cancer screening. On the other hand, prognostic discrepancy was observed within the same clinical strata such as those defined by staging or IHC subtypes. There must be some molecular heterogeneity not accounted for by these conventional prognostic factors. As a consequence, personalized precision medicine should be initiated for breast cancer based on the thorough understanding of molecular aberrations underpinning breast carcinogenesis with a hope of more therapeutic targets identified.In order to have a more accurate estimate of breast cancer recurrence risk, several gene expression profiling panels or assays have been developed and commercially available in the Western countries . HoweverThis study aimed to determine the difference in genetic profiles of subjects with breast cancer in Taiwan, and to identify the potential biomarkers for prognosis and potential therapeutics for breast cancer. These aims were achieved by analyzing the genetic profiling from a cohort of Taiwanese breast cancer subjects using the method of next-generation sequencing (NGS) with a pre-defined target panels including actionable genes.The objectives of the study were to conduct comprehensive genetic profiling of Taiwanese breast cancers by targeted sequencing and identify therapeutics ready for treatment decisions.Fig. Relevant clinical ER and HER2 status phenotype was determined as following: ER positivity was defined as at least 1% of nuclei with positive results of IHC assay. HER2 status was determined according to the ASCO and CAP guidelines; IHC 3+ and IHC 2+ with fluorescence in-situ hybridization (FISH) amplification indicated HER2 over-expression. For clarity, HER2 over-expression indicated IHC/FISH testing positive results and HER2 amplification was based on copy number variation (CNV) algorithm calls from NGS experiments.Group 1 was subdivided into: (A) subjects who were planning to receive surgery (mastectomy or breast conserving surgery) as the first-line treatment for breast cancer and followed by adjuvant therapy, and (B) subjects with breast cancer recurrence at screening, and who had received surgery for primary breast cancer within 3 years prior to screening (early relapse), and with primary tumor formalin-fixed paraffin-embedded (FFPE) tissues available. Group 2 included subjects who were planning to receive neoadjuvant therapy as the first-line treatment for breast cancer and followed by surgery. Group 3–1 included subjects diagnosed with de novo and treatment naïve stage IV breast cancer and Group 3–2 recruited: (A) stage IV patients with breast cancer recurrence beyond 3 years after surgery (late relapse), and (B) stage IV patients who had received or were currently receiving treatments for breast cancer and with progressive disease at enrollment. A retrospective cohort composed of locally advanced/metastatic breast cancers with poor outcomes or breast cancers with non-pathological complete response (non-pCR) after neoadjuvant therapy was retrieved from biobank.The study design is a mix of prospective and prospective analysis of retrospective cohort. Prospective sample recruitment was conducted in Group 1A (primary surgery), 2 (neoadjuvant therapy followed by surgery) and Group 3–1 (de novo stage IV), while Group 1B (early relapse), Group 3–2 (late relapse or progressive stage IV disease during therapy) enrolled both retrospectively collected  as well as prospectively collected (relapse or progressive disease) samples. Only the retrospective cohort  enrolled samples in an exclusively retrospective manner.> 20 years old and with a confirmed diagnosis of invasive breast cancer who were planning to receive or under treatment. All participants must be graded as being Eastern Cooperative Oncology Group (ECOG) performance status ≤3 and have a life expectancy ≥3 months. Subjects were excluded if they had primary cancer(s) other than breast cancer within 5 years prior to screening.Subjects enrolled in this study were females aged The genetic profiles or potential biomarkers were determined through NGS targeted sequencing of FFPE sample. For breast cancer relapse/progress at recruitment screening (Group 1B and Group 3–2), paired FFPE tissues (primary and recurrent tumors) from the same subject were collected and sequenced. For Group 2 patients, paired FFPE samples (diagnostic and post neoadjuvant specimens) were assayed as well.The preparation of FFPE section was performed at the clinical site following the standard procedures. The extraction of DNA and RNA as well as hematoxylin and eosin (H&E) staining were performed in the central laboratory according to laboratory manual under the guidance of a certified pathologist (CYH). Additional sections of FFPE samples of individual subject might be pursued if failed nucleic acid quality check happened. The criteria of DNA/RNA quality check followed the manual of the TMO assay requirement (see below). Approximately 7 unstained sections of tumor FFPE tissues per subject were retrieved; at least one unstained section was prepared for H&E staining, and 6 unstained sections were prepared for TMO comprehensive assay.Targeted sequencing of NGS experiments were performed with TMO comprehensive assay , which was used to detect thousands of variants across 161 genes which are relevant to cancers . The TMOAmplicon libraries were constructed with multiplex PCR primers for preparation of DNA and RNA (for fusion genes) from FFPE samples. Sequencing was performed with the Ion Gene Studio S5 System and Ion 540 Chips. Raw data process, alignment, and variant calling was performed with Torrent Suite™ Software, with variant calling using the Torrent Variant Caller plug-in. Further management was proceeded by Ion Reporter™ Software with workflow “Oncomine Comprehensive v3 - w3.2 - DNA and Fusions - Single Sample” version 5.10 selected and filter chain “Oncomine Variants” version 5.10 applied. Reference genome was hg19.The primary outcomes were actionable mutations determined by genetic profiling of Taiwanese breast cancers using TMO comprehensive assay. The frequency of targetable alterations was tabulated by the subdivisions of Groups 1A/1B, 2, 3–1, 3–2, as well as the retrospective cohort. For whole study population and subgroup analysis, MutationMapper and OncoPrinter were used for visualization purpose , 9. AddiLevel of actionability of targeted sequencing data was determined based on the ESMO Scale of clinical actionability of molecular targets (ESCAT) definition . A briefERBB2 amplification, BRCA1/2 germline mutation, and PIK3CA mutation , NTRK translocation , PTEN loss and ESR1 mutation , AKT1 mutation, ERBB2 mutation , BRCA1/2 somatic mutation, MDM2 amplification (Tier IIIA: based on prospective study), and ERBB3 mutation (Tier IIIB: without supportive data) were retrieved and analyzed.Based on these criteria, matched targets including All statistical analyses were performed using SAS statistical software . Continuous variables were summarized as the number of observations, mean, standard deviation, minimum, maximum, and 95% confidence interval as indicated. Categorical variables were presented as counts and percentages. Unless otherwise specified, all statistical assessments were performed at the significance level of 0.05.n = 213), Group 1B , Group 2 , Group 3–1 , and Group 3–2 . In addition, there were 74 subjects from retrospective biobank cohort. Eight patients were assayed twice, including 5 in Group 2 (diagnostic/post-neoadjuvant pair), 2 in Group 1B and 1 in retrospective cohort (primary/recurrent pair). Demographic and clinical features were detailed in Table In this study we presented analytical results of 380 TMO assays from 372 breast cancer patients for potential therapeutic targets. The distributions of clinical scenarios were: Group 1A  of the 380 TMO assays showed at least one actionable mutation based on ESCAT criteria. The average number of actionable genes across the whole study population was 1.0 , and the average number of called variants was 3.1 . For impacted patients, the average number of actionable genes was 1.4 , and the average number of called variant: 3.2 . Table PIK3CA (38%), followed by ERBB2 (23%), ESR1 (10%), AKT1 (6%), and BRCA2 (5%), and rare variants (less than 5% of whole population) were BRCA1 (3%), MDM2 (2.2%), and ERBB3 (1.1%). Mutual exclusivity was calculated and revealed co-occurrence of BRCA1 and BRCA2 mutations  as well as borderline mutual exclusivity between BRCA1 and PIK3CA . Additional file Figure p = 0.0011) and more called variants  than other groups. Group 1B also had the highest number of copy number alternation (CNA)  and missense mutation  while Group 2 had the highest number of truncation mutation  were observed for ER−/HER2+ breast cancer (Table p = 0.008), and truncation mutation  was found in 81 (21.3%) of assayed samples and was distributed unevenly across clinical study groups , and 11 ER+/HER2- and 4 ER−/HER2- patients were without clinical HER2 over-expression .in 81 21.% of assaERBB2 missense  and 2 ERBB2 in frame mutations  were identified in 12 patients with 11 truncating and 1 missense mutations  were identified in 17 patients (5 with concurrent BRCA1 mutations). The associated clinical phenotypes were 2 ER+/HER2+, 12 ER+/HER2-, 1 ER−/HER2+, and 2 ER−/HER2- among 5 Group 1A, 2 Group 1B, 3 Group 2, 2 Group 3–1, and 5 retrospective cohort cases. There were 22 truncating and 1 missense mutations. R3052Q constituted the only missense mutation, which occurred in one ER+/HER2- non-pCR retrospective cohort patient with co-existent R2520* nonsense mutation. One stage III ER−/HER2+ retrospective cohort patient reported both Q649* and Q2829* nonsense mutations. Another ER+/HER2- Group 1A patient harbored W194*, Q3227* nonsense and E1493fs frameshift mutations. There was one ER+/HER2+ Group 1B subject sequenced with triple nonsense mutations including Q126*, Q1124*, and Q3295*, and another triple negative Group 3–1 patient with triple frameshift mutations . The remaining nonsense mutations including W2970*, Q2499*, Q2539*, S547* and frame shift mutations including S2186fs, V1999fs, S2186fs, L1930fs, K1472fs, N588fs, F506fs, and S2186fs came from 12 patients, with each patient reported only one variant. Figure BRCA2.PIK3CA: 11 with amplification  or inframe (n = 2) mutations , H1047L (n = 50), E545K (n = 19), and E542K (n = 15), and the remaining missense mutations were N345K (n = 7), E542G, E726K, K111E, Q546K (n = 3), C420R, E453K, E545G, G1049R, H1047Y (n = 2), and D1029N, G1049S, G118D, M1043I, N1044K, N345D, N345I, P539R, Q546R, R108C, R108H, R93W, V344G, Y1021H (n = 1). Two ER+/HER2- (one Group 3–1 and one Group 3–2) reported inframe deletion in E109del. More than half of patients harbored only one mutation, with the exception of two patients with 4 variants , four patients with 3 variants , 44 with both H1047R and H1047L mutations, and remaining 9 patients with dual mutations of H1047Y/Q546K, E542K/E726K, E542G/E542K (two subjects), E545K/M1043I, G1049R/G1049S, E453K/E545K, E542K/P539R, and E453K/H1047R.There were 141 samples with variants in ion Fig. , of whicNTRK1 (n = 23), NTRK2 (n = 1), or NTRK3 (n = 2). Among them, two were ER+/HER2+, 16 were ER+/HER2-, 2 were ER−/HER2+, and 6 were ER−/HER2-. Figure There were 26 subjects with amplifications in RK1 n = 2, NTRK2 , and C136Y, of which each variant occurred in one patient and one Group 1B ER+/HER2+ case with dual mutations of G165R and G165E. On the other hand, T319* nonsense mutation was found in four patients, while for Q245* and R15*, each nonsense mutation was reported in one patient. There were 10 frameshift mutations, namely E150fs, Q214fs, E43fs, L247fs, K164fs, P246fs, E73fs, and R47fs, with each mutation occurred in one patient and one retrospective cohort stage IV, clinically ER+/HER2- case reported concurrent L247fs and D187fs frameshift mutations. It deserved notice that the case with T319* nonsense mutation also harboured Q214fs mutation  while K125T missense/R15* nonsense mutation happened in the same patient . Figure PTEN inactivating mutations.ESR1 mutations were reported in currently study (ESCAT Tier IIA). Three missense mutations, including E380Q, Y537S, and D538G, were reported form three retrospective cohort heavily pretreated ER+/HER2- stage III or IV patients . Only one case  was sequenced with L52R missense mutation, and the remaining 18 cases were with E17K hotspot pathogenic missense mutation  was identified in 8 patients, with 3 being ER+/HER2+, 4 ER+/HER2-, and 1 ER−/HER2+ and clinical study groups were 4 Group 1A, 1 Group 1B, 2 Group 2, and 1 Group 3–1  was recognized in 4 patients, of which two were with V104L (both with ER−/HER2+) and two with E928G missense mutations  breast cancer . Indeed,In current study, we adopted TMO comprehensive assay v3 as the platform for molecular profiling for Taiwanese breast cancers. The instrument is a targeted, NGS-based assay that enables the detection of relevant SNVs, CNAs, gene fusions, and indels from 161 cancer-related genes. One of the merits is that as little as 40 ng of nucleic acid from FFPE samples is feasible, which made sequencing of pre-operative needle biopsy from Group 2 subjects possible. The content driven by the Oncomine Knowledgebase also help assure coverage of key targets aligned to published evidence and experienced clinicians’ domain knowledge. Relevant guidelines and clinical trials are generated in an automatic fashion, further augment the utility of TMO platform in clinical trials such as NCI-MATCH and MDACC IMPACT –16.Another major issue impedes the applicability of precision medicine is the lack of a standardized and generalized definition of actionable mutations. The 4-tier classification system is recommended jointly by the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists for cancer somatic sequence variations based on their clinical significances, with Tier I and II  being potential therapeutically relevant . This syERBB2 amplification (ESCAT Tier IA), 15 (18.8%) were clinically HER2 negative, indicating that targeted sequencing may identify more HER2 positive breast cancers than traditional IHC/in situ hybridization assays. Considering that anti-HER2 target therapy is the mainstay of treatment for HER2 positive breast cancers, more patients might benefit from the merit of targeted therapy if NGS is performed. There is currently no evidence supporting the benefit of anti-HER2 targeted therapy for HER2 IHC/FISH-negative but ERBB2 amplified breast cancers. Further studies are warranted to shed light on discordant cases between IHC/FISH and sequencing assays. On the other hand, only 2 out of the 9 ERBB2 mutated cases were clinically HER2 over-expressed; except the putative neutral D769N, all missense and in frame mutations of the remaining 8 cases were candidates of tyrosine kinase inhibitor neratinib [Among the 81 patients with eratinib .ERBB3 mutations (ESCAT Tier IIIB) were recognized in four patients, and those with V104L missense mutation were of ER−/HER2+ phenotype, while the two with E928G were ER+/HER2- and triple negative. Hanker et al. demonstrated co-occurrence of gain-of-function mutations in various HER2 mutations and HER3 E928G missense mutation, resulting in subsequent HER-signaling and breast cancer growth [HER3 only mutation in current study, neratinib may be a choice for advanced diseases as in the SUMMIT trial [r growth . CombineIT trial .BRCA1 mutations, five were triple negative and four were ER+/HER2-. Without HER2 over-expression, these 9 patients might be responsive of FDA-approved poly (ADP-ribose) polymerases (PARP) inhibitors such as olaparib and talazoparib if germline mutation was also confirmed [BRCA2, while 5 were also tested positive of BRCA1 mutation . The corresponding mutations were R2520*/R3052Q, Q649*/Q2829*, W2970*, Q126*/Q1124*/Q3295*, and S538fs/T598fs/S973fs in BRCA2 and Q1577*, Q1867*, R1720Q, Q855*, and S1180fs in BRCA1. The clinical phenotypes were ER+/HER2-, ER−/HER2+, ER+/HER2-, and triple positive for last two. These subjects with BRCA2 mutations were also candidates for synthetic lethargy if their mutations were proved germline origin as well. It’s interesting to see if there exists a dose-dependent effect of PARP inhibition based on the dual BRCA alternations or doublet/triplet mutations of BRCA2.Of the 12 patients with onfirmed , 24. HowPIK3CA mutations/amplifications were by far the most common ESCAT alternations identified from Taiwanese breast cancers . For PIK3CA mutant subjects, 79.4% were ER+ and this mutation was observed in nearly 40 % of Group 1A patients, indicating that PIK3CA may be an earlier driver for hormone responsive breast cancer during oncogenesis. Hotspots for Taiwanese breast cancers were identified, namely H1047R (n = 74), H1047L (n = 50), E545K (n = 19), and E542K (n = 15) [PIK3CA mutant breast cancer [PIK3CA amplification remains unclear, and clinical trials enrolling PIK3CA amplified breast cancers are ongoing [BRCA1/PIK3CA mutations were mutually exclusive (P = 0.003), and no subject harboured both mutations in current study.(n = 15) , 26. Bast cancer . It’s bet cancer . The cli ongoing . BRCA1/PPTEN (ESCAT Tier IIA) is a crucial step during breast carcinogenesis as PTEN is a negative regulator of PI3K pathway; PTEN acts as a direct antagonist of PI3K through dephosphorylation of PIP3. PTEN loss results in AKT-mTOR signaling activation, and capivasertib (AZD5363), an AKT inhibitor, can suppress the aberrant pathway [PTEN mutations, and capivasertib combined with fulvestrant or chemotherapy might be a choice for refractory diseases depending on hormone receptor status. T319* and G129E were the most common PTEN mutations for Taiwanese breast cancers.Loss or inactivation mutation of  pathway . In currAKT1, and mutant AKT1 (ESCAT Tier IIB) was identified from 19 subjects, of which 15 were ER+ with E17K being the most prevalent missense mutation [AKT1E17K-mutant patients might benefit from mTOR inhibitor [AKT1 and PIK3CA variant was mutually exclusive (only 3 were mutated in both genes), and one plausible explanation comes from the fact that AKT1 is the downstream effector of PIK3CA. Recent update of IPATunity130 trial failed to demonstrate a progress-free survival advantage of ipatasertib (an AKT1/2/3 inhibitor) with paclitaxel for ER+/HER2- advanced/metastatic breast cancers, while phase II FAKTIOL showed that fulvestrant plus capivasertib improved progress-free survival irrespective of PI3K/PTEN alternation [Another actionable gene of PI3K/AKT/mTOR pathway is mutation . In addinhibitor . It deseernation . WhetherESR1 mutations, and these patients were from retrospective cohort and were heavily pre-treated ER+ stage III/IV patients. All these patients could take advantage of selective estrogen receptor degrader fulvestrant once progressed on prior endocrine therapy [ESR1 amplifications and fusions were identified and most of these subjects were ER+ while the clinical significance remains inconclusive [Only 3 cases were sequenced positive for  therapy . ESR1 amnclusive .NTRK translocations were categorized as ESCAT Tier IC alternations as certain tyrosine kinase receptor (TKR) inhibitors may be of help [NTRK1 amplification constituted the majority of NTRK variants in current study. MDM2 amplification (ESCAT Tier IIIA) is an oncogenic aberration and was observed in 8 patients (7 were ER+). MDM2 inhibitor may reactivate p53 and may act as a salvage recue for endocrine refractory breast cancers [ of help . NTRK1 a cancers .NTRK translocation were simply not under the coverage of TMO assay. As commercialized panel was adopted; the interrogated targets were limited by the pre-selected genes designated into the panel design. Second, only somatic variants of BRCA1 and BRCA2 were sequenced, while germline mutations were not assayed as only tumor tissues were tested. The clinical significance of somatic BRCA1/2 mutations in breast cancer remains inconclusive, and the ongoing NCT03344965 and VIOLETTE (NCT03330847) trials are designed to answer the question whether BRCA1/2 somatic mutation could act as a predictive marker for the PARP inhibitor olaparib response as in ovarian cancer [There were some limitations of current study. First, some actionable alternations were missed in TMO comprehensive assay. Notably, microsatellite instability (MSI), tumor mutation burden (TMB) and n cancer . Third, Our study presented an ongoing effort for breast cancer personalized therapy with precision medicine , and theAdditional file 1. A tab-separated values (Additional file Additional file 2. A scalable vector graphs (Additional file


These cells displayed the typical morphology and markers of astroglia, and were susceptible to the action of inflammatory mediators and BAF312, because expressing receptors for IL1, IL17, and S1P (namely S1P1 and S1P3). Targeting of S1P signaling by BAF312 inhibited NFκB translocation evoked by inflammatory cytokines, indicating a direct anti-inflammatory activity of the drug on the human astrocyte. Further, while glia cells exposed to IL1 or IL17 downregulated protein expression of glutamate transporters, BAF312-treated astrocytes maintained high levels of GLAST and GLT1 regardless of the presence of inflammatory mediators. Interestingly, despite potential glial susceptibility to S1P signaling via S1P3, which is not targeted by BAF312, NFκB translocation and downregulation of glutamate transporters in response to S1P were inhibited at similar levels by BAF312 and FTY720, another S1P signaling modulator targeting also S1P3. Accordingly, specific inhibition of S1P1 via NIBR-0213 blocked S1P-evoked NFκB translocation, demonstrating that modulation of S1P1 is sufficient to dampen signaling via other S1P receptors. Considering that NFκB-dependent responses are regulated by Nrf2, we measured activation of this critical transcription factor for anti-oxidant reactions, and observed that BAF312 rapidly induced nuclear translocation of Nrf2, but this effect was attenuated in the presence of an inflammatory milieu. Finally, in vitro experiments with spinal neurons exposed to astrocyte-conditioned media showed that modulation of S1P or cytokine signaling in astrocytes via BAF312 prevented neurons from astrocyte-induced degeneration. Overall, these experiments on human astrocytes suggest that during neuroinflammation targeting of S1P1 via BAF312 may modulate key astrocyte functions and thereby attain neuroprotection indirectly.Multiple sclerosis (MS) is an inflammatory neurodegenerative disease of the central nervous system (CNS) with heterogeneous pathophysiology. In its progressive course oligodendrocyte and neuroaxonal damage is sustained by compartmentalized inflammation due to glial dysregulation. Siponimod (BAF312), a modulator of two sphingosine-1-phosphate (S1P) receptors (S1P1 and S1P5) is the first oral treatment specifically approved for active secondary progressive MS. To address potential direct effects of BAF312 on glial function and glia-neuron interaction, we set up a series of  BAF312 treatment significantly hinders lesion enlargement and brain atrophy after 12 months  is a complex, highly debilitating inflammatory disease of the central nervous system (CNS) and represents the most common cause of neurological disability in young adults . Most of2 months , demonst6 cells/flask) for 24 h. The day after, proliferation medium was changed to DMEM supplemented with 1% antibiotics, 200 mM L-Glutamine, 100 mM Sodium Pyruvate (Thermo Fisher Scientific), 10% FCS and 0.3% N2  . iPSC cl 0.3% N2 . AstrocyHuman iAstrocytes were incubated with 100 nM Fingolimod , 100 nM Siponimod  or 1 μM NIBR-0213 (Merck) or vehicle (PBS or DMSO max 0.4% v/v) for 1 hour. Cells were then treated with IL1β , IL17  or S1P . Incubation times were 1 h for S1P1 internalization assay, 30 min for NFκB assay, 1, 2 or 4 h for Nrf2 assay or 24 h for glutamate transporter assay. Cells were then processed for immunofluorescence and stained with appropriate primary antibodies. For the generation of astrocyte conditioned media iAstrocytes were pre-incubated with drugs, and then exposed to the inflammatory stimuli for 8 h. Astrocyte medium was replaced with fresh neuronal medium and, after additional 24 h culture, supernatants were collected, centrifuged to remove cell debris, and stored at −80°C. Before addition to primary neurons, astrocyte supernatants were diluted down to 1:4 with medium.Total RNA was extracted by Tri Reagent Solution (Thermo Fisher Scientific) and reverse transcribed using random hexamer primers and Superscript III reverse transcriptase  following the manufacturers’ instructions. To remove contaminating DNA, RNA was treated with DNaseI enzyme (Thermo Fisher Scientific). As positive control, human peripheral blood mononuclear cells (PBMC) were isolated from a healthy donor as described in  and totaPrimary spinal neurons were obtained from 16-day-old Sprague Dawley rat embryos as described , 23. Brihttp://rsbweb.nih.gov/ij/) software was used for image analysis. To quantify nuclear NFkB and Nrf2, DAPI images were converted to 8-bit, and regions of interest (ROIs) were generated to select (DAPI positive) nuclei. Then ROIs were applied to the corresponding NFkB or Nrf2 images, fluorescence thresholds were fixed on the unstimulated condition, and the fraction of positive nuclei was assessed , blocked in PBS + 1% BSA (Merck) + 5% FCS and stained with primary antibodies. Then, cells were incubated with appropriate species-specific Alexa Fluor 488/594-conjugated secondary antibodies (Thermo Fisher Scientific), counterstained with 4′,6-diamidino-2-phenylindole  and mounted with fluorescent mounting medium (Agilent). The following primary antibodies were used: rabbit anti-GFAP (Agilent), mouse anti-nestin (Merck Millipore), mouse anti-vimentin (Abcam), rabbit anti-S100β (Abcam), rabbit anti-EDG1 (Santa Cruz Biotechnology), rabbit anti-EDG3 (Santa Cruz Biotechnology), mouse anti-IL1R (R&D), rabbit anti-IL17R (Santa Cruz biotechnology), rabbit anti- NFκB p65 (Abcam), rabbit anti-GLAST (Abcam), guinea pig anti-GLT1 (Merck Millipore), rabbit anti-Nrf2 (Abcam), mouse anti Neuronal Class III β-Tubulin (Covance). The following secondary antibodies were used: Alexa Fluor 488 donkey anti-rabbit IgG (H + L), Alexa Fluor 594 donkey anti-rabbit IgG (H + L), Alexa Fluor 488 donkey anti-mouse IgG (H + L), Alexa Fluor 594 donkey anti-mouse IgG (H + L), Alexa Fluor 488 goat anti-guinea pig IgG (H + L) . Fluorescence images were captured at fluorescence microscope (Leica DM5500B) or Leica TCS SP5 confocal laser-scanning microscope equipped with 40× oil objective. LASAF and LASX softwares were used for image acquisition, and ImageJ  or standard error of the mean (SEM) as indicated in figure legends. The exact number of independent experiments performed is reported in figure legends. Unpaired in vitro human cell model mimics the phenotype of the reactive glia cell in the human pathological tissue and may be responsive to inflammatory mediators. Human iAstrocytes did not express transcripts for S1P5, another possible target of BAF312 or FTY720 . Cells were cultured for several weeks and sampled at different time points to assess their morphology and phenotype. First, we observed a significant change in cell size and morphology during the differentiation process from iNPC, as human iAstrocytes clearly showed an increment in cell size and acquired the typical morphology of astroglia . Immunofr FTY720 .in vitro assays with our human cell system to study the effects of BAF312 and FTY720 on different astrocyte functions.We set up As these drugs were shown to induce rapid S1P1 internalization in rodent astrocytes , 25, we NFκB is a key transcription factor in cytokine and S1P signaling, and plays a pivotal role in the amplification of inflammatory and neurodegenerative processes , 26. We Maintenance of extracellular glutamate concentrations below neurotoxic levels is a critical function of glial glutamate transporters GLAST and GLT1 . Both trAstrocytes may react to the mediator S1P via S1P1 and S1P3. While FTY720 targets both receptors, BAF312 is selective for S1P1 only, leaving open the possibility of responding to S1P via S1P3. To check this hypothesis we used the NFκB assay to measure astrocyte activation in response to S1P and differentially interfered with S1P signaling via FTY720 or BAF312. As shown in All together these data indicate that triggering of inflammatory signaling cascades in glia cells may be prevented by S1P receptor modulators, and that S1P1 targeting via BAF312 is sufficient to directly dampen inflammatory activation via other S1P receptors and support physiological astrocyte functions.Activation of the transcription factor Nrf2 represents a key checkpoint for cellular antioxidant responses and its induction in astrocytes may confer neuroprotection during neuroinflammation . To asceTo test the overall impact of astrocyte mediators on neurons, we exposed human iAstrocytes to the drugs and then to the inflammatory mediators for 8 h, changed the medium to remove stimuli and collected the supernatants after a further 24 h culture. Astrocyte-conditioned media were then added to pure cultures of spinal neurons, which were then assessed for cell number and morphology via DAPI and β-tubulin stainings. While supernatants from non-treated (sCTRL) or FTY720- or BAF312- treated cultures  did not affect neuronal survival and network integrity, conditioned media from human iAstrocytes stimulated with the inflammatory factors  triggered robust degenerative responses characterized by neuronal loss and neurite fragmentation . Howeverin vitro evidence of neuroprotective effects of BAF312 via the human astrocyte during inflammation. In particular, we (i) generated human fibroblast-derived astrocytes to measure pharmacological effects of S1P receptor modulators; (ii) addressed the direct impact of BAF312 on NFkB activation and glutamate transporters in astrocytes; (iii) demonstrated the direct activation of Nrf2 by S1P receptor modulators; and (iv) performed side-by-side comparisons between BAF312 and FTY720 in all in vitro assays. Importantly, BAF312 action on the astrocyte was strong enough to inhibit neurodegeneration triggered by glial mediators generated during neuroinflammation.In this study, we provided In vivo findings from experimental neuroinflammation support the hypothesis of protective effects of BAF312 in the CNS  and FTY720 (which targets both S1P1 and S1P3). Moreover, treatment with the potent and selective S1P1 antagonist NIBR-0213 is sufficient to abolish NFκB activation.Although S1P signaling in astrocytes can be triggered by two (S1P1 and S1P3) receptors, S1P1 appears to play a major role in glial functions. In fact, S1P1-selective agonism reduces astrogliosis in experimental MS with similar efficacy to FTY720, which targets several S1P receptors . FurtherInternalization of S1P1 receptor following ligand binding has been observed in cells artificially overexpressing S1P1 , 46. ThiA pathological consequence of neuroinflammation is the dysfunction of glutamatergic transmission due to malfunctioning of glutamate transport. Glutamate is the main excitatory neurotransmitter in the CNS, but excessive glutamate accumulation in the synaptic and extra-synaptic spaces leads to neuronal death through a process called excitotoxicity . Under pAstrocyte activation is crucial in driving inflammation-induced neurodegeneration. In fact, S1P- or cytokine-activated astrocytes release factors that trigger neuronal death, as nitric oxide  or reactIn conclusion, our investigation highlights indirect neuroprotective properties for BAF312 via targeting S1P-S1P1 axis in glia cells.All datasets generated for this study are included in the article/The participants to the study provided their written informed consent approved by the Ethics Committee of Ospedale San Raffaele, Milan.in vitro experiments and statistical analyses. AD performed the iAstrocyte differentiation from human iNPCs. FR and LO generated and provided the human iNPCs. EC and CF analyzed the data and wrote the manuscript. GC and GM discussed the project and results. GM provided reagents. CF conceived and designed the experiments. All authors read and approved the submitted version.EC and CB performed GC received consulting and speaking fees from Novartis, Teva Pharmaceutical Industries Ltd., Teva Italia Srl, Sanofi Genzyme, Genzyme Corporation Genzyme Europe, Merck KGgA, Merck Serono SpA, Celgene Group, Biogen Idec, Biogen Italia Srl, F. Hoffman-La Roche, Roche SpA, Almirall SpA, Forward Pharma, Medday, and Excemed. CF received grant support from Novartis and FISM related to the work. The remaining 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.


Dear Editor,2 and chondroblastoma3, documented the transcriptomic reprogramming through the alterations of histone modifications. We recently reported the identification of a novel cancer associated mutation, the H2BG53-to-D in pancreatic ductal adenocarcinoma (PDAC)4. We showed that the H2BG53D mutation weakens the interaction between nucleosomal DNA and histone octamer, subsequently enhances transcription in vitro. We further showed that cells expressing the G53D mutant H2B acquired oncogenic phenotypes in our CRISPR-Cas9 knock-in model. However, the mechanism by which H2BG53D mutation promotes PDAC remains unknown.Histones are essential proteins in compacting genomic DNA and regulating gene expression. Previous studies on histone H3 oncohistones in pediatric brain cancers4. We identified 280 upregulated and 262 downregulated genes  when compared with the randomly shuffled peaks with the same length of G53D peaks  to arrest transcription elongation by RNA Pol II (Fig. ANXA3 primary transcript was reduced significantly in both wildtypes and H2BG53D mutants upon DRB treatment, but gradually increased after released from DRB inhibition (Fig. ANXA3 transcripts were found at all tested exon–intron junctions along the gene in time course experiments in the two H2BG53D clones, indicating increased transcription elongation on ANXA3. Conversely, SNAP47, a gene that does not have specific FLAG-H2BG53D enrichment, showed comparable recovery after DRB treatment. Together, these results suggested that the H2BG53D plays a direct role in elevating the transcription of ANXA3.The G53D-H2B occupancy at RUN Fig.  and confRUN Fig.  as well RUN Fig. . The ele II Fig. . By RT-qion Fig. . ImportaANXA3 by shRNAs reduced the cell migration abilities in both wild-type and H2BG53D clones, when compared with their respective non-targeting controls (Fig. ANXA3 expression had poor overall survival (Supplementary Fig. ANXA3 expression (Supplementary Fig. We performed oncogenic assays and found that depletion of ols Fig. . However4, we propose a model in which the H2BG53D mutation weakens the interaction between nucleosomal DNA and histone octamer, elevates the expression of cancer associated genes and leads to enhanced oncogenic properties in PDAC (Fig. Together with the findings reported earlier by usDAC Fig. . Our worSupplementary Information


S100A8/S100A9, a secreted cytokine, also acts in the nucleus as a transcriptional coactivator to promote cellular transformation. Cytokines are extracellular proteins that convey messages between cells by interacting with cognate receptors at the cell surface and triggering signaling pathways that alter gene expression and other phenotypes in an autocrine or paracrine manner. Here, we show that the calcium-dependent cytokines S100A8 and S100A9 are recruited to numerous promoters and enhancers in a model of breast cellular transformation. This recruitment is associated with multiple DNA sequence motifs recognized by DNA binding transcription factors that are linked to transcriptional activation and are important for transformation. The cytokines interact with these transcription factors in nuclear extracts, and they activate transcription when artificially recruited to a target promoter. Nuclear-specific expression of S100A8/A9 promotes oncogenic transcription and leads to enhanced breast transformation phenotype. These results suggest that, in addition to its classical cytokine function, S100A8/A9 can act as a transcriptional coactivator. Cytokines are a large and diverse group of small proteins that are important in many biological processes, such as immune responses, inflammation, and cancer. By conveying messages between cells by interacting with their cognate receptors at the cell surface, cytokines are a critical aspect of autocrine, paracrine, and endocrine signaling. Upon cytokine binding, some surface receptors activate the Janus kinase–signal transducer and activator of transcription (JAK-STAT) signaling pathway to regulate gene transcription  are calcium-binding proteins that form homodimeric, heterodimeric, and higher-order complexes that are essential for their biological activities  . These hybrid proteins localize to the nucleus , and theTo investigate the role of nuclear S100A8/A9, we performed ChIP-sequencing (ChIP-seq) using stable cell lines expressing Flag-tagged proteins at comparable levels of endogenous proteins in the absence of tamoxifen . The nucR = 0.88) between S100A8 and S100A9 binding levels . Polymerase II (Pol II) occupancy at all these three groups of genes is higher in the transformed condition as compared with the nontransformed condition . Genes wP = 4.4 × 10−11 by Fisher’s exact test; We analyzed the relationship between S100A8/A9 binding and gene expression upon transformation by comparing RNA expression levels of genes whose regulatory regions are bound by S100A8/A9 with those that are unbound. In both nontransformed and transformed conditions, the S100A8/A9 bound genes are expressed at higher levels than the unbound genes . In addiP < 2.2 × 10−16; Fisher’s exact test) with contributions from ONG (P = 2 × 10−8), TSG (P = 2 × 10−14), and OncoTSG enrichment (P = 6 × 10−6). The distinction between cancer and noncancer genes is also observed when considering S100A8/A9 target sites showing the highest level of inducible binding upon transformation , protein binding, mRNA processing, cytokine secretion, cell adhesion, and cell cycle (fig. S4). In addition, we addressed the relationship between S100A8/A9 target genes and human cancer gene annotations from UniProt . Notablyormation . At targormation .To examine the function of nuclear S100A8/A9, we measured the expression of ONG, TSG, and OncoTSG genes (six genes for each class), whose promoters and enhancers are bound by S100A8/A9 in cells expressing S100A8 or S100A9 derivatives with a nuclear localization signal (NLS) (fig. S5). When considered as a class, the ONGs show increased expression upon ectopic expression of either S100A8 or S100A9, either in the presence or absence of tamoxifen . In contAs S100A8/A9 does not have intrinsic DNA binding activity, they are presumably recruited to genomic sites by association with specific DNA binding transcription factors. To identify these transcription factors, we performed de novo motif searches in S100A8/A9 binding sites in promoter, enhancer, and repressed regions. We identified ETS (Erythroblast Transformation Specific), KLF (Kruppel-Like Factor)/SP1 (Specificity Protein 1), THAP11 (THAP domain Containing 11), AP-1, CREB (cyclic adenosine monophosphate response element–binding protein), and ZBTB33 (Zinc finger and BTB domain containing 33), motifs enriched in promoter peaks; AP-1, CTCF (CCCTC-binding Factor), CEBP (CCAAT/Enhancer-Binding Protein), and STAT3 motifs enriched in enhancer peaks; and CTCF motif enriched in repressed peaks . Using sFor each motif or selected combination of motifs located within 50 base pairs (bp) of the S100A8/A9 peak summits, we determined the fold change of S100A8/A9 binding at that site during transformation (tamoxifen:ethanol ratio). At promoters, fold increases in S100A8/A9 binding observed at sites with ETS, KLF/SP1, THAP11, CREB, or ZBTB33 motif alone or in combination are subtle, although statistically significant when compared against target sites lacking any of the listed motifs . HoweverTo examine more directly whether S100A8/A9 behaves like a coactivator, we first performed coimmunoprecipitation experiments in nuclear extracts from which nucleic acids were degraded. As expected from the motifs enriched among S100A8/A9 target sites, S100A9-Flag immunoprecipitates FOS, STAT3, C/EBPβ, and CTCF, but not SNAI1 . In accoTo examine whether S100A8/A9 has an autonomous transcriptional activation function, we fused the individual full-length proteins to the DNA binding domain of the yeast Gal4 proteins. When compared with the Gal4 DNA binding domain alone, the resulting fusion proteins confer about threefold higher levels of transcription from a Gal4-based reporter construct . These oCytokines are secreted molecules that bind and activate cell surface receptors, thereby transducing extracellular signals to effector proteins that regulate gene expression and other biological events. Many cytokines are also found in the nucleus and can be associated with chromatin, but the nuclear functions of cytokines are poorly understood. In two cases, S100A9 and interleukin-33, ChIP analysis of an individual gene has suggested that these cytokines could associate with a specific genomic region. Here, we show that S100A8/A9 associates specifically with numerous genomic regions even though the cognate receptors are absent from the nucleus. We provide evidence that S100A8/A9 functions as a transcriptional coactivator and show that nuclear-localized S100A8/A9 increases transformation efficiency.Multiple lines of evidence support the conclusion that S100A8/A9 can act as a transcriptional coactivator. First, S100A8/A9 binding sites occur primarily in promoters and enhancers, and binding is associated with active chromatin regions and Pol II transcription. Second, most DNA sequence motifs associated with S100A8/A9 binding are recognized by transcriptional activator proteins, with the level of binding being increased at motifs of activators and decreased at motifs of CTCF, an insulator protein associated with repression. Third, in nuclear extracts, S100A9 coimmunoprecipitates and, hence, interacts (directly or indirectly) with proteins that recognize the motifs enriched among genomic sites bound by S100A8/A9. Fourth, nuclear-specific expression of S100A8/A9 increases transcription of many genes tested, with a preference for ONGs. Fifth, S100A8 and S100A9 can stimulate transcription from a reporter construct when artificially recruited by a heterologous DNA binding domain.There are several possible mechanisms, not mutually exclusive, by which S100A8/A9 could function as a transcriptional coactivator upon recruitment to specific genomic sites. The artificial recruitment experiment  suggestsS100A8/A9 is up-regulated and functionally important in many forms of human cancer  were washed with phosphate-buffered saline (PBS) and treated with trypsin. The trypsinized single cells were resuspended in the upper agar phase , which was layered onto the lower agar phase (growth medium containing 1.0% agar). The solidified agar was covered with growth medium, which was changed every 3 days. Colonies (>0.01 mm2) were counted with ImageJ after 30 days.MCF-10A cells expressing ER-Src (MCF-10A–ER-Src cells) were grown in DMEM/F-12 (Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12) with 5% charcoal-stripped fetal bovine serum (Sigma-Aldrich) and supplements as previously described . For NLS-GFP, GFP with triplicated NLS (sequence copied from Invitrogen’s pCMV/myc/nuc) at both the N- and C-terminal sides was cloned from pEGFP-N1 (provided by R. Pomerantz from Temple University School of Medicine) using ultramer primers and then was subcloned into pLHCX. For NLS-GFP-A8 or NLS-GFP-A9, S100A8 or S100A9 open reading frame was subcloned to the C-terminal side of NLS-GFP in pLHCX.Retrovirus was produced by transfection of corresponding retroviral vector in HEK293T cells carrying pCL-Ampho (provided by V. Sukhatme). Retroviral supernatant from HEK293T cells was collected at 48 and 72 hours after transfection, filtered through 0.45-μm filter, and used to infect exponentially growing target cells in the presence of polybrene . Cells were passaged after 72 hours of infection, after which drug selection was performed with hygromycin B . For nuclear-specific expression of S100A8 or S100A9, cells expressing NLS-GFP, NLS-GFP-A8, or NLS-GFP-A9 were further selected by a cell sorting flow cytometer  based on GFP signals, and GFP levels for all groups of cells were kept similar on average during the selection.Firefly and Renilla luciferase activities were measured with the Dual-Glo Luciferase Assay System (Promega). Luciferase activity is calculated as Firefly normalized by Renilla.Gal4 DNA binding domain (DBD)–based constructs were generated by replacing the NLS-GFP region of NLS-GFP, NLS-GFP-S100A8, or NLS-GFP-S100A9 with Gal4DBD cloned from pCAG-GBP1-10gly-Gal4DBD . For the Gal4-based reporter assays, Gal4-based constructs (1 μg), pUAS-luc2 , and pCMV-RL  were electroporated together into human breast MCF7 cells by the 4D-Nucleofector System with the SE Cell Line 4D-Nucleofector X Kit (Lonza). Forty-eight hours after the electroporation, g for 10 min. For cytosol/nuclear fractionation, cells were incubated in buffer A  for 15 min, followed by mixture with Triton X-100  for 1 min, and the cytosol fraction was cleared by centrifugation at 16,000g for 5 min. The nuclear pellet was briefly washed in buffer B , dissolved in 1× RIPA buffer, and briefly sonicated, and the nuclear fraction was cleared by centrifugation at 20,000g for 10 min.Cells were lysed in 1× radioimmunoprecipitation assay (RIPA) buffer  supplemented with protease inhibitor cocktail (Sigma-Aldrich), followed by brief sonication, and the lysate was cleared by centrifugation at 20,0002, 0.4% NP-40, 25% glycerol, and protease inhibitor cocktail) for 1 hour, diluted to 100 mM NaCl strength with buffer D , and treated overnight with Benzonase (Millipore), and the nuclear extract was cleared by centrifugation at 20,000g for 10 min. Coimmunoprecipitation and Western blot analyses were performed as previously described –fluorescein isothiocyanate and anti-mouse IgG–Texas Red. Clinical immunohistology data of S100A9 and S100A8 were obtained from The Human Protein Atlas database , and S100A9 (HSC.RNAI.N002965.12.1) were obtained from Integrated DNA Technologies. siRNA-mediated knockdown was performed as previously described (20 and SuperScript III (Invitrogen). Analysis of relative RNA expression was performed by quantitative polymerase chain reaction in real time. The relative RNA levels of indicated genes were normalized to the average RNA level of two control genes rRNA18S and FAM50A in the same samples. Data presented were obtained from four independent experiments.RNA was extracted with QIAzol (QIAGEN) and precipitated with isopropanol. cDNA was generated from 3.5 μg of total RNA template using oligo(dT)Ribosome profiling and RNA sequencing (RNA-seq) experiments were described previously  for sonication and subsequent immunoprecipitation . The sheared chromatin fragments were enriched around 200 to 300 bp. For ChIP and corresponding chromatin input samples, DNA was isolated and used to prepare libraries, which were sequenced on Illumina NextSeq 500 or HiSeq 2000. Other ChIP-seq  and DNase sequencing (DNase-seq) experiments mentioned in this study were performed in transformed or nontransformed cells as previously described , 1.5 mM disuccinimidyl glutarate (DSG), and 1.5 mM disuccinimidyl suberate (DSS) in PBS] for 45 min at room temperature. Then, protein-DNA cross-linking with 1% formaldehyde (methanol free) was performed for 15 min at room temperature, and the reaction was quenched by 125 mM glycine. For Pol II ChIP, standard formaldehyde cross-linking was performed. After fixation, nuclei were isolated and dissolved in sonication buffer  peaks called by MACS2 were identified in at least two biological replicates; (iii) at least five raw reads were aligned at the peak region. Depending on the comparisons indicated, peak regions from different datasets were merged if overlapping at least 1 bp. Confident Pol II ChIP-seq peaks were defined as MACS2 paired-end peak-calling P value ≤1 × 10−8 against genomic background of 1-Mb genomic region around. In ChIP-seq data of histone modifications and DNase-seq data, confident sites were defined as previously described  in hg19 refGene collection were downloaded from UCSC  Genome Browser. Classification of S100A8/A9 peaks was determined by patterns of histone modifications. For promoter-bound S100A8/A9 peaks, genes were assigned by mapping the nearest annotated TSS to the peak summit if the peak summit was within 1500 bp upstream and 250 bp downstream of the TSS. If the peak summit was located downstream of the TSS even though within the 250-bp range, we further required that the distance of the peak summit to TSS be <5% of the gene length because the promoter region of a given gene should be around TSS but not largely extended to gene body region. For enhancer-bound S100A8/A9 peaks, genes were assigned either if their TSSs were within 50 kb the of peak summit or if the gene regions overlapped with the peak summit.Promoter proximal was defined as ±250 bp around TSS, and gene body was defined as the remaining length of gene. Calculation of Pol II density as reads per million per kilobase (RPM/kb) was carried out in promoter proximal and gene body of each gene longer than 251 bp. The Pol II density ratio of promoter proximal and gene body was defined as pausing index or traveling ratio  were compared with JASPAR CORE collection of vertebrate motifs (De novo motifs were searched with HOMER (We collected and consolidated annotations of human cancer genes from the following sources: (i) 420 human cancer genes from UniProt . Molecular subtypes of breast cancer were defined by PAM50 classification  online server with default knowledgebase mapping  and are accessible through GEO accession number GSE155421.


These mutations were in the polymerase PA subunit and acted cooperatively with the E627K mutation in the PB2 polymerase subunit to provide higher replication in contemporary clade 2.2.1.2 viruses than in ancestral clade 2.2.1 viruses. These data indicated that ongoing clade 2.2.1 dissemination in the field has driven PA mutations to modify viral replication to enable host range expansion, with a higher public health risk for humans.Clade 2.2.1 avian influenza viruses (H5N1) are unique to Egypt and have caused the highest number of human H5N1 influenza cases worldwide, presenting a serious global public health threat. These viruses may have the greatest evolutionary potential for adaptation from avian hosts to human hosts. Using a comprehensive phylogenetic approach, we identified several novel clade 2.2.1 virus polymerase mutations that increased viral replication  in vitro in human cells and in vivo in mice compared to ancestral clade 2.2.1 viruses. The PA mutations acted cooperatively to increase viral polymerase activity and replication in both avian and human cells, with the effect being more prominent in human cells at 33°C than at 37°C. These results indicated that PA mutations have a role in establishing contemporary clade 2.2.1.2 virus infections in poultry and in adaptation to infect mammals. Our study provided data on the mechanism for PA mutations to accumulate during avian influenza virus evolution and extend the viral host range.Adaptive mutations and/or reassortments in avian influenza virus polymerase subunits PA, PB1, and PB2 are one of the major factors enabling the virus to overcome the species barrier to infect humans. The majority of human adaptation polymerase mutations have been identified in PB2; fewer adaptation mutations have been characterized in PA and PB1. Clade 2.2.1 avian influenza viruses (H5N1) are unique to Egypt and generally carry the human adaptation PB2-E627K substitution during their dissemination in nature. In this study, we identified other human adaptation polymerase mutations by analyzing phylogeny-associated PA mutations that H5N1 clade 2.2.1 viruses have accumulated during their evolution in the field. This analysis identified several PA mutations that produced increased replication by contemporary clade 2.2.1.2 viruses IMPORTANCE Clade 2.2.1 avian influenza viruses (H5N1) are unique to Egypt and have caused the highest number of human H5N1 influenza cases worldwide, presenting a serious global public health threat. These viruses may have the greatest evolutionary potential for adaptation from avian hosts to human hosts. Using a comprehensive phylogenetic approach, we identified several novel clade 2.2.1 virus polymerase mutations that increased viral replication in vitro in human cells and in vivo in mice. These mutations were in the polymerase PA subunit and acted cooperatively with the E627K mutation in the PB2 polymerase subunit to provide higher replication in contemporary clade 2.2.1.2 viruses than in ancestral clade 2.2.1 viruses. These data indicated that ongoing clade 2.2.1 dissemination in the field has driven PA mutations to modify viral replication to enable host range expansion, with a higher public health risk for humans. The H5N1 subtype of highly pathogenic avian influenza (AI) viruses was first reported in Egypt in 2006 and has been enzootic since then , 4. The Influenza virus replication is mediated by a trimeric polymerase complex composed of the viral PB2, PB1, and PA proteins. The polymerase complex binds viral RNA (vRNA) and NP to form a ribonucleoprotein. For human infections, the polymerase complex needs to adapt to replicate in human cells through gene mutation or reassortment , 8. In t9–13–In contrast to PB2, there has been limited information about human adaptation mutations in PB1 and PA , 19. Rec20–AI viruses circulating in Egypt have a characteristic feature: they have accumulated representative human adaptation mutations during their dissemination in bird populations in the field. The Egyptian H5N1 clade 2.2.1 strains in poultry species generally carry the PB2-E627K mutation , 23, 24.In this study, we investigated the phylogeny of mutations in PA, the polymerase subunit that increased the polymerase activity of contemporary H5N1 clade 2.2.1.2 viruses, and the effect of these mutations on the biological properties of the H5N1 virus, including its adaption to infect mammals. Our results provide data for understanding how PA mutations accumulate during AI virus evolution to affect the viral host range.In our previous study, we isolated H5N1 clade 2.2.1.2 viruses from chickens in Egypt in 2013 and found that one of the viruses, A/chicken/Egypt/CL69/2013 (denoted EG13 here), replicated at a high level in human cells and with high pathogenicity in mice , which mTo determine the effect of each polymerase subunit on EG13 polymerase activity, the activity of polymerase complexes, in which the polymerase subunits were reassorted between the EG13 and D1 viruses, was measured by minigenome assays in 293T and DF-1 cells. In the EG13 background, PA from the D1 virus (denoted D1/PA here) significantly decreased the viral polymerase activity in both human cells and avian cells, but the D1/PB2 reassortant had only a minimal increase in polymerase activity  to C. InTo investigate the effect of each subunit on complex formation of the reassorted polymerases in this study, the reassortant polymerase complexes were analyzed by Western blotting with and without immunoprecipitation of PB2-Flag. Similar amounts of PA in the polymerase complexes were coprecipitated with PB2  and H, cTo trace the evolution of the PA gene in clade 2.2.1 viruses, we analyzed the phylogeny of PA genes in ancestral clade 2.2.1 viruses  and contemporary viruses  . The virEach of the substitution mutations that were identified in the PA gene was introduced into the EG13/PA gene as a reverse mutation and into the D1/PA gene as a forward mutation. All these mutants had similar levels of PA expression  to D, inTo investigate the effect of the PA mutations on polymerase activity, we measured the activity of the polymerase complexes, each with a PA subunit carrying one of the mutations, using minigenome assays in DF-1 cells and 293T cells. In the EG13 background, the G18E, R388S, and E448A reverse mutations each decreased the polymerase activity, although the decrease was greatest with the reassorted D1/PA (denoted EG13-D1/PA here) that was included as a control  to C. ThBased on the effects of the single mutations, we selected three PA substitutions  for further study to investigate whether they might act cooperatively on polymerase activity. Multiple mutations were combined in their putative phylogenetic order, i.e., the G18E, R388S, and E448A reverse mutations and the A448E, S388R, and E18G forward mutations. The R388S/E448A reverse mutations were also included in this study because they were associated with the high polymerase activity in ancestral clade 2.2.1 viruses described below.The three reverse mutations acted cooperatively to decrease EG13 polymerase activity by >3 logs in human cells  to C, wiG18E/R388S/E448A here) had the lowest progeny virus yield in both cell lines, in particular by >2 logs in human cells at early times postinfection at 33°C, with the R388S/E448A mutation producing the greatest effect.We investigated the effect of the PA-A448E, -S388R, and -E18G substitution mutations on the replication kinetics of clade 2.2.1 viruses using recombinant viruses carrying these mutations. Avian DF-1 cells and human airway epithelial Calu-3 cells were infected with EG13 viruses carrying the PA reverse mutations, and the viral replication kinetics was measured. In both cell types, the PA mutants produced lower progeny virus yields than wild-type EG13 virus  to C, wiA448E/S388E/E18G had the greatest progeny virus yield in both cells: D1/PA-wt < A448E < A448E/S388R < A448E/S388R/E18G, which was comparable to that of wild-type EG13.The effect of the forward PA mutations in the D1 background on viral replication was also studied. For these studies, we used a recombinant D1 virus carrying a reassorted polymerase complex composed of D1/PA with the other genes  from EG13 (denoted D1/PA virus here) to assess PA-specific effects on viral replication. This experimental strategy was chosen since previous studies suggested that influenza virus polymerase genes may coevolve in the field , 27, 28.Together, these results suggested that during the evolution of contemporary clade 2.2.1.2 viruses, the A448E and S388R mutations acted cooperatively to provide a more fit phenotype for replication in human cells at a lower temperature and that the E18G mutation produced strain-specific higher replication in EG13.in vitro effects of the A448E/S388R/E18G mutation to in vivo infections, BALB/c mice were inoculated intranasally with serial dilutions of D1/PA-wt virus and D1/PA viruses carrying representative forward mutants  or with EG13-wt virus and the EG13G18E/R388S/E448A reverse mutant. The body weight and survival of each infected mouse were monitored for 2 weeks.To assess the relevance of the 3 focus-forming units (FFU) of wild-type D1/PA virus caused slight weight loss in infected mice, and all 6 of the infected mice survived (50) was 2.5 × 104 FFU, which was in agreement with the MLD50 of ancestral clade 2.2.1 viruses reported previously , and virus titers in the lungs were measured by focus-forming assays. Virus titers in the infected mice were consistent with their polymerase activity and replication kinetics in human cells in vitro (A448E/S388R(/E18G) being comparable to those of contemporary EG13  or EG13-wt. The degree of pathological change corresponded to the amount of H5 antigen detected by immunohistochemistry in the alveolar areas of the lungs , A448E, S388R, and E18G were located on the outer side of the polymerase complex (6QPF as the modeling template), our model indicated that PA-A448E, located on helix α16, created a hydrogen bond to PA-N444 , we generated models of the D1/PA virus trimeric polymerase complex structure. The influenza virus polymerase complex is inherently flexible and can adopt several conformations depending on the presence or absence of specific viral RNA –32. Our – complex  and B. P complex . There w complex . PA-E18  complex . In the  PA-N444 , presumaAdaptive mutations and/or reassortments in the avian influenza virus polymerase complex are major factors enabling the virus to overcome the species barrier between birds and humans , 7, 34. Recently, it has become apparent that PA plays a role in the host adaptation of AI viruses , 38, wit39–44–47–The H5N1 clade 2.2.1 viruses that are unique to Egypt generally carry the human adaptation PB2-E627K mutation in the field and have no history of reassortment during long-term dissemination. This gave us the opportunity to identify novel human adaptation mutations in the polymerase complex, other than PB2-E627K, that have accumulated during viral evolution in nature, especially in PA and PB1.In this study, we traced the evolution of the PA gene in clade 2.2.1 influenza viruses and found that multiple PA mutations accumulated during viral evolution in the field. Several of these PA mutations  acted cooperatively to increase polymerase activity and viral replication in both avian and human cells to7 andThe PA protein is structurally separated into N-terminal (PA-N) and C-terminal (PA-C) domains, which are connected by a narrow linker (residues 257 to 276) . PA-N r–53, and –The PA-S388R and -A448E mutations, which were identified in this study as main contributors to the higher polymerase activity of contemporary clade 2.2.1.2 viruses, were both localized in PA-C. In the crystal structure, PA residue 388 lies within the PA arch domain, which forms an integral part of the 5′ vRNA binding site together with the adjacent PB1 β-hairpin domain . In our PA residue 448 is in helix α16 in the polymerase complex structure and is one of the key residues that make direct contact with the Pol II C-terminal domain for transcription . Helix αThe effect of PA-E18G was different from those of the above-described two mutations. PA-E18G is in PA-N and was characterized here as an auxiliary mutation for increasing polymerase activity and unique to an EG13 virus strain. PA-E18G was found in H1N1 pdm09 virus during a marmoset infection (nonhuman primate model)  and duriThe number of human H5N1 cases has declined considerably in Egypt since 2017. Nonetheless, the persisting circulation of clade 2.2.1 viruses in poultry poses an ongoing risk to humans . ContempNine-day-old embryonated chicken eggs were purchased from Shimizu Laboratory Supplies, Japan. All animal experiments were conducted in compliance with Japanese legislation  and guidelines under the jurisdiction of the Ministry of Education, Culture, Sports, Science, and Technology in Japan. Animal care, housing, feeding, sampling, observation, and environmental enrichment were approved by the Animal Experiment Committee of the Kyoto Prefectural University of Medicine .+ (BSL3+) at the Kyoto Prefectural University of Medicine. All studies with recombinant DNA were conducted under the relevant laws in Japan and approved by the Biological Safety Committee of the Kyoto Prefectural University of Medicine  after risk assessments were conducted by the Living Modified Organisms Committee of the Kyoto Prefectural University of Medicine and, when required, by the Ministry of Education, Culture, Sports, Science, and Technology of Japan.All experiments with live H5N1 viruses were performed at enhanced biosecurity level 3293T cells were obtained from the Riken BioResource Center Cell Bank. Human bronchial epithelial  cells and chicken fibroblast (DF-1) cells were obtained from the American Type Culture Collection. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum (FCS). AI viruses A/duck/Egypt/D1Br/2007 (denoted D1 here) and A/chicken Egypt/CL69/2013 (denoted EG13 here) are representative ancestral and contemporary strains of H5N1 clade 2.2.1, respectively. The details of the two viruses were described previously , 63. AllRecombinant viruses were generated using a plasmid-based reverse-genetics system in the EG13 and D1 genetic backgrounds as described previously , 24, 63.Renilla luciferase was also cotransfected to measure transfection efficiencies. The transfected cells were incubated at 33°C or 37°C and collected with cell lysis buffer at 24 h posttransfection. The firefly luciferase activities were expressed relative to the Renilla luciferase activity.293T and DF-1 cells were transfected with pCXN2 plasmids carrying the PB2, PB1, PA, and NP genes of EG13 or D1 and a human or chicken polymerase I-driven plasmid expressing firefly luciferase as described previously , 64. A phttp://www.gisaid.org). A phylogenetic tree was reconstructed using MEGA6 software for the neighbor-joining method with the EG13 and D1 nucleotide sequences. The confidence level for the phylogenetic tree was calculated by performing 1,000 bootstrap replicates.PA gene sequences of the H5N1 clade 2.2.1 virus strains, isolated in Egypt in 2006 to 2015, were obtained from the GISAID database . The PA proteins were detected with anti-influenza virus PA antibody (GeneTex) and horseradish peroxidase (HRP)-conjugated secondary antibody. The Amersham ECL Select Western blotting reagent was used for band visualization. The band intensities were quantified using Amersham Imager 600 analysis software .Calu-3 cells and DF-1 cells were seeded on a 24-well plate (90% confluent) and infected with the indicated viruses at multiplicities of infection (MOIs) of 0.03 and 0.003, respectively. After 1 h of incubation at 37°C, the cells were washed twice with phosphate-buffered saline (PBS), maintained in DMEM–F-12 medium containing 0.2% bovine serum albumin (BSA), and incubated at 37°C or 33°C. The supernatants were collected at the indicated times, and progeny virus titers were measured by focus-forming assays as described above.1 to 105 FFU) of viruses in PBS. The body weight and survival of each mouse were monitored daily for 2 weeks. Mice that lost more than 30% of their original weight within a few days were humanely euthanized. Lungs of mice infected with 103 FFU of virus were collected at 6 dpi, and virus FFU titers were assayed. Immunohistochemical staining of the viral H5 antigen was performed as described previously , under mixed anesthesia (medetomidine-butorphanol-midazolam), were inoculated intranasally with 50-μl samples of 10-fold serial dilutions . MOE was used for software, and most of the modeling conditions were described previously . Statistically significant differences between virus pairs were determined by analysis of variance (ANOVA) with Tukey’s multiple-comparison test.