Nearly half of the human genome is made of transposable elements (TEs) whose activity continues to impact its structure and function. Among them, Long INterspersed Element class 1 (LINE-1 or L1) elements are the only autonomously active TEs in humans. L1s are expressed and mobilized in different cancers, generating mutagenic insertions that could affect tumor malignancy. Tumor suppressor microRNAs are ∼22nt RNAs that post-transcriptionally regulate oncogene expression and are frequently downregulated in cancer. Here we explore whether they also influence L1 mobilization. We show that downregulation of let-7 correlates with accumulation of L1 insertions in human lung cancer. Furthermore, we demonstrate that let-7 binds to the L1 mRNA and impairs the translation of the second L1-encoded protein, ORF2p, reducing its mobilization. Overall, our data reveals that let-7, one of the most relevant microRNAs, maintains somatic genome integrity by restricting L1 retrotransposition. Human Long INterspersed Element class 1 (LINE-1) elements are expressed and mobilized in many types of cancer, contributing to malignancy. Here the authors show that the tumor suppressor microRNA let-7 targets the LINE-1 mRNA and reduces LINE-1 mobilization. However, the only TE that remains autonomously active nowadays is a non-Long Terminal Repeat (non-LTR) retrotransposon known as Long INterspersed Element class 1 (LINE-1 or L1), whose mobilization continues to impact our genome2. LINE-1s comprise >20% of our DNA1 but only about 80–100 of the ~500,000 L1 copies present in the average human genome are full-length elements that retain the ability to mobilize and are thus called Retrotransposition-Competent L1s (RC-L1s)3. RC-L1s belong to the human-specific L1Hs subfamily, are 6 kb long and encode two proteins (L1-ORF1p and L1-ORF2p) that are indispensable for retrotransposition4. However, ORF2p is expressed at a significantly lower level than ORF1p6, and these differences are thought to be controlled at the level of translation7. L1-ORF1p is a 40 kDa RNA binding protein with nucleic acid chaperone activity9, whereas L1-ORF2p is a 150 kDa protein with Endonuclease (EN) and Reverse Transcriptase (RT) activities11. RC-L1s mobilize by a “copy-and-paste” mechanism, involving reverse transcription of an RNA intermediate and insertion of its cDNA copy at a new site in the genome (reviewed in2). Briefly, retrotransposition starts with the transcription of a full-length RC-L1 bicistronic mRNA, which is exported to the cytoplasm and translated, giving rise to L1-ORF1p and L1-ORF2p that bind preferentially to the same L1 mRNA to form a ribonucleoparticle (RNP)12. The RNP gains access to the nucleus where retrotransposition occurs by a mechanism known as Target Primed Reverse Transcription (TPRT)14. During TPRT, the endonuclease activity of L1-ORF2p nicks the genomic DNA, and its reverse transcriptase activity uses the L1 mRNA as a template to generate a new copy of the element in a different genomic location. L1 can target all regions of the genome, but integration is locally dictated by the presence of a consensus sequence 5′-A/TTTT-3′, which is recognized by L1 endonuclease activity and allows annealing of L1 mRNA poly(A) to the target DNA16. Other non-autonomous retrotransposons such as Alu and SINE-R/VNTR/Alu (SVA) may hijack the L1-encoded proteins and be mobilized in trans18. Furthermore, L1-encoded proteins can sporadically generate pseudogenes using cellular mRNAs as templates19.Transposable elements (TEs) account for nearly half of the human genome20. Genomic alterations caused by L1 activity have resulted in several human disorders21. Among these alterations, new L1 insertions can disrupt a gene unit, induce changes in splicing patterns, or interfere with transcription (reviewed in refs. 2 and21). Remarkably, new L1 insertions accumulate not only during early embryogenesis and in the germline, being transmitted to the next generation, but also in cancer cells, which are characterized by genome instability33 (thoroughly reviewed recently in refs. 35). In fact, L1s are highly expressed and mobilized in a wide range of human epithelial cancers24, and high levels of L1 mobilization are found in lung and colorectal cancers29. Interestingly, several reports have shown that somatic L1 insertions can drive tumorigenesis and may even have initiated the tumor in normal cells36. Transcriptional control through methylation of the L1 promoter is one of the main defence mechanisms against L1 activity38, and it has been demonstrated that hypomethylation of specific RC-L1s is associated with retrotransposition in early tumorigenesis29. However, additional post-transcriptional mechanisms that silence and reactivate L1 in somatic normal and tumor cells are not completely understood yet.TEs can affect genome stability in several ways, including the accumulation of insertions and rearrangements40. Hundreds of bona fide miRNAs exist in humans and each of them is predicted to target many mRNAs41. Therefore, miRNAs could be influencing essentially all human developmental, physiological, and pathological processes39. In particular, overall miRNA dysregulation has been described in cancer43. Interestingly, it was previously shown that mouse embryonic stem cells (mESCs) lacking mature miRNAs (DGCR8 or Dicer knockout) accumulate LINE-1 mRNA46. Whereas the increase in LINE-1 mRNA levels in the absence of DGCR8 was attributed to reduced noncanonical functions of the Microprocessor, which cleaves stem-loops present in L1 elements45, it remains possible that miRNAs regulate L1 expression levels. Consistently, a previous study reported that miR-128 represses engineered L1 retrotransposition in cultured cells47. Thus, we hypothesized that some miRNAs could control L1 retrotransposition and that their misexpression in tumors could contribute to increased LINE-1 mobilization.MicroRNAs (miRNAs) are small RNAs that are loaded into the Argonaute (AGO) proteins to form the RNA-Induced Silencing Complex (RISC) and post-transcriptionally repress gene expressionTo test this possibility, we first analyze whole genome sequencing data from a panel of human lung tumor/normal pairs and miRNA expression data from the same tumor samples. Notably, we find that samples containing tumor-specific L1 insertions express reduced levels of several members of the tumor suppressor miRNA let-7 family, suggesting that this miRNA could influence retrotransposition in vivo. Indeed, we further demonstrate that let-7 binds directly to the L1-mRNA impairing L1-ORF2p translation, and reduces L1 retrotransposition in cultured tumor cells. Altogether, our results uncover a role for let-7 in maintaining genome integrity and provide mechanistic insight into how downregulation of let-7 miRNAs in tumors may unleash L1 activity, causing genome instability and driving tumor genome evolution.33. We selected all the samples (45 patients) for which whole-genome sequencing data from tumor and matched normal lung tissue, together with tumor miRNA-seq data, were available. We computationally identified tumor-specific somatic L1 retrotransposon insertions from whole-genome sequencing data using the MELT (Mobile Element Locator Tool) software48. Briefly, MELT detects Mobile Element Insertions (MEIs) by searching for discordant reads pairs and split reads that are enriched at genome positions containing new, non-referenced insertions48. First, to rule out possible biases produced by different coverage or quality of sample pairs, we analyzed the polymorphic germline L1 insertions identified by MELT both in tumor and normal tissue. We excluded four samples where this common reference polymorphic calls number was abruptly reduced under 10% after filtering. In the 41 selected samples, the number of polymorphic L1 insertions found in tumor/normal DNA pairs was similar and at least 63% of them were common to both DNAs  from The Cancer Genome Atlas (TCGA), as endogenous L1s are known to retrotranspose efficiently in this tumor type50  < 0.01 . Thus, even though we cannot rule out a possible bias in the analysis due to sample variability and the limited number of cases available, these data suggest that let-7 and miR-34a might control the accumulation of new L1 insertions in human lung cancer samples. Next, we used SQuIRE (Software for Quantifying Interspersed Repeat Elements)55 to quantify L1Hs expression in RNA-seq data from these tumor samples, available in TCGA. As expected, L1Hs RNA levels were significantly increased in samples with tumor-specific L1 insertions  or absence (0) of tumor-specific L1 insertions Fig. . Using a50 Fig. . Interes.01 Fig. . This co.01 Fig. . Althougons Fig. . As a co23 . Remarkably, the expression levels of let-7 family members (let-7a and let-7e) and miR-34a were again significantly reduced in those tumors containing tumor-specific L1 insertions when the 26 miRNAs related to lung cancer were analyzed  as well as when all the miRNAs expressed in lung were included  with miRNA mimics or inhibitors. Briefly, in this assay, cells are transfected with a plasmid containing an RC-L1 tagged with a reporter cassette . Strikingly, overexpression of miR-34 did not affect L1 mobilization or cell clonability in this assay  and zebrafish L2-1 and L2-2 (L2 clade). Structures of the different LINEs are shown in the left panel of Fig. The aforementioned regulation could occur either by a direct interaction between let-7-guided RISC and L1 mRNAs, or by an indirect effect, since let-7 could be regulating any host factor involved in the multiple steps of the retrotransposition cycle39, although 5′UTR and coding sequence (CDS) binding sites have been described and validated62. Thus, to find out where the putative let-7 binding site was located in L1 mRNA, we performed the same retrotransposition assays but using an engineered human RC-L1 (L1.3) lacking either the 5′or the 3′UTR . For this, we used a human embryonic teratocarcinoma cell line (PA-1), characterized by high levels of endogenous LINE-1 mRNA and L1-ORF1p65 and very low levels of let-7 miRNAs  and 4596-4616 (bs2), respectively, in the consensus L1Hs sequence . In order to validate them, five tandem copies of each binding site (bs) were cloned in the 3′UTR region of the Renilla luciferase (Rluc) gene in the psiCHECK-2 vector, which also encodes a Firefly luciferase (Fluc) gene to correct for transfection efficiency , between the RT and Cysteine-rich domains of this protein  we generated a mutant sequence . However, the fact that mutating this binding site reduced but not abolished the effect of let-7 in L1 mobilization suggests that additional mechanisms mediated by let-7 may work to restrict human L1 retrotransposition. Overall, these results suggest that there is at least a functional let-7 binding site in the ORF2 region of human L1 mRNA.To further corroborate the functionality of “bs2rh” in the context of retrotransposition, we generated an allele mutated RC-L1 construct containing a mutated “bs2rh” site  were reduced , pSA500. Strikingly, we observed an increase in ORF2p upon let-7 depletion and a decrease upon let-7 overexpression in HeLa cells , the binding site (“bs2rh”) and a modified bs2 that contains a canonical 8-mer site for let-7 (“8mer”)  and the inefficient translation of ORF2p-mCherry7, we obtained an insufficient number of double positive cells to enable a quantitative analysis by microscopy. Therefore, we turned to a more sensitive and quantitative approach: flow cytometry. We co-transfected pVan583 with let-7 inhibitor in HeLa cells and analyzed EGFP+ cells (i.e. >3500 cells per sample). Notably, we found that depleting let-7 led to an increase in the number of mCherry+ cells in the EGFP+ population suggesting an increase in the synthesis of L1-ORF2p-mCherry  where tumor miRNAs expression data was available were retrieved for lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). High coverage (28-95x) WGS files aligned to hg19 from primary tumor and solid tissue normal samples, and miRNA gene quantification files from primary tumor were downloaded for LUAD (17 patients) and LUSC (28 patients).Both WGS , miRNA expression quantification and RNA-seq raw sequencing data files from TCGA were obtained from the Genomic Data Commons (GDC) Legacy Archive using the GDC Data Transfer Tool48. To discard possible sequencing artifacts, candidate somatic insertions were further filtered including calls supported with a minimum of three split-reads, with the highest accuracy assessment for breakpoint detection and passing all internal filters (MELT parameters ASSESS = 5 and FILTER = PASS). Polymorphic insertion calls were found using a curated database included in TEBreak software (https://github.com/adamewing/tebreak) and excluded from final results.Putative somatic LINE-1 insertion calls for both normal tissue (NT) and primary tumor (PT) were obtained using MELT version 2.1.5Several quality values were checked as a measure of filtering effects on original (unfiltered) MELT results. First, somatic insertions found in NT alone, and NT and PT simultaneously were expected to be zero, and only a maximum of one insertion was allowed for these values. All samples passed this additional filtering.Furthermore, polymorphic L1 insertions after MELT filtering were controlled, requiring that a similar number was found for PT and NT samples, and that this number were uncorrelated with sample coverage. Four samples in LUSC  and LUAD  were excluded from analysis because only a low number (<10%) of polymorphic insertions passed all filters.Filtered LINE-1 calls were considered tumor somatic insertions if detected in primary tumor filtered results and absent in unfiltered solid normal tissue insertion set within a range of 100 bp.Samples were divided into two groups depending on whether putative somatic insertions were or were not found in the primary tumor. Only miRNAs with medium-high expression (over 100 reads per million (RPM) mapped reads) were considered. For some of the analysis, expression of specific miRNAs known to be involved in the development and progression of lung cancer was analyzed.t test adjusted by FDR = 1%. Results were confirmed using a rank-sum test. RPMs were normalized to the highest expression value of each miRNA to enable visualization of all miRNAs in the same graph. Tumor-suppressor miRNAs and oncomiRs related to lung cancer used for this analysis were described in a recent revision50. As a control, L1 insertion numbers were randomly reassigned to each sample and analysis was repeated. Moreover, analysis was done with L1 insertions determined by Helman and col. using Transpo-seq in lung and breast cancer samples obtained from TGCA as well23. Data processing and analysis were performed as described above.For each miRNA, outliers were discarded . Differentially expressed miRNAs were identified applying an unpaired two-tailed 55 (Software for Quantifying Interspersed Repeat Elements). SQuIRE quantifies expression at the subfamily level. It outputs read counts and fragments per kilobase transcript per million reads FPKM. Linear correlation between L1Hs RNA levels and miRNAs expression was calculated using Pearson Correlation Coefficient in GraphPad Prism 6.To analyze global TE expression in RNA-seq experiments, we use SQuIRE79.HEK293T, PA-1, HeLa, and U2OS cells were originally obtained from ATCC and were provided by Drs Jose Luis Garcia-Perez  and John V. Moran . Lung cancer cell lines  were provided by Dr Pedro Medina . Stable Flp-In-293 cells expressing T7-tagged L1-ORF1p were previously generated for a different studyHEK293T, HeLa, U2OS, A549, and SK-MES1 cells were cultured in high-glucose Dulbecco’s Modified Eagle’s Media  supplemented with GlutaMAX, 10% fetal bovine serum  and 100 U/mL penicilin–streptomycin (Invitrogen).PA-1 cells were cultured in Minimal Essential Medium  supplemented with GlutaMAX, 10% heat-inactivated FBS (Gibco), 100 U/mL penicilin-streptomycin (Invitrogen) and 0.1 mM Non-Essential Amino Acids (Gibco).2. The absence of Mycoplasma spp. in cultured cells was confirmed at least once a month by a PCR-based assay (Minerva). Cell identity was confirmed at least once a year using the STR genotype .All cells were maintained in humidified incubators at 37 °C with 5% CO83 were performed and are described below56. In the Neo/Blast assays, 2 × 105 HeLa JVM cells were plated in 6-well tissue culture plates. Within 24 h, cells were co-transfected with 0.5-1 µg of L1 plasmid and 60 nM of let-7 mimic or 40 nM of let-7 inhibitor and their respective controls, scr, and c-, using Dharmafect DUO (Dharmacon) following manufacturer’s instructions. For Neo assays, selection with 400 µg/mL of G418 (Life) was started 48 h post-transfection. For Blast assays, selection with 10 µg/mL of blasticidin (Millipore) was started 5 days post-transfection. In both cases, medium was changed every 2 days. Between 12 and 14 days after transfection, cells were washed with 1× PBS (Gibco), fixed , and stained with 0.5% crystal violet. Colonies were manually counted. The number of antibiotic-resistant colonies was used to quantify retrotransposition levels in cultured cells. Clonability assay was performed with 0.5 µg pU6i Neo and 1 × 105 HeLa cells84.Modified versions of previously established L1 retrotransposition assays5 HEK293T/SK-MES-1 or 8 × 104 A549 cells were plated in 24-well tissue culture plates. Next day, 200 ng of pYX014, pYX015, or pYX017 were co-transfected with 60 nM of let-7 mimic or 40 nM of let-7 inhibitor  using Lipofectamine 2000 (Life). Luciferase activity was measured 96 h post-transfection using Dual-Luciferase Reporter Assay System (Promega), in a GloMax Luminometer (Promega). Untransfected cells were used to correct for luciferase background.Luciferase retrotransposition assays were performed as follows. 1 × 105 HEK293T cells were plated in 6-well tissue culture plates. Next day, cells were co-transfected with 1 µg 99-UB-LRE3 and 40 nM of let-7 inhibitor or its control, c-, using Lipofectamine 2000 (Life). Retrotransposition (EGFP+ cells) was quantified 8 days post-transfection in a FACS Canto cytometer (BD).EGFP-based retrotransposition assays were performed as follows. 4 × 10Let-7a/b mimic (C-300473-05 and C-300476-05), miR-34 mimic (C-300551-07), let-7a hairpin inhibitor (IH-300474-07), and their respective controls scr and c- (CN-002000-01-05 and IN-002005-01-05), were purchased from Dharmacon. They were resuspended in 1× siRNA Buffer (Thermo) to a working concentration of 20 µM and kept at −80 °C.6 PA-1 cells were transfected in 10 cm tissue culture plates with 4 µg of FLAG-AGO2 and 25 nM of scr/mimic let-7 using lipofectamine 2000 (Life). Transfection with pBSKS (an empty plasmid) was used as a negative control for the IP. 48 h post-transfection, cells were washed with ice-cold 1× PBS, scraped, and transferred to a 1.5 ml tube. After centrifugation at 200g for 2 min, cells were resuspended in 200 µl of cold resuspension buffer (20 mM Tris (pH = 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA) containing 1U/µL RNAsin Plus (Promega) and lysed adding 800 µl of cold lysis buffer , 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethyl-sulfonyl fluoride , 1X cOmplete EDTA-free Protease Inhibitor cocktail (Roche) and incubating for 10–30 min on ice. After centrifugation , 10 µL of RQ1 Dnase (Promega) was added to the supernatant. Immunoprecipitation of FLAG-AGO2 was performed with Dynabeads Protein G (Life) and anti-FLAG M2 mouse  for 3 h at 4 °C with rotation. After five washes with lysis buffer, 10% of sample-beads were used for protein extraction and western blot by adding LDS sample buffer and DTT and heating the samples at 70 °C for 20 min. The 90% of sample beads were incubated with RQ1 DNAse for 30 min for later RNA extraction with Trizol LS (Ambion).2 × 105 HEK293T cells were seeded per well. Within 24 h after seeding, cells were co-transfected with 10 ng of each siCHECK plasmid and 50–80 nM scr/let-7 mimic using Lipofectamine 2000 (Life). 24 h post-transfection, Firefly and Renilla luciferase measurements were performed in a GloMax Luminometer (Promega) using Dual-Luciferase® Reporter Assay System (Promega), following manufacturer’s instructions.In 24-well plates, 1 × 10Binding site mutant “bs2rhmut” was generated using an established protocol. Briefly, two sequential PCRs were performed, using an active L1.3 as a template. First, two PCRs were performed using the following primers under standard conditions: Let7-ORF2PCRafw/Let7-ORF2PCRa_PG2rv and Let7-ORF2PCRarv/Let7-ORF2PCRa_PG2fw. The products of both reactions were purified, mixed in equal amounts, and used as a template for a second PCR using primers Let7-ORF2PCRafw/ Let7-ORF2PCRarv. Conditions for this PCR were: 95 °C 5 min, 10 cycles with , 25 cycles with , 72 °C 10 min. The resulting product contained the mutated sequence in ORF2. This product was purified and cloned into a plasmid containing an active L1 (pJCC5/L1.3) using EcoNI and BsaBI sites, generating pJCC5/bs2mutL1.3. This mutant L1 was then cloned into pJM101 using NotI and BstZ17I sites, generating pJM101/bs2mutL1.3.Binding site mutant 8-mer (pSA500 ORF2-8mer) was generated using the same protocol described above. For the first two PCRs, primers used were Let7-Bcl1-ORF2bs-PCRaFw/Let7-ORF2PCRa_8mer and Let7-ORF2PCRb_8mer/pCEP4_Rv. The products of both reactions were purified, mixed in equal amounts, and used as a template for a second PCR using primers Let7-Bcl1-ORF2bs-PCRaFw/pCEP4_Rv. The product was purified and cloned into pSA500 using BclI and BstZ17I sites.Restriction enzymes were purchased from New England Biolabs (NEB).Sequences were ordered as oligos flanked by a BstZ17I site  supplemented with 1× Complete EDTA-free Protease Inhibitor cocktail (Roche), PMSF (Sigma), 0.25% β-mercaptoethanol (Sigma) and incubated for 10 min on ice. Extracts were then centrifuged  and debris-free supernatants were transferred to new tubes. Protein concentration was determined using the Micro BCA Protein Assay Kit (Thermo) following the manufacturer’s instructions.Cells were washed with 1× PBS, trypsinised and pelleted at 200To control the transfection efficiency in pSA500 western blots, three fractions of the cells were pelleted in different tubes after trypsinization, and DNA, RNA, and protein extractions were performed simultaneously.Proteins were resolved on an SDS-PAGE gel and transferred to a nitrocellulose membrane (BioRad). In L1-ORF2p western blots, proteins were resolved in a 4–15% Mini-PROTEAN TGX Precast Gels (BioRad), and transferred to a PVDF membrane using Trans-Blot Turbo Mini PVDF Transfer Packs (BioRad) and the Trans-Blot Turbo Transfer System (BioRad).For western in Supplementary Fig. For blotting we used the following antibodies: a polyclonal rabbit anti-L1-ORF1p , a polyclonal rabbit anti-L1ORF1p , anti HMGA2 , anti DICER , anti-tubulin , anti-actin , anti-GFP . For chemiluminescent detection, we used anti-rabbit HRP  or anti-mouse HRP , and Clarity ECL Western Blotting Substrate (BioRad) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo). Images were acquired with an ImageQuant LAS4000 and quantified using ImageJ software. For Odyssey analysis, anti-rabbit and anti-mouse fluorescent antibodies (LI-COR) were used at 1:10000 dilution, and detection and quantification were performed in Odyssey (LI-COR).To control plasmid transfection in L1-ORF2p western blots, transfected cells were lysed in a buffer containing 10 mM Tris pH=8.2, 10 mM EDTA, 200 mM NaCl, 0.5%SDS and 200 µg/ml proteinase K, and incubating for 3 h at 56 °C with agitation. Afterwards, DNA was purified with phenol:chloroform:isoamyl alcohol  following standard procedures. 50 ng of each DNA sample were used per qPCR reaction , and an untransfected control was used to discard plasmid contamination. qPCR method is as follows: 5 min at 95°, and 40 cycles of 15 s at 95° followed by 60 s at 60 °C. Plasmid levels were quantified using CMV and EBNA-1 primers, and normalization was performed using genomic GAPDH primers.85. Transfected L1 mRNA was quantified using NEOjunct2 primers designed to exclusively amplify the spliced NEO cDNA86 , following the manufacturer’s instructions. 1 µg of RNA was subsequently treated with RQ1 DNAse and purified by a phenol/chlorophorm extraction. cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems), and used for qPCR  using standard protocols. Two controls were used to verify the absence of contaminating gDNA: no-RT and no-template. Endogenous L1 mRNA was quantified using N51 primersFor mature miRNAs quantification, a RT-qPCR was used. 1 µg of total RNA isolated with Trizol was polyadenylated and then cDNA was synthesized, using qScript miRNA cDNA synthesis Kit (QuantaBio). Quantitative PCR was performed using a universal primer against poly(A) and a miRNA-specific primer that allows the specific detection of polyadenylated mature miRNA and not its precursors (QuantaBio). qPCR was performed with primers Let-7a, Let-7b, and miR-34a.5 HeLa cells were seeded per well. Next day, cells were transfected with 1 µg of pVan583 and 40 nM c-/let7inh using lipofectamine 2000 (Life). Seventy-two hours post-transfection, cells were washed with 1× PBS (Life), detached with TrypLE Express (Gibco) for 5–10 min at 37 °C, pelleted 4 min at 200g, resuspended in 1× PBS with 5% FBS and 5 mM EDTA, and passed through a 70 µM filter. After incubation with 10ug/mL 7AAD (Sigma) for 10 min, fluorescence was quantified in a FACSAria (BD) cytometer. For each replicate, 105 cells were passed through the cytomerer. Only live and transfected cells  were used for %mCherry analysis, which was performed using FlowJo software (LLC) version 10. Controls were used to set the threshold for each fluorescent channel of detection: untransfected cells, and cells expressing either GFP only or mCherry only.In 6-well plates, 2 × 105 U2OS per well were seeded in 6-well tissue culture plates. Next day, cells were transfected with 1 µg of DNA and 60 nM scr/mimic using lipofectamine 2000 (Life), following standard protocols. Twenty-four hours post-transfection, cells were detached and re-seeded in 24-well plates where a UV-sterilized glass slide had previously been placed. Forty-eight hours post-transfection, cells were washed in 1× PBS, fixed in paraformaldehyde (4% in 1× PBS) for 30 minutes at room temperature, and slides were mounted with Slow-Fade Gold Antifade reagent with DAPI (Life). Slides were imaged using a Zeiss LSM-710 confocal microscope (Leica).2 × 1069. Briefly, the region of L1Hs identified as “bs2” with RNA22 was paired to the mature sequence of let-7, using default parameters.The potential structure formed by let-7 and WT L1 or its binding site mutant was analyzed by RNAHybrid83, JM101/L1.370, JM105/L1.34, JJ101/L1.387 and TAM102/L1.380, 99-UB-LRE388, Tgf21-Neo89, Zfl2-1-Neo, and Zfl2-2-Neo90 have been previously described. JJ101/L1.3Δ3’UTR is a derivative of JJ101/L1.3 but the L1 lacks the 3′UTR45. JM101/L1.3bs2mut was generated by cloning a bs2mut L1 into JM101/L1.3 (cloning strategy described in the “Site-directed mutagenesis” section of the methods). FLAG-Ago1 and FLAG-Ago2 were a gift from Edward Chan (Addgene plasmid #21533 and #21538)91. pSA500 is as pAD50063 where the TAP epitope was replaced by 3 consecutive copies of the FLAG epitope tag obtained from PJCC5 ORF1T7 ORF3XFLAG using BclI and BstZ17I restriction enzyme sites. pVan583 is a derivative of JM101/L1.3 where EGFP and mCherry were cloned in frame with the last amino acid of L1-ORF1 and L1-ORF2, respectively. All psiCHECK2 constructs were generated by cloning sequences synthesized and cloned in pUC57 (Genescript) into psiCHECK2 using XhoI and NotI restriction enzyme sites (Promega).pYX014, pYX015 and pYX017Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1


Graphene is a material with outstanding properties and numerous potential applications in a wide range of research and technology areas, spanning from electronics, energy materials, sensors, and actuators to life-science and many more. However, the insolubility and poor dispersibility of graphene are two major problems hampering its use in certain applications. Tethering mono-, di-, or even poly-saccharides on graphene through click-chemistry is gaining more and more attention as a key modification approach leading to new graphene-based materials (GBM) with improved hydrophilicity and substantial dispersibility in polar solvents, e.g., water. The attachment of (poly)saccharides on graphene further renders the final GBMs biocompatible and could open new routes to novel biomedical and environmental applications. In this review, recent modifications of graphene and other carbon rich materials (CRMs) through click chemistry are reviewed.  Graphene holds great potentials in numerous applications ranging from electronics and optoelectronics to mechanical reinforcement technologies, energy materials, sensors and actuators and many more. Properties of these materials such as aqueous stability and biocompatibility are essential, especially in the arena of biological and medical applications ,2. Yet, For instance, graphene oxide (GO) which can be obtained by chemical oxidation of graphene exhibits excellent dispersibility in various solvents . VariousCovalent and non-covalent functionalization of graphene with mono-, di- or even poly-saccharides (PSs) has recently attracted a great deal of interest owing to the exceptionally nontoxic, hydrophilic, biocompatible, and biodegradable nature, of saccharides ,14. The There are various routes allowing for (poly)saccharide functionalization of graphene. One of the most prominent functionalization approaches of that type which to the best of the authors’ knowledge has not been reviewed so far, is the click chemistry approach . The terThe various approaches leading to polysaccharide-modified graphene can be categorized into three types see . In the These molecules can bear a functionality which is desired for a specific application. The role of click chemistry here is to enable covalent attachment of this functionality on a molecule which shows proven affinity to the π-conjugated surface of graphene, e.g., a polycyclic aromatic hydrocarbon (PAH) .Various complementary methods to click chemistry are known see . In manyDirect functionalization of graphene through click chemistry can also be achieved via Diels Alder reactions or other types of cycloadditions see . Some re1 in In recent years functionalization of graphene with chitosan (Structure 3) was then treated with propargylamine (in the presence of dicyclohexylcarbodiimide) to afford transformation of the edge carboxy groups of GO to propargyl amide groups (4). The two counterparts 2 and 4 were clicked together via a Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition and chitosan-functionalized graphene oxide (5) was finally isolated. The described methodology enables selective functionalization of GO at its edges since the carboxy groups (lying at the edges of GO) were selectively alkyne-modified prior to the click reaction with the azide-modified chitosan.In a first step, the azido moiety was introduced via a reaction of the chitosan with azido-epichlorhydrin  leading to polymer 2. GO (10) derivative is achieved. The relevance of this synthetic strategy is high as it clearly proves the orthogonality of click chemistry . In. In56]. In this paragraph substitution reactions of graphene with mono- and di- saccharides were reviewed and the required preparations of the graphene and saccharide precursors were discussed. A Cu(I) catalyzed Huisgen 1,3-dipolar cycloaddition was identified as the click method mostly used for this type of graphene covalent modifications. This is presumably because of the ample existing knowledge on the azide- or alkyne-modifications of mono- and di-saccharides . In the Another way to modify graphene is through the so-called non-covalent functionalization approach. In this case, the functionalities are not bound to graphene by means of a covalent bond but instead they are typically bound to a compound which can stay on the surface of graphene by means of relatively weak interactions, e.g., through π-π interactions between a PAH and graphene. This strategy is often preferred when it is desired that the electrical and electronic properties of graphene are retained after functionalization and this can be ensured by means of methodologies which retain intact the fully π-conjugated honeycomb structure of graphene ,57. Ιn t25) scaffold which they initially non-covalently functionalized GO and in one step they achieved reduction of GO to rGO merely using sonication. The reducing capacity of the az-TTF is such that allowed for the GO to rGO reduction. The result is a GBM involving non-covalent az-TTF-functionalization. Moving a step further it was possible to apply a CuAAC chemistry to tether a monosaccharide unit (mannose) which was pre-alkyne modified. The final material was thus a modified graphene involving a monosaccharide prepared in two steps (structure 28). What is even more interesting is that the mannose-TTF units can reversibly be detached form the graphene surface merely by applying the tetracationic cyclophane cyclobis(paraquat-p-phenylene): 29 which efficiently binds mannose-TTF. The process was nicely monitored by means of Atomic Force Microscopy (AFM)  see C.32 and 33) varying on the type of monosaccharide employed . Alkyne-modified rhodamine B was attached to each azido-monosaccharide through a CuAAC reaction. The π-conjugated nature of the rhodamine matches well the partly π-conjugated honeycomb structure of GO. Assembly of 32 and 33 on GO occurs efficiently leading to the glycosheet materials 34–35 respectively fluorene (TBF) see  60]. Th. Th60]. ar to 37 . In termWu et al. , some yePyrene among other PAHs is an indispensable part of many non-covalent functionalizations to graphene, CNTs and other CRMs ,64,65. T6 [In this paragraph, focus is placed on click approaches allowing for covalent functionalization of CRMs other than graphene. One of the most prominent allotropes of carbon which during the last decades has attracted a great deal of interest is CNTs . CNTs co6 ). A vast6 . Yet, a 6 ,72. Many6 ,78. Clic6 . Variousp-amino-O-propargylphenol and isoamylnitrite to afford product 42 (42) efficiently reacted with azide-functionalized β-CD (43) to form β-CD-CNTs (44). The method described led to a new type of functional material. As an application, the binding ability of the β-CD-CNT fluorescence spectroscopic study was conducted towards quinine which was found to efficiently enter the β-CD cavity of 43 [Guo et al.  reportedoduct 42 A. The sity of 43 . (It is ty of 43 ). Notablty of 43   with chitosan (1) [46. The latter reaction has recently been applied on CNTs [Click strategies other than the CuAAC chemistry allowing for the grafting of CNT-involving materials with polysaccharides have also been reported. Yadav et al. for example reported on the functionalization of multiwalled carbon nanotubes (MWCNTs osan (1) . The syn on CNTs ,83,84 wi47) was as high as 1.8 chitosan polymeric chains every 1000 carbon atoms of MWCNTs. When it comes to the properties of the final material it was shown that it exhibits enhanced mechanical properties and antimicrobial activity when compared to the parent materials.From a sugar-CNTs-functionalization perspective, what makes the click reaction by Yadav et al. attractive, is the fact that only one derivatization of the polysaccharide with a linker encompassing an azide-terminus is required. The CNTs are then used directly, i.e., without any pre-derivatization. The reaction is brought about at temperatures typically higher than 150 °C in solvents like N-methyl-2-pyrrolidone (NMP). The reported degree of substitution on MWCNTs in the final product . The techniques used are the same as described in corresponding graphene modifications. Nonetheless, different pre-modification methods are applied depending on the CRM-substrate.To the best of the author’s knowledge, very few examples on click-sugar modified CRMs other than CNTs and GBMs can be found. There are various examples involving PAHs saccharides enabled by click chemistry. Emphasis was given to click chemistry approaches which have been shown to allow for either covalently or non-covalently graphene functionalizations. The review expands to CRMs like CNTs and includes a range of examples and potential applications of the (poly)saccharide modified materials. In most cases reviewed, it was shown that (poly)saccharide modifications increased the hydrophilicity and dispersibility of CRMs in water or other solvents. Moreover, the materials thus produced exhibit multifunctional behavior and are proposed as important candidates for various new applications.


We show that the degree of oxidation of graphene oxide (GO) can be obtained by using a combination of state-of-the-art ab initio computational modeling and X-ray photoemission spectroscopy (XPS). We show that the shift of the XPS C1s peak relative to pristine graphene,  Graphene oxide (GO) is an amorphous , non-stoichiometric, functionalized form of graphene bearing different oxygen functional groups ,2,3. BecHere, we present a technique based on the analysis of the XPS peak energies that can quantitatively determine the oxygen content of GO. The main result is shown in ΔEC1s=A(cGraphene oxide dispersions with different degrees of oxidation were prepared by direct oxidation of graphene flakes using a modified Hummer’s method (see Methods section) . The evoThe functional groups responsible for the C–O, C=O, and O–C=O peaks in XPS can be narrowed down to four candidates. These include the epoxy and hydroxyl groups at the basal plane , as welThe C1s core level shifts for the four functional groups just discussed have been calculated using density functional theory, using a self-consistent ‘approach ,11. Prevapproach . Our pseapproach .Several approximations are involved in the ‘This includes the effects of valence electron relaxation after the core ionization has taken place, but it does not include the relaxation of the lattice, which is expected to happen in a much slower time scale.The ground-state energy of the system in the presence of the core hole is computed using a carbon pseudo-potential where the atabase,  with theatabase, . AdditioThe C1s level shift of a C1s state hole near the oxygen functional group is compared to that of graphene. This eliminates the problem of lack of a common energy reference when different pseudopotentials are used. Further, it is directly comparable to the distance between the peak under consideration and the C=C peak for the same concentration.Previous DFT works ,16 have We have therefore calculated the C1s core level shifts up to the fourth nearest neighbor for basal plane functional groups, and up to the third nearest neighbor for the edge carboxyl a–d. In eThe results agreed well with the experimental spectra for graphene, namely for the C−O peak, which was the highest oxygen-related peak and was 1.48 eV higher in energy than the C-C The more oxidized the graphene is, the less metallic it becomes, and fewer free electrons are available to screen ionized cores. This induces the drift of the core level shifts that we report in this letter. We thus carried out calculations in a GO model consisting of a series of oxidized 32-atom graphene supercells, with randomly positioned, parallel-oriented epoxy oxygen, in a concentration of up to 33% atomic fraction . The useThe shifts of the C–O. peak relative to graphene . The percentage of oxygen was obtained from the oxygen peak intensity in the survey spectra [Samples were drop-casted on silicon (Si) substrates for XPS analysis. The measurements were performed with Kratos AXIX Ultra  equipment with a mono-chromatic source, Al K spectra .First-principles calculations were based on the framework of DFT, as implemented in the Quantum Espresso package . The PBEMP) grid  for all


Pulmonary involvement occurs in up to 95% of sarcoidosis cases. In this pilot study, we examine lung compartment-specific protein expression to identify pathways linked to development and progression of pulmonary sarcoidosis. We characterized bronchoalveolar lavage (BAL) cells and fluid (BALF) proteins in recently diagnosed sarcoidosis cases. We identified 4,306 proteins in BAL cells, of which 272 proteins were differentially expressed in sarcoidosis compared to controls. These proteins map to novel pathways such as integrin-linked kinase and IL-8 signaling and previously implicated pathways in sarcoidosis, including phagosome maturation, clathrin-mediated endocytic signaling and redox balance. In the BALF, the differentially expressed proteins map to several pathways identified in the BAL cells. The differentially expressed BALF proteins also map to aryl hydrocarbon signaling, communication between innate and adaptive immune response, integrin, PTEN and phospholipase C signaling, serotonin and tryptophan metabolism, autophagy, and B cell receptor signaling. Additional pathways that were different between progressive and non-progressive sarcoidosis in the BALF included CD28 signaling and PFKFB4 signaling. Our studies demonstrate the power of contemporary proteomics to reveal novel mechanisms operational in sarcoidosis. Application of our workflows in well-phenotyped large cohorts maybe beneficial to identify biomarkers for diagnosis and prognosis and therapeutically tenable molecular mechanisms. It affects 45–300/100,000 individuals in the US, all ages, races, and both sexes3. Diagnostic delays are frequent as sarcoidosis is a diagnosis of exclusion, with no confirmatory test currently available. Despite a greater understanding of sarcoidosis pathogenesis5, the mechanisms contributing to the heterogeneity of disease manifestations and predictors of disease outcomes are poorly defined6. The annual mortality is approximately 2.8/million people1 and rising. Sarcoidosis-related mortality is attributed to four high-risk manifestations that include: treatment-resistant pulmonary sarcoidosis, multi-organ sarcoidosis, cardiac sarcoidosis, and neurosarcoidosis7. Respiratory failure from progressive pulmonary disease is the leading cause of sarcoidosis-related mortality in the US8. While remission is common, it is not known if systemic anti-inflammatory therapy decreases the risk of progressive pulmonary disease. Another current knowledge gap is the absence of validated markers to predict which patients with pulmonary sarcoidosis will progress.Sarcoidosis is a multisystem immune-mediated disease of unknown cause with widely variable disease manifestations, severity, and outcomes9. While the exact details are not known, it appears that exposure to a yet unidentified antigen(s) results in an exuberant adaptive immune response with CD4 + T cells10, regulatory T cells (Tregs), and high levels of Th1 cytokines TNF–α, IFNγ, and IL–2. Additionally, an abnormal innate immune response is seen in bronchoalveolar lavage (BAL) cells in sarcoidosis. A less robust immune response is apparent in remitting disease compared to the exuberant response in progressive sarcoidosis, likely due to different T cell populations and abnormal counter-regulatory immune measures. Overall, the immune response is aberrant in sarcoidosis and compartmentalized to the lung with much higher response noted in the lung cells compared to blood cells12. The whole blood transcriptional profile of active sarcoidosis overlaps with tuberculosis and chronic beryllium disease, and inactive sarcoidosis overlaps with controls14. Genes with differential expression in sarcoidosis map to IFN-signaling, TLR signaling, and Fcɣ receptor-mediated phagocytosis16. In chronic progressive sarcoidosis, the gene expression in peripheral blood mononuclear cells demonstrates differential expression of genes participating in CXCL9 and TCR-mediated responses17. Transcriptional studies in BAL cells revealed that pathways linked to adaptive immune response, T-cell signaling, and chemokine signaling such as IFNγ, IL-12, 1L-17, and IL-23 are involved in sarcoidosis18. In lung tissue, gene networks engaged in cell movement, immune function, and in Th1-type responses such as signal transducer and activator of transcription 1 (STAT1), IL-5, IL-7, CXCR5, and CXCR9 were overexpressed in sarcoidosis lung tissues11. However, the approach of examining comprehensive protein changes that result from these differences in transcription is underutilized and has not been well evaluated using contemporary techniques.The pathologic hallmark of sarcoidosis is the formation of epithelioid granuloma associated with infiltration of CD4 + T cells and scattered macrophages, giant cells, with CD8 + T cells and B cells around the granuloma20, 2-dimensional electrophoresis (2DE)23, and top-down24 as well as shotgun proteomics27 to examine variable sarcoidosis phenotypes including Lofgren’s syndrome, non-Lofgren’s chest x-ray (CXR) stage I, and stage II/III pulmonary sarcoidosis and compared them to subjects with asthma, IPF, tuberculosis or healthy smoking and non-smoking controls. These studies have identified differences in protein spots on 2DE22, differentially expressed proteins28 and also possible mechanisms that could explain the development of sarcoidosis27. In a large study that utilized SELDI-TOF MS to compare BAL fluid (BALF) from sarcoidosis subjects with Lofgren’s syndrome and different CXR stages of pulmonary sarcoidosis (n = 65) with healthy controls, 40 differentially expressed peaks were identified compared to healthy controls and included 27 peaks that were specific for a particular CXR stage24. A study using affinity planar antigen microarray proteomics examining BALF and reported that mitochondrial ribosomal protein L43, nuclear receptor coactivator 2, adenosine diphosphate-ribosylation factor GTPase activating protein 1 and zinc finger protein 688 demonstrated higher reactivity in sarcoidosis lungs20. Another study reported several differentially expressed BALF proteins in nine sarcoidosis patients with stage II/III sarcoidosis compared to healthy controls analyzed by 2DE followed by MALDI-TOF MS25. The differentially expressed proteins mapped to canonical PI3K/Akt/mTOR signaling, MAP kinase, hypoxia response, and pluripotency-associated transactional factor pathways. These studies support rigorous evaluation of well-characterized, clinically-meaningful sarcoidosis phenotypes by contemporary techniques to identify novel mechanisms of sarcoidosis which can provide tenable treatment targets and biomarkers for personalized care.Prior studies have used protein microarraysOur goal is to couple contemporary proteomics with data-driven analytics for unbiased discovery of novel disease mechanisms in pulmonary sarcoidosis and progressive pulmonary disease, a known high-risk manifestation of sarcoidosis. As a critical first step in evaluating the proteome in sarcoidosis, we focus on BAL cells as alveolitis is seen in patients with active pulmonary sarcoidosis and immune cells provide an ex vivo model for biological mechanisms in inflammatory lung diseases. The BALF is the most proximate fluid to the site of injury, and thus has a high likelihood to identify disease-specific and potentially pathogenic changes. For this proof-of-concept study, we performed label-based MS for measuring protein abundance to gain insights into the intracellular protein interactions in sarcoidosis. We also employed label-free quantitative proteomics on BALF from controls and untreated sarcoidosis cases who, on follow-up, either were found to have progressive or non-progressive pulmonary disease. We found significant differences in BALF and cellular proteins between cases and controls and progressive versus non-progressive cases suggesting that this approach may find useful application in larger studies.We characterized the proteins in BAL cells from four controls and four sarcoidosis cases. There was no difference in age, sex, race and smoking status for the two groups. The BAL leucocyte count was not significantly different but the sarcoidosis cases had more lymphocytes and a lower number of macrophages Table . For theThe liquid chromatography (LC)-tandem mass spectrometry (MS/MS) identified 23,837 spectra at the given thresholds; 16,890 (71%) were included in quantitation. From these spectra, we identified 4,365 proteins , and SERPINA1.We used a stringent permutation testing and identified 272 differentially expressed proteins controlling for an FDR of ≤ 5%, Fig. To determine the biological significance of the differentially expressed proteins, we performed IPA core analysis to identify the canonical pathways that map to these proteins. The pathways that met the statistical threshold  and the proteins assigned to each canonical pathway are listed in Table We examined BALF from seven control and ten sarcoidosis subjects. All BALF samples were analyzed by label-free mass spectrometry in triplicates. We identified 1,293 BALF proteins at an FDR of ≤ 1% , interleukin 6 receptor, programmed cell death 1 ligand 2, and aquaporin-1. The proteins with most significant differences with a higher abundance in sarcoidosis or controls are listed in Table We identified 293 differentially expressed proteins in sarcoidosis (n = 10) compared to the seven control subjects , chitotriosidase 1, ICAM 1, tropomyosin, integrin beta-2, pulmonary surfactant protein B and D, fatty acid binding protein, and HLA class II histocompatibility antigen DQ-α. The proteins with most significant differences with a higher abundance in cases with progressive disease compared to non-progressive disease are listed in Table When we compared the BALF proteins between progressive vs. non-progressive sarcoidosis subjects n = 5 each), there were 121 differentially expressed proteins. The proteins that differed between phenotypes included heat shock protein 90, glutathione- each, th29. We implemented an approach that coupled state-of-the-art mass spectrometry based proteomics with novel bioinformatics for a comprehensive characterization of the protein changes in the lung compartment in well-phenotyped cases. In the absence of well-characterized animal models, the examination of BAL cells provides an ex vivo model of the immune response in sarcoidosis. While proteomic studies have been conducted previously28, no prior study has comprehensively characterized mixed BAL cells and BALF. In addition, we established workflows for comprehensive characterization of BALF to obtain unprecedented coverage and detect proteins that originate from diverse cellular and extracellular sources. Ultimately, our approach to characterize mixed BAL cells captured the complex interplay between inflammatory cells in sarcoidosis. Specifically, in BAL cells and fluid we identified several pathways present in macrophages such as clathrin-mediated endocytic signaling and other phagocytic processes as well as redox-related pathways that were previously reported to be upregulated in sarcoidosis30. We also identified novel pathways implicated in sarcoidosis such as signaling by integrin-linked kinase, IL-8, and caveolar-mediated endocytic signaling in our studies comparing BAL cells from controls and sarcoidosis cases. The studies in BALF showed higher levels of chitotriosidase, a potential biomarker and an investigational agent for therapy32 when comparing cases to controls. Several of the biological pathways identified in the BAL cells were also identified in the BALF, suggesting that BALF is a useful biofluid to investigate mechanistic processes in sarcoidosis. In our comparison of cases with progressive vs. non-progressive sarcoidosis, we identified several novel pathways that may be involved in progression in sarcoidosis. These included CD28 signaling and PFKFB4 signaling. These results suggest that a systematic characterization of BALF may prove fruitful to develop disease models and classifiers with diagnostic and prognostic utility, while BALF and the cellular proteome will provide insight into the mechanisms underlying sarcoidosis as well as the processes that promote progressive disease.Use of ‘omics’ tools to improve the understanding of sarcoidosis has been recognized as a high priority area of research in sarcoidosis34, along with inappropriate counter regulatory responses. Previous studies investigating protein changes in alveolar macrophages26 and gene expression changes in peripheral blood mononuclear cells35 found phagocytosis-related pathways to be upregulated in sarcoidosis subjects such as Fcγ receptor-mediated phagocytosis and clathrin-mediated endocytic signaling. We identified differences in cellular proteins mapping to phagosome maturation and clathrin-mediated endocytic signaling in sarcoidosis vs. controls BAL cells. Phagocytosis is crucial for innate and adaptive immune response and plays an essential role in antigen presentation, supporting the notion that sarcoidosis results from the response to an unknown external exposure requiring antigen processing and presentation for the development of disease. Similar to previous reports, we observed that the proteins involved in clathrin-mediated endocytic signaling differ in sarcoidosis cases when compared to controls. Additionally, caveolar-mediated endocytic signaling was also different between the two comparison groups. While both these pathways play a role in endocytic internalization of a variety of particles, again implicating exposure in disease ontogeny, these pathways also play a role in signal transduction and the regulation of many plasma membrane activities that have not been studied in sarcoidosis as well as have an influence on the immune response in alveolar macrophages37 and peripheral blood mononuclear cells38. In fact, the role of clathrin and caveolar pathways in the development of sarcoidosis has not been systematically studied. Thus, our findings suggest new pathways for investigation of potential disease pathogenesis and or cell regulation in sarcoidosis.We examined BAL cells as the inflammatory response is aberrant in sarcoidosis with (a) yet unknown antigen(s) triggering an exuberant although dysfunctional immune response with CD4 + T cells, Tregs, high levels of Th1 cytokines TNF-α, IFNγ, and IL-239. The downstream signaling cascade of PI3K/AKT activation includes activation of mTOR25, which is implicated in the development and the progression of sarcoidosis and has been proposed as a potential therapeutic target40. Thus, ILK-mediated mTOR activation could be a possible mechanism mediating inflammation in a subset of sarcoidosis cases. ILK signaling also activates c-Jun N-terminal kinase (JNK) via transcription-factor activator protein 1 (AP1) and regulates the gene expression of MMP941 and also IL-8 signaling42. IL-8 is a chemokine in the CXC family and is produced by non-leucocytic and leucocytic cells including macrophages, and binds to CXCR1 and CXCR2 surface receptors43. Several cytokines such as TNF-α induce the production of IL-844. Higher levels of IL-8 have been reported sarcoidosis BALF45 and serum, with the latter correlating with pulmonary46 and chronic disease47. IL-8 signaling has recently been reported to directly regulate adaptive T cell reactivity48 and phagosome function. Thus, our findings are not surprising but suggest that future studies investigating IL-8 signaling could improve the understanding of sarcoidosis pathogenesis and potentially phenotypes. They also highlight the importance of comprehensive characterization of the BAL cell protein changes in providing insight into sarcoidosis development and or progression, an approach that offers promise and is underutilized thus far in sarcoidosis research.With an unbiased approach, we identified several canonical pathways mapping differentially expressed proteins that have not been previously linked to sarcoidosis, but would be likely to play a role in disease pathogenesis. These include integrin-linked kinase (ILK) signaling, IL-8 signaling, and inhibition of matrix metalloproteinases. ILK is an intracellular protein that primarily functions to connect integrins to the cytoskeletal proteins. The intracellular domain of ILK interacts with different proteins and regulates the phosphorylation of protein kinase B (PI3K)/AKT1 and glycogen synthase kinase 3B49. Binding of the adaptor proteins to the activated motif, in turn phosphorylates and activates CDC-4250, culminating in the activation of JNK51. While we did not identify enrichment of canonical JNK pathways, BALF may only reflect some of the processes involved in sarcoidosis pathogenesis with secreted proteins. Regardless, the finding of differentially expressed BALF proteins mapping to CDC-42 and CD28 signaling suggests that they may possibly be involved in disease progression. Additionally, CD28 controls differentiation of Tregs from naïve CD4 T cells, providing novel mechanisms that may explain progression or remission of sarcoidosis. Interestingly, we identified PFKFB4 and IL-12 signaling also mapping to proteins that are differentially expressed in progressive vs. non-progressive cases. PFKFB4 is a bifunctional glycolytic enzyme that synthesizes and degrades fructose 2,6,-biphosphate. PFKFB4 regulates glucose metabolism and cell fate of dendritic cells52 and may provide a link for immunomodulatory effects by 1,25-dihydroxyvitamin D3  D3). Vanhewegan et al., identified PFKFB4 as a master regulator of 1,25 (OH)2 D3 induced DC tolerogenicity and inhibition of PFKFB4 signaling promotes secretion of proinflammatory cytokines including TNF-α53. The exact role of these pathways in the progression of pulmonary disease remains to be investigated, but our study suggests further investigation should be undertaken.The examination of BALF revealed many proteins that are represented by canonical pathways that were also found in BAL cells. This indicates that biological mechanisms that contribute to the development of sarcoidosis can be identified in the BALF. When we compared BALF from sarcoidosis subjects to controls, similar to the findings from BAL cells, we identified several pathways that are linked to the inflammatory response. These included phagosome formation/maturation, clathrin- and caveolar-mediated endocytic signaling, LXR/RXR activation, IL-8 signaling, fatty acid oxidation, NRF2-mediated oxidative stress response and tryptophan degradation. Several of these pathways are also assigned to the proteins that are differentially expressed between progressive and non-progressive sarcoidosis cases. Some BALF pathways map to proteins that are only differentially expressed between progressive and non-progressive sarcoidosis. Specifically, we identified proteins mapping to CD28 signaling in T-helper cells, PFKFB4  signaling and IL-12 signaling and production in macrophages. CD28 is a stimulatory immune checkpoint molecule of B7-CD28 superfamily with diverse roles in naïve and CD4 + T cells. The cytoplasmic tail of CD28 contains signaling motifs that are phosphorylated in response to TCR and CD28 stimulation54 and BALF55. In our study, the examination of mixed BAL cells indicated altration in redox balance in newly-diagnosed sarcoidosis subjects. Specifically, the mitochondrial l-carnitine shuttle pathway which is involved in fatty acid and lipid degradation, was mapped by proteins with differential abundance in controls compared to  sarcoidosis, suggesting that the mitochondrial metabolism is altered. Furthermore, we found differentially expressed proteins in pathways related to β-oxidation of fatty acids and mitochondrial dysfunction. We also identified several cytoprotective enzymes that mapped to NRF2 mediated oxidative stress response were differentially abundant in sarcoidosis compared to controls. NRF2 regulates mitochondrial redox homeostasis by several mechanisms such as detoxification of peroxides, regeneration of GSH, increased synthesis of GSH and NADPH and via the NRF2-Keap 1 response. Mitochondrial dysfunction occurs when the reactive oxygen species (ROS)-mediated stress overpowers the antioxidant defense system56. Bleomycin challenge in NRF2 knockout mice results in increased inflammatory makers, lower level of antioxidant enzymes, a bias towards Th2 response and increased fibrosis57. Taken together, these findings suggest that in sarcoidosis abnormal fatty acid and lipid degradation in the mitochondria cause the production of oxidants, with altered redox balance. It is possible that the detoxification mechanisms are overwhelmed causing mitochondrial dysfunction, production of reactive oxygen species that contribute to the inflammatory response seen in the lungs. NRF2 activators such as curcumin, sulforaphane, resveratrol, and quercetin counteract increased oxidant stress have a potential benefit in acute respiratory distress syndrome58, chronic obstructive pulmonary disease59, asthma60 and idiopathic pulmonary fibrosis61 and could be tested as a possible therapeutic strategy in sarcoidosis. The proteins differentially expressed between controls vs sarcoidosis and progressive vs. non-progressive sarcoidosis cases also mapped to Aryl hydrocarbon receptor signaling. AhR signaling is emerging as an important regulator of immunity in response to endogenous and exogenous ligands62 including tryptophan and serotonin metabolism. The differentially expressed proteins in both of the comparisons mapped to tryptophan /serotonin degradation but only reached statistical significance in the sarcoidosis vs control comparison. AhR signaling controls adaptive immunity by regulating T cell differentiation and by effecting antigen-presenting cells63. AhR regulates T cell response at multiple levels including T cell fate64. AhR is linked to induction of CD4 + Treg or Th1765 and Th22 cell differentiation directing the balance between effector and regulatory T cells. AhR signaling is implicated in other diseases with granulomatous inflammation. In Crohn’s disease, AhR RNA transcripts were markedly downregulated in the inflamed tissue and in the CD4 + T cells66. AhR signaling is also implicated in particulate induced granulomatous inflammation such as silicosis 67.In pulmonary sarcoidosis, higher oxidant stress is reported in inflammatory cells in the lung68. Another interesting protein with differential expression higher in the BALF in sarcoidosis compared to controls, programmed cell death 1 ligand 2 (PD-L2), is a ligand for programmed death-1 (PD1) receptor. PD-L2 is a transmembrane protein that is involved in immune checkpoint activity of PD1. In sarcoidosis, PD1 has been linked to the development of T cell exhaustion69 and a blockade of the PD1 pathway restored sarcoidosis CD4 proliferative capacity70. The notion that the PD1 pathway is involved in sarcoidosis is also strengthened by reports of sarcoidosis-like illness in patients receiving PD1 immune checkpoint modulators71. While the presence of individual proteins in our dataset is encouraging, we expect that the systematic examination of global protein changes in the BALF coupled with statistical approaches to construct a parsimonious model consisting of an orthogonal set of proteins will be the best approach for diagnosis and prognostication in sarcoidosis.While we observe differences in the biological processes annotated to the differentially expressed proteins, systematic investigation of the BAL could provide the yet elusive biomarkers with diagnostic and prognostic value in sarcoidosis. In our dataset, we found higher BAL levels of chitotriosidase in sarcoidosis cases compared to controls. Chitotriosidase is a monocyte-macrophage-derived protein that is elevated in plasma and BALF and has been associated with sarcoidosis severity72. Networks are represented as a collection of features (nodes) and links (edges) that connect pairs of nodes. The ‘guilt-by-association’ principle73 implies shared biology of pathways. Moreover, biological networks demonstrate scale-free behavior75 indicating that they have a relatively large number of low-connectivity nodes and only a few high-connectivity nodes, called ‘hubs,’ that are likely to be essential to network function. In the analysis of cellular proteins, IL-8 signaling, leucocyte extravasation signaling, ILK signaling, glucocorticoid receptor signaling and clathrin-mediated endocytic signaling demonstrated high-connectivity  for diagnosis and prognosis and also identify therapeutically tenable treatment targets in sarcoidosis. Investigating the cellular and BALF protein changes provides an opportunity to examine the complex interplay of protein interactions response for the development and progression of sarcoidosis as well test the validity of protein participating in these biological processes as biomarkers for disease diagnosis and predict progression. The novel mechanisms identified in our pilot study will need to be evaluated with conventional structure function study to determine causal links in sarcoidosis.The study was approved by the University of Minnesota (UMN) IRB  and all studies were conducted under the relevant guidelines/regulations. Study participants provided informed consent for the collection of BAL fluid and cells for these studies.3. Subjects without presence of another disease that could significantly affect patient immune response were also enrolled as healthy controls. Bronchoscopy and bronchoalveolar lavage were performed per standard protocol at UMN and NJH77. Four newly diagnosed sarcoidosis subjects were enrolled for examination of BAL cells at UMN , the European Respiratory Society (ERS) and the World Association of Sarcoidosis and Other Granulomatous Disorders (WASOG)MN Table . LeftoveMN Table . After c79. Non-progressive pulmonary disease cases had stable disease and met the following criteria on up to two-years follow-up or more: (1) ≤ 10% decline in FVC or FEV1 and a ≤ 15% decline in DLCO and a stable CXR, and/or (2) ≥ 10% improvement in FVC or a ≥ 15% improvement in DLCO or improved CXR AND (3) no indication for immunosuppressive therapy. Progressive pulmonary disease cases met any of the following criteria from diagnosis/initial evaluation on at least two-year follow-up: (1) ≥ 10% decline in FVC and/or FEV1; or a ≥ 15% decline in DLCO; or (2) worsening CXR as determined by the interpreting radiologist/ investigator; and/or (3) start of immune-suppressive therapy.For the BALF studies, the sarcoidosis subjects were divided into two distinct phenotypes: those with non-progressive pulmonary disease and those with progressive pulmonary disease using criteria previously establishedg for 15 minutes and the supernatant was collected for further processing. To increase the protein recovery, the pellet from this step was resuspended in a buffer (containing 7 M urea and 2 M thiourea in 0.4 M triethylammonium bicarbonate at pH 8.5), freeze-thawed, vortexed and centrifuged at 15,000g for 15 min at 20 °C. The supernatants from the two centrifugation steps were combined and concentrated using an Amicon 3-MWCO filter (Millipore). An equal amount of protein was processed for in-gel cleanup and digestion (EMBL Method), reduced with dithiothreitol (Sigma-Aldrich), treated with iodoacetamide (Sigma-Aldrich) to block cysteine residues, digested with trypsin (Promega) and cleaned with an MCX stage tip (3M-Empore 2241). Isobaric labeling of digested peptides was carried out with TMT-10Plex (Thermo Fisher Scientific) reagent followed by MCX and SPE cleanup with appropriate buffer exchanges, and offline fractionation on Shimadzu Prominence with Xbridge 150 × 2.1 mm column (Waters) with two-minute fractions at a flow rate of 200µL/min, and peptide amounts of 15mAU-equivalent aliquots from fractions 7–38 were concatenated. LC–MS data was acquired for each concatenated fraction using an Easy-nLC 1,000 HPLC  in tandem with an Orbitrap Fusion (Thermo Fisher Scientific) MS instrument.BAL cells were resuspended in hypotonic lysis buffer with HALT protease inhibitor cocktail (Thermo Fisher Scientific), and lysed using sequential cell disruption techniques including a freeze–thaw at 98 °C with vortexing and sonication (Sonics) on ice before buffering with 1 M triethylammonium bicarbonate (Sigma). The lysed cells were then centrifuged at 20,00081. Briefly, BALF was sonicated (Sonics), centrifuged for 15 min at 14,000g at 4 °C and filtered with pre-rinsed (5% methanol and water) syringe  and 0.22um PES filter to remove remaining particulates. The fluid was then concentrated and desalted using Amicon 3-MWCO filters, and a Bradford assay (Bio-Rad) was used to quantify protein. High-abundance proteins were removed using a Seppro IgY 14 spin-column (Sigma-Aldrich) with appropriate buffer exchanges. Equal amount of enriched medium- and low-abundance protein was processed for in-gel cleanup and digestion similar to the BAL cells above. LC–MS data was acquired for each concatenated fraction using an Easy-nLC 1,000 HPLC in tandem with an Orbitrap Fusion using settings similar to BAL cells analysis with minor differences. The differences were (1) The column was heated to 50 °C and (2) the dynamic exclusion was set to 15 s with a 10-ppm high and low mass tolerance.The BALF was processed using our previously published protocolThe BAL cell quantification was accomplished using TMT reagent, and the BALF dataset was analyzed using MS1 spectral quantification.https://www.thegpm.org/crap/). Scaffold Q +  was used to perform TMT-based peptide quantitation and protein identification. The threshold of peptide identifications was set at an FDR < 0.5% by the Scaffold Local FDR algorithm. The protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least one peptide82. Channels were corrected according to the algorithm described in i-Tracker83. Normalization was performed iteratively (across samples and spectra) on intensities, as described in Statistical Analysis of Relative Labeled Mass Spectrometry Data from Complex Samples Using ANOVA84. Medians were used for averaging. Spectra data were log-transformed, pruned off those matched to multiple proteins and those missing a reference value, and weighted by an adaptive intensity-weighting algorithm. Of 23,837 spectra in the experiment at the given thresholds, 16,890 (71%) were included in quantitation. The proteins that matched to the cRAP or the decoy sequence were removed from analysis.The spectral dataset was searched against the target-decoy version of the Human UniProt database  along with the contaminant sequences from the cRAP database  along with the cRAP database using the MaxQuant 1.6.10.43 algorithm. Default search parameters were used as follows: peptide spectral matching and proteins with 1% FDR modifications include fixed carbamidomethyl of C, variable oxidation of M, and N-terminal acetylation. BALF samples were quantified in label-free quantification (LFQ) mode, and spectra were “matched between runs”.https://galaxyp.org) framework for implementing the Peptide-level Expression Change Averaging (PECA)-procedure76 using the Bioconductor package (https://www.bioconductor.org/packages/release/bioc/html/PECA.html). This method differs from the common approach, where protein expression intensities are precomputed from the peptide data and an expression change between two groups of samples is first calculated for each measured peptide. The corresponding protein-level expression changes are then defined as medians over the peptide-level changes. For this analysis, we determined the modified t-statistic, which is calculated using the linear modeling approach in the Bioconductor limma (linear models for microarray data) package85. To identify differential expression in the BALF dataset, the comparability of relative expression changes between alternative peptides was investigated by considering signal log-ratios by a two-sample t-test with a p-value ≤ 0.05 corrected for multiple hypotheses testing. For the intracellular proteins, given the substantially higher number of proteins detected, we used a conservative permutation test to decrease the possibility of type 1 error rate with an unadjusted significance level p ≤ 0.05 corrected by the Benjamini–Hochberg method for testing multiple hypotheses.The peptide-level data for the BALF was imported into the GalaxyP (https://www.quiagen.com/ingenuty)]. This analysis was performed on proteins with an FDR corrected p-value ≤ 0.05 as the cutoff for differential expression for both BAL cell and fluid datasets. The IPA core analysis was performed using the difference of the weighted log fold change between comparison groups. We focused on canonical pathways that met a Benjamini and Hochberg (B-H)–corrected p-value obtained using the right-tailed Fisher exact test of ≤ 0.05 , as done previously81. We also examined on Overlapping Canonical Pathways functionality in IPA which is designed to visualize the shared biology in pathways through the common features (genes/proteins) in the pathways. The network of overlapping pathways shows each canonical pathway meeting the statistical threshold of −log  ≥ 1.3 as a single “node”. An edge connects any two pathways when there are at least two common proteins shared between the pathways.To gain insight into the biological significance of differentially expressed proteins, we performed functional analysis using Ingenuity Pathway Analysis [IPA (IPA QIAGEN, Redwood City Supplementary informationSupplementary Table S1Supplementary Table S2Supplementary Table S3


All the tested concentrations of neem leaf extract and the highest concentration of commercial neem product significantly reduced the sporangial germination under in vitro conditions. In in vivo experiment, 3-days old pre-treated seedlings with both concentrations of neem leaf extract and the highest concentration of commercial product showed a significant reduction in the infection indicating possible systemic effect of neem. When the seedlings were treated following the infection with P. halstedii , only the highest concentrations of neem leaf extract and the commercial product showed a significant reduction in the infection indicating curative effect of neem. Possibilities for the control of P. halstedii with neem-derived pesticides are discussed.Two neem-derived pesticides were examined under  Helianthus annuus L. is one of the most important oilseed crops in the world. Downy mildew is a major disease caused by the biotrophic oomycete Plasmopara halstedii (Farl) Berlese et de Toni. This pathogen affects sunflower yield losses, more than 85%, as well as the cost of sunflower crop protection and resistance breeding [P. halstedii as the 16th most important oomycete [P. halstedii have been studied extensively, especially in North America and Europe.Sunflower breeding ,2,3. Frooomycete ,5. The dPlasmopara halstedii infection in the sunflower usually takes place in the below ground plant parts by direct penetration in the roots [P. halstedii occurs much more readily through hypocotyls than through roots [This disease is mostly initiated by the soil-borne oospores and occasionally from infected seeds. In this context, the most susceptible stage of host development is between germination and emergence . Plasmophe roots . Systemigh roots . Seedlingh roots ,10.Plasmopara halstedii rapidly develop races (pathotypes) that can break down the resistance genes in sunflowers [nflowers ,12. Sincnflowers .PI resistance genes) [Downy mildew of sunflower can be controlled by using resistant cultivars, agrotechnical methods, and chemical treatment (with fungicides) of the seeds with Mefenoxam . Howevere genes) . Azadirachta indica A. Juss) plant protection products are known to possess antifeedant, antifungal [Plasmopara viticola (Berk. and M.A. Curtis) Berl. and de Toni in grapevine and found that there was a protective treatment efficacy of over 90%.Widespread fungicide research has led to finding alternate solutions, which are sustainable, economical, and eco-friendly. Plant material and their respective by-products/compounds offer a great scope as an alternative to these chemicals . Neem  and a commercial neem-based pesticide product NeemAzal T/S (denoted as AZA) (containing only 1% azadirachtin) on P. halstedii under in vitro and in vivo conditions in sunflower. Different possible reasons for how the neem-derived pesticides work to control P. halstedii are discussed.No previous records (or literature) are available about the effects of neem-derived pesticides on The chromatogram of the analysis of the standard azadirachtin can be seen in Peak 1 from the spiked and non-spiked neem leaf extract sample was confirmed to be azadirachtin A when compared to the standard azadirachtin A, as they had the same spectrum  and retention time . There wP. halstedii isolate used in the test is shown in Pl6 was not effective against the examined isolate. There were no infected plants among the rest of the differential lines, so these were considered as resistant to the P. halstedii isolate examined. Summing the scores given according to the reactions of the differential lines by each triplet, the examined P. halstedii isolate could be identified as pathotype 704. The result of pathotype characterization of the p < 0.05).The microscopical examination of sporangia was done 24 h after treatment with neem leaf extract and NeemAzal T/S (1% azadirachtin). The statistical analysis shows that all the neem-derived pesticide treatments showed a significant inhibition on the total number of empty sporangia  at reducing the total number of empty sporangia  was not significant in reducing the sporulation as compared to control , in the case of pre-treatment, plant height measured for AZA 0.01% was significantly shorter compared to other treatments of neem leaf extracts, AZA 0.1%, and Mefenoxam, but was not significantly different from the infected control (seed plus inoculum). Plants pre-treated with different concentrations of neem leaf extracts, AZA 0.1%, and Mefenoxam and then inoculated with ed water .In the case for post-treatment , the lowP. halstedii after a period of 19 days . No chlorosis was observed in any of the plants inoculated with It is evident from our result that the azadirachtin A content in the leaves is very low (0.2 µg/g = 1 mg/5 g leaves). The same result was observed by Ghimeray et al. , where tPlasmopara halstedii, the causal agent of sunflower downy mildew in vitro and in vivo conditions. Under in vitro conditions, all the treatments, except the lowest concentration of NeemAzal T/S, showed a significant reduction in the sporangial germination. Our results are similar to those reported by Mirza et al. [Phytophthora infestans (Mont.) de Bary (P. infestans). They found that all the neem products, namely crude neem seed oil, nimbokil , crude neem seed oil terpenoid extract, and neem leaf decoction were effective against mycelial growth, sporangial germination, and sporangium production of P. infestans. Rashid et al. [P. infestans. They found that all the neem products tested significantly inhibited the different developmental stages of this oomycete. Our results are also consistent with Mboussi et al. [Thevetia peruviana seeds  on P. infestans, and concluded that both extracts had the same effects as Ridomil Gold Plus, a chemical fungicide with effective ingredients Mefenoxam and copper, against Phytophthora megakarya, Brasier and M.J. Griffin. The results of this experiment are also in line with previous reports of Ngadze [Azadirachta indica (Neem) was found to be effective against P. infestans both under in vitro and in vivo conditions. The effect of two different neem-derived pesticides, namely neem leaf extract and NeemAzal T/S, was tested against a et al.  where thd et al.  observedi et al.  after thf Ngadze , where Ain vivo tests, in the pre-treatment experiment, we found in our study that both the selected concentrations of neem leaf extract and highest concentration of NeemAzal T/S successfully reduced the infection in sunflower. Our results contradict the findings of Rovesti et al. [P. infestans, but they are in line with the results found by Ngadze [in vivo experiment suggested that extracts of both Allium sativum and neem were effective to control P. infestans in potatoes. Our results were also consistent with Achimu and Schlösser [Plasmopara viticola (Berk. and M.A. Curtis) Berl. and De Toni, 1888, in grapevine. Moreover, Krzyzaniak et al. [P. viticola using plant extract. It can be said that these results might be due to the presence of different biologically active compounds such as azadirachtin in neem leaves and other plant parts. Shakywar et al. [in vivo conditions and they stated that the inhibitory action in the neem leaf extract might be due to azadirachtin present in all parts of the plant. The reduction of the infection in the pre-treatment might be the result of the sensitizing of sunflower defense response towards P. halstedii, which was also reported by Fernandez et al. [Bupleurum gibraltarium against P. halstedii. They reported that the oil pre-treatment might activate the defense response of the seedlings against P. halstedii.During i et al. , where ny Ngadze  where anchlösser , where nk et al.  found thk et al. , where tr et al.  also fouz et al. , where tP. halstedii infection could be the systemic effect of neem. Systemic effect of neem is validated by various studies conducted by Naumann et al. [Betula spp.), respectively. Goel et al. [Pseudomonas syringae pv. tomato and Bhuvaneshwari et al. [Drechslera graminea. One of the possible reasons of controlling n et al.  who founn et al.  and Marin et al. , where tl et al.  demonstri et al.  demonstrin vitro results of this experiment. Perhaps azadirachtin in the neem leaf extract alone may or may not cause this inhibitory action. There might be more than one biologically active compound working synergistically to control the infection that are different from azadirachtin and related substances [In the case of post-treatment, highest concentrations of both the neem-derived pesticides inhibited the infection. This might be attributed to the curative effect of neem-derived pesticides. The curative effect of neem products was observed by Achimu and Schlösser , who stabstances ,33. P. halstedii. It is a naturally available fungicide (the neem tree) and a promising alternative to chemical pesticides for controlling downy mildew in sunflower by whole seedling treatment.Plant extracts possessing different properties against pest and pathogens can prove to be beneficial where chemical pesticides fail; hence, a thorough and extensive research in this field is needed. This is the first report of neem leaf extracts and commercially available azadirachtin exhibiting strong antifungal activity against P. halstedii pathotype 704 under in vitro and in vivo conditions were examined for the first time through this research. Our results proved that neem-derived pesticides can be a valuable fungicide in controlling downy mildew of sunflower. Although it is the first step towards testing neem-derived pesticides against P. halstedii, further research is needed to test the effect of neem extracts and neem-derived commercial products on different pathotypes of P. halstedii under in vitro, glasshouse, and field conditions with different modes of application. In addition, an investigation on the systemic and/or curative effect of neem-derived pesticides against P. halstedii in sunflower by measuring different enzymatic activities in the plants needs to be done. It is highly recommended to test the freshly harvested seeds from the field that were previously treated with neem-derived pesticides to check the presence of different biologically active compounds of neem in them. The effects of neem-derived pesticides on w/v) were prepared by suspending 10 g and 20 g of neem leaf powder, respectively, in 100 mL of distilled water overnight, followed by filtration through a cheesecloth to remove the coarse leaf materials. The filtered extract was centrifuged at 5000 rpm for 5 m, to remove the remaining particles and obtain a clear extract.Air-dried neem leaves were obtained from the local market of Mumbai, India. The leaves were ground into powder using an electric blender. The methodology for preparing neem leaf extract was followed according to Doshi et al.  and PetrA working concentration of 0.01% and 0.1% were prepared of NeemAzal T/S obtained from Trifolio Gmbh, Germany, containing (1% azadirachtin), a registered plant protection commercial product in the European Union, by dissolving 1 mL and 10 mL NeemAzal T/S in 100 mL of distilled water, respectively.To prepare Mefenoxam as a positive control, Apron XL 350 FS  was prepared as per the EU registered rate (3 mg/kg seeds) by homogeneously coating the seeds and keeping them at room temperature until they were dried.To determine the azadirachtin content in the leaves of neem plant, an HPLC analysis was conducted in the Food Analysis laboratory of the Szent István University Gödöllő, Hungary. Five grams of ground leaves were extracted by shaking for 15 min with 100 mL HPLC grade methanol or water followed by subjection to ultrasonication in a water bath ultrasonic device (Raypa Model UCD-150) at a maximum frequency of v = 230 and W = 450 for 5 min. The mixture was stored overnight in a refrigerator. The supernatant was first filtered through filter paper and finally through a 0.22 µm, 25 mm hydrophilic PTFE syringe filter, before injection onto HPLC instrument. For the standard (Std), azadirachtin A  was used for comparison.The HPLC runs and data processing were operated by EZChrom Elite software; external standard solution of 250 µg/mL in methanol was used for the quantitative determination of azadirachtin and their possible derivatives. Peak identification was based on comparing retention time and spectral characteristics with those of standard material.In order to find the factor, which is needed to calculate the azadirachtin content, the following formula was used:The amount of azadirachtin A and the other peaks (which are suspected to be the derivatives or isomers of azadirachtin) in the leaf extracts were calculated by multiplying the area of the peaks with the factor. The final amount was calculated in mg/5 g neem leaves. Plasmopara halstedii were collected in the Jász-Nagykun-Szolnok county of Hungary. The isolate was stored at −70 °C in a deep freezer. Preparation of inoculum and pathotype characterization of the isolate was performed by the internationally standardized method of Trojanová et al. [P. halstedii were used . Seeds of each line (20 seeds/lines) were surface sterilized and germinated between wet filter papers at 21 °C for 3 days. Sporangia from infected sunflower leaves were washed off in bidistilled water. The concentration of inoculum was measured by hemocytometer and adjusted to 50,000 sporangia/mL. The whole seedling immersion (WSI) method by Cohen and Sackston [Sunflower leaves infected by á et al.  and Bán á et al. . Nine suere used   and identified as pathotype 704 according to the international method for pathotype identification by Trojanová et al. [in vitro and in vivo experiment. The á et al. . DetaileInfected sunflower leaves stored in deep freezer were soaked in 30 mL double distilled water to release the sporangia. One milliliter of sporangia suspension was diluted/mixed with 1 mL of each tested concentrations of neem leaf extract or azadirachtin solutions, and with 1 mL of Mefenoxam for positive control in an Eppendorf tube. It was agitated gently to mix uniformly and avoid bursting of sporangia, and was incubated at 16 ± 1 °C for 24 h in the dark in a thermostat. After a 24 h incubation period, samples were observed with a microscope at 200× magnification, to check the effect of neem derived pesticides on the sporangia morphology and release of zoosporangia. Microscopic examination was done for each tested treatment by counting first 50 sporangia/treatment. The experiment was replicated five times with each treatment. Microscopic examination of sporangia in bidistilled water (BW) served as a negative control. Based on the microscopic examination, we establish a germination scale (from 0–2) to identify the morphology of sporangia, wherein, 0 = Completely full sporangia, 1 = Partial empty sporangia, 2 = Completely empty sporangia. This scale is built and developed on the hypothesis that every single released zoospore is capable of infecting the host plant.Plasmopara halstedii sporangial suspension.Seedlings inoculated with Seedlings treated with bidistilled water (BW).Plasmopara halstedii sporangial solution.Treated seeds (Mefenoxam) inoculated with Treated seeds (Mefenoxam) inoculated with bidistilled water (BW).Plasmopara halstedii sporangial solution.Seedlings pre-treated with AZA 0.1% inoculated with Seedlings pre-treated with AZA 0.1% inoculated with bidistilled water (BW).Plasmopara halstedii sporangial solution.Seedlings pre-treated with AZA 0.01% inoculated with Seedlings pre-treated with AZA 0.01% inoculated with bidistilled water (BW).Plasmopara halstedii sporangial solution.Seedlings pre-treated with NLE 10% inoculated with Seedlings pre-treated with NLE 10% inoculated with bidistilled water (BW).Plasmopara halstedii sporangial solution.Seeds pre-treated with NLE 20% inoculated with Seedlings pre-treated with NLE 20% inoculated with double distilled water (BW).The Whole Seedling Immersion (WSI) method, Cohen and Sackston , was useP. halstedii by immersing them in the sporangial suspension, which was adjusted to 50,000 sporangia/mL using a hemocytometer, and then incubated at 16 °C overnight in a dark place. For negative control, germinated seeds were first immersed in different treatments for 2 h, followed by immersion in bidistilled water (BW) for 24 h. Germinated seeds were planted in pots placed in a tray containing the moistened perlite with 5 seeds/pot, and with the five repetitions, and placed in the growth chamber with the controlled conditions . Seedlings were watered regularly for 10 days. After 10 days, when the plants developed true leaves of about 1 mm in length, bidistilled water was sprayed using a hand sprayer on the plant leaves, enclosed in trays with a lid, covered in a dark polyethylene bag (to saturate it with moisture), and kept overnight at 19 °C under completely dark conditions to induce sporulation. The next day after sporulation, the first evaluation was done based on the cotyledons bearing sporangia (white growth). Plant growth characteristics, such as height, was measured as well. Plants were kept back in the growth chamber at 22 °C, with the photoperiod of 12 h, RH = 60%, and watered regularly. After 19 days post-sowing, the second evaluation, i.e., presence or absence of chlorosis, damping-off of seedlings, was done and recorded.Three-day old germinated seedlings of susceptible sunflower var. Iregi szürke csíkos (25 seedlings) were firstly immersed in NeemAzal T/S  or neem leaf extract  solutions, respectively, for 2 h. These treated seedlings were further inoculated with Plasmopara halstedii sporangial suspension.Seeds inoculated with Seeds inoculated with bidistilled water (BW).Plasmopara halstedii sporangial solution.Pre-treated seeds (Mefenoxam) inoculated with Pre-treated seeds (Mefenoxam) inoculated with bidistilled water (BW).Plasmopara halstedii sporangial solution followed by AZA 0.1% solution.Seeds inoculated with Seeds inoculated with bidistilled water followed by AZA 0.1% solution (BW).Plasmopara halstedii sporangial solution followed by AZA 0.01% solution.Seeds inoculated with Seeds inoculated with bidistilled water followed by AZA 0.01% solution (BW).Plasmopara halstedii sporangial solution, followed by NLE 10% solution.Seeds inoculated with Seeds inoculated with bidistilled water followed by NLE 10% solution (BW).Plasmopara halstedii sporangial solution followed by NLE 20% solution.Seeds inoculated with Seeds inoculated with bidistilled water followed by NLE 20% solution (BW).The Whole Seedling Immersion method, Cohen and Sackston , was usePlasmopara halstedii sporangial suspension for 24 h, which was adjusted to 50,000 sporangia/mL using a hemocytometer, followed by respective treatments, for 2 h. For negative control, germinated seedlings were first treated in bidistilled water (BW) for 24 h followed by respective treatments for 2 h. Germinated seedlings were planted in pots placed in a tray containing the moistened perlite with 5 seeds/pot, and with the five repetitions, and placed in the growth chamber with the controlled conditions . Plants were watered regularly. After 10 days of sowing, when the plants developed true leaves of about 1 mm, bidistilled water was sprayed onto the seedlings and enclosed in trays with a lid, covered in a dark polyethylene bag (to saturate it with moisture), and kept overnight at 19 °C under completely dark conditions to induce sporulation. The next day after sporulation, first evaluation was done based on the cotyledons bearing sporangia. Plant growth characteristics, such as height, was measured as well. Plants were kept back in the growth chamber at 22 °C, with the photoperiod of 12 h RH = 60% and watered regularly. After 19 days of sowing, a second evaluation, i.e., presence or absence of chlorosis on true leaves, damping-off of seedlings, was done and recorded.Three-day old germinated seedlings of susceptible sunflower var. Iregi szürke csíkos were first inoculated with in vitro and for the in vivo experiments, ANOVA followed by a post-hoc Tukey test was performed to compare the different treatments in R software v 3.4.0 R Core Team [For both, the ore Team , while g