Drosophila melanogaster is a simple and useful model to study the pathophysiological role of this protein in vivo. It has been demonstrated that the depletion of the TDP-43 fly ortholog (tbph) induces deficient locomotive behaviors and reduces life span and anatomical defects at the neuromuscular junction. In this study, using the known binding specificity of TDP-43/tbph for (UG) repeated sequences, we performed a bioinformatic screening for fly genes with at least 6 (TG) repeats in a row within the 3'-UTR regions in order to identify the genes that might be regulated by this factor. Among these genes, we were able to identify RhoGAPp190 as a potential target of the tbph-mediated neurodegeneration. RhoGAPp190 is a negative regulator of Drosophila RhoA, a GTPase protein implicated in the fine modulation of critical cellular processes including axon branch stability and motor axon defasciculation at muscle level and cognitive processes. We were able to demonstrate that the RhoGAPp190 expression is upregulated in a tbph-null fly model, providing evidence that this deregulation is associated to tbph silencing. Our results introduce RhoGAPp190 as a novel potential mediator in the complex scenario of events resulting from in vivo tbph loss-of-function.TDP-43 is an ubiquitous and highly conserved ribonucleoprotein involved in several cellular processes including pre-mRNA splicing, transcription, mRNA stability and transport. Notwithstanding the evidence of TDP-43 involvement in the pathogenesis of different neurodegenerative disorders (i.e. ALS and FTLD), the underlying mechanisms are still unclear. Given the high degree of functional similarity between the human and fly orthologs of TDP-43,  Drosophila melanogaster ortholog of TDP-43  shares a number of structural and functional features with the human protein. Not only tbph is able to preferentially bind the same (UG)n rich sequences [TDP-43 is an ubiquitously expressed nuclear factor, implicated in several aspects of cellular biology including mRNA splicing, transport and stability . In 2006equences , it is aequences , 11. Thuequences , 13. In Drosophila melanogaster genes (BDGP6) in the ENSEMBL Genes 86 database by selecting (among attributes) the search for sequences of the 3'-UTR of the transcripts. The database interrogation returned 29173 entries. After the removal of 3401 items whose sequence was unavailable, the remaining records were sorted by the number of (TG) repetitions (ranging between 15 and 6). The first step in our investigation on the role of TDP-43 in the pathogenesis of neurodegeneration was to identify genes whose expression could be influenced by this nuclear factor. Given the known binding specificity of TDP-43/tbph for (UG) repeated rich sequences , we sougDrosophila heads than in bodies [Drosophila model lacking tbph expression, thereby suggesting that this nuclear factor could be essential to keep RhoGAPp190 expression finely controlled in physiological conditions. According to this hypothesis, the tbph protein might negatively regulate the RhoGAPp190 mRNA steady state levels.In order to verify if tbph is actually able to influence the expression of these genes, we compared their expression levels in the w1118 line (used as a control) and a previously generated tbph-null allele fly model (tbphΔ23/tbphΔ23) . Startinn bodies , this scn bodies , we analTo further verify whether tbph is involved in the regulation of RhoGAPp190 mRNA levels, we tested the effects of tbph overexpression in neurons of tbph-null flies on RhoGAPp190 transcripts level. To this end, the UAS-GAL4 system was used to drive ectopic expression of a gene of interest under the control of the GAL4 transcription factor . In vivoTo understand whether the functional link between RhoGAPp190 and tbph was direct, we tested the protein-mRNA physical interaction. Two different approaches (RNA-co-immunoprecipitation and RNA-GST-pull down) were used to test this hypothesis. The co-immunoprecipitation (co-IP) experiments were carried out with head extracts deriving from flies expressing a Flag-tagged form of wild -type tbph selectively in neurons, or from flies overexpressing tbph-FL, a variant of tbph mutated within the RNA binding domain  which doRhoGAPp190 mRNA was initially identified as a possible target of tbph/TDP-43 binding because of the UG-repeats stretch (TG)6TA(TG)11 located 1470nt downstream of the stop codon  to discrTo investigate the role of TDP-43 in regulation of protein expression possibly dependent on the presence of the (TG)6TA(TG)11 sequence, equal amounts of these vectors were co-transfected in Hek293 cells after TDP-43 silencing by RNAi (siTDP-43). The RNAi against luciferase (siLUC) acted as the control condition. The anti-GFP immunoblotting showed that the EGFP expression derived from the ΔTG construct #2  lane 1. Drosophila melanogaster RhoGAPp190 factor as an additional player in the complex scenario of tbph-related neurodegeneration. Using a fly model lacking tbph expression, we provide evidence of the genetic interaction between tbph and RhoGAPp190. In addition, RhoGAPp190 seems to be linked to the TDP-43 protein through a regulatory function exerted by the interaction of this nuclear factor with the RhoGAPp190 transcripts. Interestingly, multiple UG-rich sequences, known as preferred binding sequence of tbph/TDP-43, are present within the RhoGAPp190 3’UTR. Apparently, the regulatory effect of TDP-43 on the fly RhoGAPp190 transcript is possibly mediated by these UG-rich sequences, as suggested by combining the results of in silico, in vivo, in vitro and ex vivo analyses. RhoGAPp190 is a Rho GTPase activating protein, initially identified as a binding partner of p120 RasGAP in Src-transformed cells [Drosophila melanogaster, RhoGAPp190 has also been shown to be involved in processes crucial for the proper functioning of nervous system, i.e. the regulation of axon branch stability [In the present manuscript, we identify the ed cells , 24 and ed cells . RhoGAPped cells . In Drostability , the contability .This is particularly meaningful because of the established RhoGAPp190 involvement in processes that are crucial for proper synaptic contacts and nerve signalling  and the The fly lines used are as follows:Drosophila Stock Center. The RNAi efficiency in the used lines was confirmed by qPCR. The tbph RNAi efficiency in the used lines was previously tested [(w1118), (OregonR),  ,  and RNA was extracted according to manufacturer’s instruction. The resulting RNA samples were digested with 1U of DNase RNase-free for 20 min at RT. For cDNA synthesis 500ng of RNA/sample were retrotranscribed with M-MLV reverse transcriptase  and hexameric random primers or poly-(T)20 primer . Specific primer pairs were designed to analyze gene expression levels by qPCR, using sybr green technology . Expression of the house-keeping gene RpL32 was utilized to normalize gene expression of targets. qPCR amplifications were performed using a CFX96 real-time PCR detection system . The relative expression levels were calculated using the 2^-ΔΔCT method [Adult fly males were collected within 24 hours after eclosion, frozen in liquid nitrogen and stored at -80°C. For RNA extraction heads and bodies were separated. Larval brains were dissected from third instar larvae. 10 T method . Statist5’-CGCCTGCCTCCGCTGTTC-3’; Rev RhoGAPp190, 5’-GTTGGAATATGTATTTGAGTATGG-3’; For RpL32, 5’-GCCCAGCATACAGGCCCAAG-3’; Rev RpL32, 5’-AAGCGGCGACGCACTCTGTT-3’.Primer pair sequences were the following: For RhoGAPp190, The primer sequences for the other tested genes are available on request.Newly eclosed flies were transferred to fresh feeding tubes (20 flies/genotype/tube). Four days later they were transferred, without anesthesia, to a 50 ml glass cylinder and their climbing ability was calculated by the percentage of flies able to reach the top of the cylinder in 15s. The test was repeated three times for each fly group and the final percentage of top-scoring flies was calculated from the resulting average values. A minimum of 60 flies/genotype were tested.After rinsing protein G magnetic beads  with PBS+0.02% Tween, anti-FLAG M2 monoclonal antibody  was added to the beads. Fly heads  extracts were prepared by homogenization in Lysis buffer  followed by centrifugation for 5 min at 0.4 rcf. The (UG)9 RNA , 34 was 5′-GGTATAAACTGCTGCGGAAG-3′, and 5′-GACACTGATGATGCGGTAC-3′.homer: 5′-GAAAATAACAAAGATCTGCTTGGCC-3′ and 5′-AAGTGGCCCTTGGGCTTCAG-3′.rpl-52: 5′-AGCGGGCAGTGAGCGCAACGCA-3′ was used for specific reverse transcription of (UG)9 RNA. Amplification of (UG)9 transcripts was carried out with the following oligos: pBSKS 667_687s 5′-TGGCGGCCGCTCTAGAACTA-3′ and pBSKS 903_924s 5′-ATGTGAGTTAGCTCACTCATTA-3′.The pBSKS 929_950as oligo The enrichment fold was calculated by normalizing all data vs the respective inputs then followed by the subtraction of controls Δub (tbph-FL or GST) from GST) n experimental sample overexpressing tbph-wt or w1118 flies. The results were derived from three independent immunoprecipitation experiments and error bars represent standard errors. The statistical significance of differences observed between control- and specific- immunoprecipitation samples were determined by t-test.For GST-pull down, 30μl/sample of GSH beads  were used. Beads were pre-cleared by washing twice with 1 ml of HEGN buffer . 5 μg of each GST-fusion protein was added to the washed beads in a final volume of 1000 μl of HEGN buffer containing 0.5 mg/ml BSA. After 2 hrs of incubation at 4°C, the mix was washed 5 times with HEGN buffer . Then, the GST-resin pellet was resuspended in Trifast reagent (for RNA extraction purposes) or in SDS sample buffer (for Western blot purposes). The data presented are representative results of three independent experiments. The statistical significance of differences observed between control- and specific- precipitation samples were determined by t-testing.For protein analysis, extracts and supernatants were diluted in Laemmli buffer  glycerol, 8% (w/v) SDS, 9.8% (v/v) β mercaptoethanol and 0.1% (w/v) bromophenol blue), boiled at 95°C for 5 min and loaded in 15% SDS-PAGE. Protein samples were transferred to Whatman Protran nitrocellulose blotting membranes  and probed with primary antibodies: rabbit anti-tbph , mouse anti-tubulin CP06 . The membranes were incubated with the secondary antibodies: HRP-labeled anti-mouse or anti-rabbit . An anti-GST antibody, HRP Conjugate  was used to detect GST fusion proteins. Protein detection was assessed with ECL Western Blotting Substrate .Drosophila melanogaster genomic DNA using the following primer couple:Expression plasmids were obtained by cloning the sequences of interest in the pEGFP-C2 vector . RhoGAPp190 3’UTR sequence was amplified by PCR from 5’-ACGGAGCTCAAGCTTGCCTAGATCCCCGCCAGGCCATGGCA-3’; RhoGAPp190_3UTR_EcoRI/KpnI_Rev, 5’-ACGGAATTCTGCAGTCGACGGTACCCAAATGCTTCACAAATATTTGGAAAAT-3’. The restriction enzymes used in the 3’UTR cloning step were SacI and KpnI. Upstream of the SacI cloning site a stop codon for EGFP protein expression was inserted by quick-change site-directed mutagenesis , according to manufacturer’s instructions, by using the following primer couple:RhoGAPp190_3UTR_SacI/HindIII_For, 5’-GGCCGGACTCAGATCTGATCGAGCTCAAGCTTGC-3’;EGFPstopRho_For, 5’-GCAAGCTTGAGCTCGATCAGATCTGAGTCCGGCC-3’.EGFPstopRho_Rev, When required, a 4xFLAG-tag coding sequence followed by its own stop codon was inserted downstream of the EGFP ORF, by using the Sac I cloning site. Cloning directionality was checked by sequencing. Quick-change site-directed mutagenesis  was alsoΔTGs_RhoGAPp190_3UTR_For,5’-TCTGTCTCTCAAAAAACCAAAACCAAATTCTACCGTATCGAATTAGTGAAAAACAACCAC-3’;ΔTGs_RhoGAPp190_3UTR_Rev,5’-GTGGTTGTTTTTCACTAATTCGATACGGTAGAATTTGGTTTTGGTTTTTTGAGAGACAGA-3’.The 4 resulting expression plasmids were the following: EGFP-RhoGAPp190-3’UTR, EGFP-RhoGAPp190-ΔTGs-3’UTR, EGFP-4xFLAG_RhoGAPp190-ΔTGs-3’UTR, EGFP-4xFLAG_RhoGAPp190-3’UTR  glycerol, 50 mM Tris HCl pH = 7.5, 150 mM NaCl, 1% (v/v) TritonX100 and protease inhibitors . Cells were sonicated for 10 min (sonication cycles: 30 sec on/30 sec off) at medium intensity . Then, the solution was centrifuged at 1000 x g for 10 min at 4°C. 12μl of the resulting supernatant protein samples were diluted in Laemmli buffer  glycerol, 8% (w/v) SDS, 9.8% (v/v) β-mercaptoethanol and 0.1% (w/v) bromophenol blue), boiled at 95°C for 5 min and loaded in 15% SDS-PAGE. Protein samples were transferred to Whatman Protran Nitrocellulose blotting membranes  and probed with primary antibodies: mouse anti-GFP , mouse anti-tubulin . The membranes were incubated with the secondary antibody: HRP-labeled anti-mouse . Protein detection was assessed with ECL Western Blotting Substrate . Protein bands were quantified using NIH ImageJ software [Hek293 cells were cultured in Dulbecco’s modified Eagle’s medium  medium supplemented with 10% (v/v) heat inactivated fetal bovine serum (FBS) and 1X antibiotic-antimycotic . For transfection assays, one day before transfection 400,000 cells were seeded in each well of a 6-well plate. Cells were transfected (2 μg plasmid /well) with Effectene reagent , according to the manufacturer’s instructions, and were then incubated at 37°C in a COsoftware . The aveS1 TableList of all the Drosophila melanogaster transcripts and genes containing at least 6 (TG) repeats in a row within their 3’UTR region, identified in the ENSEMBL Genes 86 database.(XLSX)Click here for additional data file.


MT-ND4, MT-ND4L and MT-ND6 subunit genes of Complex I. The pathogenic potential of these mtDNA haplotypes is supported by multiple evidences: first, the LHON phenotype is strictly inherited along the maternal line in one very large family; second, the combinations of mtDNA variants are unique to the two maternal lineages that are characterized by recurrence of LHON; third, the Complex I-dependent respiratory and oxidative phosphorylation defect is co-transferred from the proband’s fibroblasts into the cybrid cell model. Finally, all but one of these missense mtDNA variants cluster along the same predicted fourth E-channel deputed to proton translocation within the transmembrane domain of Complex I, involving the ND1, ND4L and ND6 subunits. Hence, the definition of the pathogenic role of a specific mtDNA mutation becomes blurrier than ever and only an accurate evaluation of mitogenome sequence variation data from the general population, combined with functional analyses using the cybrid cell model, may lead to final validation. Our study conclusively shows that even in the absence of a clearly established LHON primary mutation, unprecedented combinations of missense mtDNA variants, individually known as polymorphisms, may lead to reduced OXPHOS efficiency sufficient to trigger LHON. In this context, we introduce a new diagnostic perspective that implies the complete sequence analysis of mitogenomes in LHON as mandatory gold standard diagnostic approach.We here report on the existence of Leber’s hereditary optic neuropathy (LHON) associated with peculiar combinations of individually non-pathogenic missense mitochondrial DNA (mtDNA) variants, affecting the  Leber’s hereditary optic neuropathy (LHON) is a common cause of maternally inherited vision loss. In the large majority of cases LHON is due to mitochondrial DNA (mtDNA) point mutations, clearly distinct from common polymorphisms normally found in the general population, affecting the mitochondrial function, thus defined as pathogenic. For the first time, we here demonstrate, on the genetic and functional ground, that unusual combinations of otherwise polymorphic and non-pathogenic mtDNA variants are sufficient for causing low-penetrance maternally inherited optic neuropathy in pedigrees fitting the LHON clinical diagnosis. Our findings bridge the blurry border between “pathogenic” and “neutral” mutations in an overall continuum that truly depends on the specific and sometime unique combination of variants characterizing each mitogenome. As a result, we conclude that, for an accurate diagnosis of LHON and possibly of other mitochondrial diseases, the only approach that can disclose all possible causative sources is complete mitogenome sequencing. MT-ND4, m.3460G>A/MT-ND1 and m.14484T>C/MT-ND6 . The. TheMT-Nino acid ,12 was rino acid ,14, and utations ,16. As c variant .MT-ND6 and MT-ND1 have been highlighted as LHON mutational hotspots [More recently, hotspots ,20, sinchotspots . Furtherhotspots ,23. Simihotspots –27. ThusWe here present evidence that unusual combinations of otherwise polymorphic and non-pathogenic mtDNA missense mutations may be sufficient for causing low-penetrance maternally inherited optic neuropathy fitting the LHON clinical diagnosis in independent pedigrees. Our findings bridge the blurry border between “pathogenic” and “neutral” mutations in an overall continuum that truly depends on the specific and sometime unique combination of variants characterizing each mitogenome.MT-ND4, m.3460G>A/MT-ND1 and m.14484T>C/MT-ND6. Clinical histories of affected individuals are reported in detail in Supporting Information , within an invariant stretch of 16 amino acids in primates  variant has been previously reported in seven different haplogroups, being diagnostic for haplogroup H4a. In these databases, the sample classified as GenBank: KC878720 is from our Family 1 and it was previously published [MT-ND6 with the m.14582A>G/MT-ND6 variants is, however, unique to Family 1, when compared to all the other reported cases , finding the m.14258G>A/MT-ND6 variant in two further families, carrying the m.11778G>A /MT-ND4 (haplogroup T1a1) and m.14484T>C/MT-ND6 (haplogroup L2a1a1) LHON mutations, respectively  were RFLP surveyed for the m.14258G>A/MT-ND6 and m.14582A>G/MT-ND6 variants, which appeared always homoplasmic. In Family 2, the proband’s mother and sister were also homoplasmic for all missense variants .In Families 1a-b-c all available individuals along the maternal lines (MT-ND6 and m.14582A>G/MT-ND6) and Family 2  we generated cybrids using enucleated fibroblasts derived from the probands of Families 1a, 1b and 2, as cytoplast donors. As detailed in Material and Methods, different cell clones were obtained harboring each the two combinations of homoplasmic variants and used for subsequent investigations. In MT-ND4L variant in isolation, also displayed a defective respiration with a similar magnitude as LHON cells  of ND6. The m.10680G>A/MT-ND4L variant harbored by Family 2 affects the amino acid A71 of ND4L both in human and ovine CI. This amino acid lies in TM3 of the ND4L subunit. Lastly, the m.12033A>G/MT-ND4 induces the amino acid substitution p.N425S in the loop between TM13 and TM14 of ND4, which faces the mitochondrial matrix. Interestingly, with the only exception of the latter amino acid change, all the other variants affect positions around the putative E-channel of CI [The variant m.14258A>G/el of CI , suggestMT-ND6 and m.14582A>G/MT-ND6 variants on a K1a haplogroup background (n = 14) of this very large family are males, indicating that most likely the combination of missense mutations on a K1 mitogenome characterizing this family is not sufficient to reach and trespass the threshold for LHON in females. Furthermore, general penetrance of these 14 affected members was 13% over the total number of individuals on the maternal line, and 25% over the total number of males. This latter percentage is well below the usual quote of average 50% penetrance in males reported in literature [The current study provides genetic and functional evidence that specific and previously unreported combinations of missense mtDNA variants, which individually obey the definition of population polymorphisms, may exert a sufficient pathogenic potential for being causative of low-penetrance LHON. This, as confirmed by a few other cases retrieved from the literature, now firmly establishes that LHON is a disease that may be determined by a very mild respiratory chain dysfunction, possibly close to the boundary between functional and pathological variability, which depends on the mitogenome sequence variation ,37, and ckground . On the MT-ND6, a mutation previously not recognized as associated with LHON. By screening the entire cohort of LHON families diagnosed in our Institute, we found this variant also in two LHON pedigrees carrying one of the three common LHON primary mutations  or in the ND6 subunit (m.14484T>C/MT-ND6), which are closely assembled with ND4L according to the CI structure , thus with an extremely low possibility of co-occurrence by chance, and consequently further remarking their non-pathogenicity when isolated  and succinic dehydrogenase (SDH) activity staining, were performed . RespiraTotal DNA was extracted by standard methods from blood cells, urinary sediment epithelium and skeletal muscle after informed consent and approval of the internal review board at University of Bologna. Direct sequence analysis of the entire mtDNA molecule was performed on total DNA extracted from skeletal muscle, by Sanger  or Next http://www.mitomap.org) and HmtDB (http://www.hmtdb.uniba.it) [www.phylotree.org) [All the mutations are relative to the revised Cambridge Reference Sequence . The complete mtDNA sequence of the three maternally linked probands of Family 1 (GenBank: KC878720) as well as that from the proband of Family 2 (GenBank: MF039863) have been deposited. All variants of interest were confirmed in all their available maternal relatives by restriction fragment length polymorphism (RFLP) analysis (primers and conditions are available upon request). Mitochondrial DNA copy number was evaluated by qRT-PCR, as previously reported . Populatniba.it) ,48. Haplree.org) .in silico protocol [www.cgl.ucsf.edu/chimera/) on the entire ovine respiratory Complex I (PDB file 5LNK) [Protein conservation analysis and pathogenicity prediction were carried out applying a previously detailed protocol  and MitIprotocol . Positiole 5LNK) .- cells, as previously described [MT-ND4L in isolation (haplogroup I1b), identified after screening our entire fibroblast biobank from patients without mtDNA-based neurological disorders. Cybrids from control fibroblasts were previously generated  and used in this study after the closest mtDNA haplogroup matching with LHON patients cybrids .Cybrid cell lines were generated from patient’s skin fibroblasts  and 143B.Tkescribed . A furth2 at 37 °C. All the experiments were performed using haplogroup-matched wild type controls.Cybrids were grown in complete medium Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum , 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, in an incubator with a humidified atmosphere of 5% CO4 cells/cm2) were seeded in 24 well plates and incubated for different times in complete medium or in glucose-free DMEM supplemented with 5 mmol/L galactose, 5 mmol/L Na-pyruvate and 5% FBS . Viability was determined using the colorimetric sulforhodamine B (SRB) assay [3 Multilabel Plate Counter .For viability experiments, cells (4x10B) assay , by measIsolation of mitochondrial-enriched fraction and assessment of Complex I activity were carried out as previously described . Rotenon24 Extracellular Flux Analyzer , as previously described [Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in adherent cells were measured with an XFeescribed . OCR andThe rate of mitochondrial ATP synthesis was measured in digitonin-permeabilized cybrids using the previously described luciferin/luciferase assay, with minor modifications . Rates w2O2 levels were performed by flow cytometry or fluorescent microscopy using H2DCFDA and MitoSox fluorescent dies , as previously detailed [Quantification of mitochondrial superoxide and Hdetailed .Statistical significance was defined as p-value≤0.05 with Student’s t-test unless otherwise indicated.S1 Text(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S3 Fign = 3; mean ± SD). B. Cell viability in galactose (5 mM) medium for different times . Data are expressed as percentage of T0 .A. Cell viability in complete medium (25 mM glucose) for different times . Data are expressed as percentage of T0 ((TIF)Click here for additional data file.S4 Fig2/min, after the injection of 1μM oligomycin (O), 0.2μM FCCP (F), 1μM rotenone (R) and 1μM antimycin A (AA) (mean ± SD). Asterisks indicate statistical significance .OCR traces as pmol O(TIF)Click here for additional data file.S5 FigA. Mitochondrial superoxide anion production determined by epifluorescence microscopy using MitoSOX fluorescent dye. Cells were visualized with a digital imaging system, using an inverted epifluorescence microscope (magnification x63/1.4 oil objective) at 580nm. Images are representative of 3 different experiments. B. Hydrogen peroxide levels were measured using H2DCFDA by flow cytometry, as described in materials and methods. Data are mean ± SD (n = 3).(TIFF)Click here for additional data file.S6 FigA-B) [C-D) [A and B, the ovine amino acid Ala71  is shown as red labelled sphere, and this variant is associated in each family with Met65 , Ala147 , Leu81  or Val113 , shown as green labelled spheres. In panels C and D, the ovine amino acids Ala147 , His304  and Tyr30  are shown as green labelled spheres. In all panels, residues Glu143/ND1, Glu192/ND1, Glu34/ND4L, Tyr60/ND6, the key residues for the E-channel (near Q-site), are shown as blue labelled spheres [A-B) and adaptive variants for high altitude in Tibet (C-D) are displayed as front (A-C) and upper (B-D) views. Light blue arrows indicate the proposed proton translocation pathway.Positioning of the combinations of variants in three LHON Chinese families (A-B) ,31,32 anB) [C-D) ,42 on stB) [C-D) , using t spheres . The bac(TIFF)Click here for additional data file.


Molecular and cellular mechanisms of neuropeptide-Y (NPY)-mediated gender-difference in blood pressure (BP) regulation are largely unknown.Baroreceptor sensitivity (BRS) was evaluated by measuring the response of BP to phenylephrine/nitroprusside. Serum NPY concentration was determined using ELISA. The mRNA and protein expression of NPY receptors were assessed in tissue and single-cell by RT-PCR, immunoblot, and immunohistochemistry. NPY was injected into the nodose while arterial pressure was monitored. Electrophysiological recordings were performed on nodose neurons from rats by patch-clamp technique.1R, not Y2R protein expression, however, both were upregulated upon ovariectomy and canceled by estrogen replacement. Immunostaining confirmed Y1R and Y2R expression in myelinated and unmyelinated afferents. Single-cell PCR demonstrated that Y1R expression/distribution was identical between A- and C-types, whereas, expressed level of Y2R was ∼15 and ∼7 folds higher in Ah- and C-types than A-types despite similar distribution. Activation of Y1R in nodose elevated BP, while activation of Y2R did the opposite. Activation of Y1R did not alter action potential duration (APD) of A-types, but activation of Y2R- and Y1R/Y2R in Ah- and C-types frequency-dependently prolonged APD. N-type ICa was reduced in A-, Ah- and C-types when either Y1R, Y2R, or both were activated. The sex-difference in Y1R expression was also observed in NTS.The BRS was higher in female than male and ovariectomized rats, while serum NPY concentration was similar among groups. The sex-difference was detected in YSex- and afferent-specific expression of Neuropeptide-Y receptors in baroreflex afferent pathway may contribute to sexual-dimorphic neurocontrol of BP regulation. Several lines of evidence also imply the central mechanisms of NPY in BP regulation and potentially differential role of its receptor activation at different level of baroreflex afferent pathway, such as nodose ganglion (NG) and NTS. Firstly, significant effects of gender on the central actions of NPY on vasopressin and BP have been reported [2R mRNA expression is dramatically increased in the NTS at hypertensive condition, whereas it is decreased in the NG under the same experimental condition [1R locus has implications for heritable autonomic control of the circulation and hypertension, suggesting novel pathophysiological links among the Y1R locus, autonomic activity, and BP [2R expression is upregulated in spontaneously hypertensive rats [2R is also documented in neuroendocrine cells and neuroendocrine tissues including the brainstem of a rodent model of hypertension [The pressor responses induced by neuropeptide Y (NPY) are greater in males compared with age-matched females  and the response , 4, whilii (NTS) , indicatreported ; secondlondition , suggest, and BP . Y2R expive rats  and endortension .1R and Y2R in baroreflex afferent pathway including NG and NTS under normal or hypertensive condition. Moreover, a low-threshold and sex-specific distribution of myelinated Ah-type baroreceptor neurons (BRNs) housed in NG and NTS has been identified [2) [1R and Y2R in NG and NTS at tissue and single-cell level, the effect of direct injection of Y1R or Y2R agonist into NG on the mean arterial pressure (MAP), and the ion channel mechanism of neuroexcitation induced by Y1R and Y2R activation.Collectively, all published records indicate that NPY plays a pivotal role in BP regulation and development of hypertension through either peripheral or central pathway with gender-specific manner. However, there is no published record showing the sex-specific expression and distribution of Yentified –14. The fied [2) –17 and ified [2) , which mfied [2) . Therefovs. 266.4 ± 125.3 pg/ml, P > 0.05, n = 10). In addition, surgical removing of the ovaries did not affect serum NPY content . These data suggested that serum NPY itself may not be the causal factor for the different baroreflex afferent function of males and female.To explore whether the depressor reflex modulation of blood pressure is female hormone-dependent, the baroreceptor sensitivity (BRS) was tested in adult males, age-matched females, as well as ovariectomized (OVX) female rats by measuring the mean arterial pressure (MAP) in the presence of phenylephrine (PE) or sodium nitroprusside (SNP) . The results showed that Y1R expression was lower (P < 0.01) in females compared with age-matched males, which was slightly but not significantly upregulated by ovariectomy . Nevertheless, Y1R was remarkably down-regulated by 17β-E2 treatment   and reversed (P < 0.05 vs. OVX) by 17β-E2 treatment  Figure  and 2C. t Figure  and 2D e1R , which was further downregulated by OVX . There was no difference between males and age-matched females in Y2R/HCN1-positive, OVX dramatically upregulated Y2R level . In the case of HCN1-negative populations, the difference in fluorescent intensity for Y1R was not established among groups. However, the intensity for Y2R of females was higher  and completely reversed by OVX . Even though the averaged results of fluorescent analysis do not completely match with molecular observations, the difference might be explained by the YR whole tissue detection in molecular analysis and the afferent-specific quantification in fluorescence. The YR expression in cells other than neurons such as the satellite cells around the neurons  in A- and C-types, whereas very low expression level of Y1R was found in only 1 of 22 tested Ah-type BRNs  of Y2R  were investigated when NPY and selective agonists of Y1R, Pro-34 and Y2R, NPY13-36 were directly injected into NG , whereas 5 μg NPY-13-36 decreased BP . Most importantly, the averaged data  compared with females with either NPY or Pro-34, suggesting Y1R activation-mediated BP upregulation at the level of NG. Intriguingly, the sex-difference in Y2R-mediated reduction of BP was not conformed and the effect of Y1R was much stronger than that of Y2R, suggesting that Y1R and Y2R activation play an opposite action in BP regulation at the 1st-order neurotransmission of baroreflex afferent pathway, and NPY-mediated upregulation of BP by Y1R stimulation presumably masks BP downregulation due to its Y2R activation.We then tested if Ya Figure  showed t1R in A-type BRNs, the effect of Y1R activation on action potential (AP) trajectory and N-type calcium currents (ICa) was investigated. Firstly, Y1R activation by Pro34 (100 nM), Y1R selective agonist, showed no effects on AP waveshape and discharge profiles , a Y1R selective antagonist and PTX 100 nM, the blocker for G-protein coupled receptor, respectively . This phenomenon may attribute to lacking of the co-localization between KCa1.1 and N-type Ca2+ channels in myelinated A-type cells even though the expression of theses channels could be identified.Upon the expression profile for Ys Figure  but signy Figure . Even th-synapse . Similar2R in sexual dimorphism in BRx afferent function is expected considering its extremely higher expression in low-threshold and sex-specific myelinated Ah-type BRNs , a selective Y2R agonist, markedly prolonged AP duration (APD50) and slowed the maximal downstroke velocity (DVMAX) with increase in AP firing frequency (APFF)  Figure , notably) Figure  and inhia Figure . These d1R  compared with male group, and was completely downregulated in OVX to the equivalent level to males .The NTS is the center converged visceral afferent inputs from Vagus and aortic depressor nerve (ADN) relayed at the NG. The clarification of the expression and distribution of Ya Figure  indicates Figure . The mRN1R and Y2R are observed in baroreflex afferent pathway including NG and NTS by the use of immunoblotting and immunohistochemistry, as well as single-cell RT-PCR technique in identified baroreceptor neurons. Additionally, activation of Y1R and Y2R mediate differential neuroexcitation and Ca2+ channel modulation in myelinated A-, Ah- and unmyelinated C-type BRNs identified by electrophysiological validations. These results suggest that NPY and its receptor system play a crucial role in sexual dimorphism of BRx afferent function and neurocontrol of BP regulation.The major contribution of the current investigation is to demonstrate for the first time that sex- and afferent-specific expression and distribution of Y1R in females and the upregulation by lacking of estrogen at NG level, which may explain at least partially why the BP is lower in females vs. age-matched males and significantly increased by OVX procedure observed in our previous observation [1R mediates significant sympathetic vasoconstriction [2R expression is not sex-specific, it was upregulated by OVX procedure and downregulated by estrogen treatment. Considering that Y1R and Y2R often mediate an opposite response of MAP in the present study , strongly suggesting the Y2R activation-mediated KCa1.1 inactivation [2+ channel inhibition through the coupling mechanism.Since the sex-specific distribution , the keyoadening , 37 was tivation , 36 indi1R and Y2R and an opposite action of Y1R and Y2R activation in blood pressure regulation. Additionally, the difference in peripheral and central mechanism in neurocontrol of circulation mediated by NPY has been identified [1R and Y2R activation in NTS , which was a stark contrast compared with by Y1R and Y2R activation in NG . Even though the central hypotensive action of NPY is led by Y1R activation [1R protein expression is markedly higher in females than that in age-matched male rats, which is downregulated by OVX, and similar expression pattern for Y2R is detected in NTS. This result may contribute, at least in part, to the sex-difference in BP of animal [1R and Y2R from this study would favor the explanation for the sex-difference in IbTX-mediated discharge profiles in the 1st-order BRNs [Even though intriguing observations from the cardiovascular literature have provided quantitative evidence that myelinated and unmyelinated cardiovascular afferents evoke not only different frequency-dependent reflex responses but also potential and distinctly different sensory information processing mechanisms , 39. Theentified , 20, manf animal , 29 and f animal  with an f animal , 42 to rder BRNs , 36 of B1R expression in tissue level of NG is slightly increased but statistical significance is not established between female and OVX model perhaps due to the smaller mass of ganglion tissue leading to a relatively large variation and supported by the notion that Y1R expression could be further downregulation after estrogen treatment. Whereas, in case of Y2R, the expression is identical between sexes but the OVX-mediated upregulation is restored with the treatment of estrogen, implying that OVX led Y2R upregulation would be explained as the compensatory mechanism at NG level to counteract elevated Y1R expression when lacking of estrogen. In addition, immunofluorescence showed that the Y1R expression is further downregulated while Y2R upregulation in HCN1-positive populations and together with the opposite pressor response mediated by Y1R and Y2R, the compensatory neuromodulation of NPY through its receptors in neurocontrol of circulation and BP regulation is sex-dimorphic and estrogen-dependent.Although Y1R and Y2R at either NG or NTS level as well as an opposite pressor response of Y1R and Y2R would greatly impact on a sexual dimorphism of neurocontrol of circulation and BP.Taken together, we conclude that NPY would be a key player in either peripheral or central pathway in the regulation of blood pressure, and collaborative expression pattern between YAn expanded methods section is available in the online-only data supplement.Various doses of phenylephrine (PE) and sodium nitroprusside (SNP) were injected intravenously to measure the sex difference in baroreceptor sensitivity (BRS).1R and Y2R.Western Blot analysis was performed for testing relative expression of YDue to the afferent-specific expression, the antibody against for HCN1 was selected in this experiment as the fluorescent marker for myelinated afferents , 43, so,1R agonist), and NPY13-36(Y2R agonist) were injected, respectively. The net changes in mean arterial pressure (MAP) were collected and analyzed by using the software of Labchart 7.As described in the literature , after rIn order to test the target mRNA examination in afferent-specific manner, qRT-PCR was carried out with identical procedures as previously described  in singlAfferent fiber types of isolated neurons were classified as myelinated A-, Ah-, and unmyelinated C-types according to electrophysiological and pharmacological validations  as well


Enterococcus faecalis and Streptococcus gordonii to understand the role of bacterial proteases and cell wall modifications in bacterial resistance. GL13K was derived from the human salivary protein BPIFA2. Minimal inhibitory concentrations were determined by broth dilution and a serial assay used to determine bacterial resistance. Peptide degradation was determined in a bioassay utilizing a luminescent strain of Pseudomonas aeruginosa to detect peptide activity. Autolysis and D-alanylation-deficient strains of E. faecalis and S. gordonii were tested in autolysis assays and peptide activity assays. E. faecalis protease inactivated L-GL13K but not D-GL13K, whereas autolysis did not affect peptide activity. Indeed, the D-enantiomer appeared to kill the bacteria prior to initiation of autolysis. D-alanylation mutants were killed by L-GL13K whereas this modification did not affect killing by D-GL13K. The mutants regained resistance to L-GL13K whereas bacteria did not gain resistance to D-GL13K after repeated treatment with the peptides. D-alanylation affected the hydrophobicity of bacterial cells but hydrophobicity alone did not affect GL13K activity. D-GL13K evades two resistance mechanisms in Gram-positive bacteria without giving rise to substantial new resistance. D-GL13K exhibits attractive properties for further antibiotic development.Antimicrobial peptides represent an alternative to traditional antibiotics that may be less susceptible to bacterial resistance mechanisms by directly attacking the bacterial cell membrane. However, bacteria have a variety of defense mechanisms that can prevent cationic antimicrobial peptides from reaching the cell membrane. The L- and D-enantiomers of the antimicrobial peptide GL13K were tested against the Gram-positive bacteria  Antimicrobial peptides (AMPs) have been investigated for several decades in an effort to develop alternatives to traditional antibiotics, which face increasing levels of bacterial resistance –5. Natur2), which is derived from the human salivary protein BPIFA2  [Pseudomonas aeruginosa and contributes to their eradication in combination with the aminoglycoside tobramycin [We have developed the antimicrobial peptide GL13K (GKIIKLKASLKLL-NH20orf70) , 15. L-G20orf70) , althoug20orf70) . L-GL13Kbramycin . Studiesbramycin , 18.Streptococcus gordonii, causing cell rupture, reminiscent of autolysis, under continuous nutrient flow conditions [Enterococcus faecalis and S. gordonii. The enantiomers showed significant differences in their effects, suggesting that D-GL13K kills bacteria by evading bacterial resistance without causing new resistance.Immobilized L-GL13K is active against the Gram-positive bacteria nditions . To testnditions  and a prnditions  were tesE. faecalis strains OG1RF (wild-type); a D-alanylation mutant (dltA) of OG1RF (TX5427); a protease negative, autolysis impaired mutant  of OG1RF (TX5128); vancomycin-resistant E. faecalis V583 and S. gordonii DL1 (wild-type) have previously been described [escribed –24. BactS. gordonii DL1 atlS (SGO_2013) mutant was constructed by adapting the marker-less mutagenesis system developed for Streptococcus mutans by Xie et al. [S. gordonii ldh promoter and mutated pheS gene (pheS*) and ermAM antibiotic resistance marker were synthesized and cloned into pUC57 . The S. gordonii pheS* gene contains an A316G mutation and silent mutations after codon 316 to prevent recombination at the chromosomally encoded pheS gene, resulting in the final plasmid, pJHMD1 . This approach avoids off target effects and effects of antibiotics are null. The mutants retain all 5’ and 3’ transcriptional regulators.The e et al. . BrieflyatlS in-frame deletion strain was constructed by a two-step transformation protocol. The linear JHMD1 mutagenesis cassette was PCR amplified from pJHMD1 using the JHMD1For/JHMD1Rev primer pair  to generate a 3.1 kb atlSup-JHMD1-atlSdown PCR product, transformed into S. gordonii DL1, plated on THA containing 5 mg/L erythromycin and incubated anaerobically at 37°C for 48 hours. For the second transformation, two primer pairs, atlS-upFor/atlSupRev and atlS-dnFor/atlS-dnRev, were used to amplify the same 0.5 kb upstream and downstream regions. The upstream product contains a complementary sequence to the downstream product. The PCR products were purified and mixed in equimolar concentrations for SOE-PCR. The 1 kb product was transformed into DL1:atlSup-JHMD1-atlSdn, plated on THA containing p-Cl-Phe and incubated anaerobically at 37°C for 48 hours. The mutation was verified by diagnostic colony PCR. The phenotype of a S. gordonii atlS mutant has previously been described [A markerless mer pair . Furtherescribed .dltA mutant followed the equivalent protocol with the primers listed in The construction of the DL1 2) [L-GL13K (Gly-Lys-Ile-Ile-Lys-Leu-Lys-Ala-Ser-Leu-Lys-Leu-Leu-NH2)  and D-GL2)  were pur5 CFU/ml in 90 or 100 μl THB was added to 10 or 20 μl of a 2-fold serial peptide dilution in 0.01% acetic acid and incubated for 20 h at 37°C in 96-well polypropylene plates. The OD at 600 nm (OD600) was determined in a Synergy HT plate reader . Control wells for growth without peptide and growth medium without bacteria were included. Bacterial cell numbers in the inoculum were verified by colony count.The minimal inhibitory concentration (MIC) assays were performed essentially as described for cationic AMPs , but usiE. faecalis OG1RF or TX5128 were spotted on THA containing 3% gelatin. The plates were incubated at 37°C overnight and gelatinase activity was detected as a turbid halo surrounding the bacterial colonies.Aliquots (5 μl) of overnight cultures of 8 CFU) of bioluminescent Pseudomonas aeruginosa Xen41  that had been washed three times in 0.9% NaCl . Viability of the indicator bacteria was determined after 15 min incubation at 37°C by determining bioluminescence in a Synergy HT plate reader. The luminescence detected in the absence of added peptide was used as a measure of 100% proteolysis.To test proteolytic processing of GL13K peptides, overnight bacterial cultures were centrifuged  and the conditioned supernatants were sterile-filtered . Filtered supernatant (16.8 μl) was incubated with 3.2 μl (3.2 μg) peptide at 37°C for 2 h to allow bacterial proteases in the supernatant to process the peptide. To determine residual peptide activity, 70 μl of 10 mM sodium phosphate buffer, pH 7.4 was added to each sample and then supplemented with 10 μl  were added to the peptides diluted 2-fold (128 to 4 mg/L) in 45 μl of 10 mM sodium-phosphate, pH 7.4. The samples were incubated at 37°C for 2 h and 10 μl were then spotted on THA plates to assess bactericidal activity.These assays were performed according to our established protocol  with somE. faecalis OG1RF, TX5427 or V583 were cultured in a Calgary device for 24h in THB. Biofilms were treated with peptides (100 mg/L) for 4h and surviving cells were sonicated and plated on THA for enumeration of CFUs.To investigate potential resistance development against the GL13K peptides, dilutions of the peptides were prepared and incubated as described for the MIC assay. The next day (day 0), the MIC was recorded and the culture in the well with the highest peptide concentration that allowed growth (i.e 0.5 x MIC) was diluted 1:100 and used as an inoculum for the next round of selection in a new MIC assay and so forth until a stable plateau of resistance was reached .Data were analyzed as described in the figure legends, using Graphpad Prism v. 6.07 .8 CFU/ml wild-type E. faecalis at 15±10 mg/L (N = 4) while 48±23 mg/L (N = 3) of L-GL13K were required for complete killing. This difference was reflected in the inhibition of bacterial growth. D-GL13K showed an average MIC of 13 mg/L, while L-GL13K did not inhibit growth of wild-type E. faecalis at concentrations up to 512 mg/L  and protease-deficient E. faecalis (TX5128) and residual peptide activity tested against P. aeruginosa, which is killed by both peptide enantiomers [E. faecalis caused almost complete proteolysis of L-GL13K but not D-GL13K. In contrast, the protease-deficient strain TX5128 did not show significant proteolysis of either peptide enantiomer   . L-GL13KE. faecalis showed similar differences in susceptibility to the GL13K enantiomers . Although the untreated V583 biofilm contained fewer CFUs than that of the wild-type strain, D-GL13K reduced the viable biofilm CFUs by 3log10 in both strains  appears to regain resistance to L-GL13K while remaining highly susceptible to D-GL13K.To test if n rounds . SimilarLactococcus lactis [E. faecalis, wild-type and mutant bacteria were tested for adhesion to hydrocarbon. Wild-type OG1RF were highly hydrophilic with only 5% adhering to hexadecane  [dltA mutant  . Un-induA mutant . DespiteA mutant . These rE. faecalis, the related oral commensal bacteria S. gordonii were tested. S. gordonii are highly hydrophobic with over 90% binding to hexadecane . Consistent with the results for E. faecalis, wild-type S. gordonii are resistant to L-GL13K while a dltA mutant shows an MIC of 11 mg/L to L-GL13K  than the protease-susceptible L-enantiomer of the peptide. Similarly, a difference in MIC between the L- and D-enantiomers was reported for the synthetic peptide IK8 [E. faecalis TX5128 showed that the activity of L-GL13K also was lower than that of D-GL13K in the absence of bacterial proteases, suggesting that additional resistance mechanisms may function in these Gram-positive bacteria.Secreted proteases are effective defense mechanisms against unmodified peptides. Indeed, the protease-resistant D-GL13K is much more effective against ruginosa , S. gordtide IK8 , whereastide IK8 . Many antide IK8 . HoweverE. faecalis.Although previous results with immobilized GL13K had suggested a role for autolysis in bacterial killing , the prePorphyromonas gingivalis [dltA mutants of both planktonic bacteria and biofilms of S. gordonii and E. faecalis than the wild-type strains. In contrast, D-GL13K was highly effective against both mutant and wild-type bacteria. This could be relevant for potential clinical applications since Gram-positive bacteria have been shown to upregulate the dlt-operon to mask lipoteichoic acids with D-alanine in response to the challenge by antimicrobial peptides [A second major bacterial defense mechanism against cationic AMPs involves the reduction of the overall negative surface charge of outer membrane (Gram-negative) or cell wall (Gram-positive) components. Several Gram-negative species, including ngivalis , reduce ngivalis , 48. Grangivalis –51. Indepeptides , 53. A pdlt mutant to regain resistance to L-GL13K but not D-GL13K further strengthens the clinical potential of the latter peptide.In addition to D-alanylation, the D-enantiomer of GL13K was also able to overcome the resistance mechanisms in the vancomycin resistant strain V583. The ability of the


EGFR T790M-mediated resistance to first- and second-generation EGFR-TKIs. Third-generation EGFR-TKIs, such as osimertinib and nazartinib, are effective for patients with the EGFR T790M mutation. However, there are no direct comparison data to guide the selection of a third-generation EGFR-TKI for patients with different EGFR mutations. We previously established an in vitro model to estimate the therapeutic windows of EGFR-TKIs by comparing their relative efficacies against cells expressing mutant or wild type EGFRs. The present study used this approach to characterize the efficacy of third-generation EGFR-TKIs and compare them with that of other EGFR-TKIs. Treatment efficacy was examined using human lung cancer-derived cell lines and Ba/F3 cells, which were transduced with clinically relevant mutant EGFRs. Interestingly, mutation-related differences in EGFR-TKI sensitivity were observed. For classic EGFR mutations , osimertinib showed lower IC50 values and wider therapeutic windows than nazartinib. For less common EGFR mutations (G719S or L861Q), afatinib showed the lowest IC50 values. For G719S+T790M or L861Q+T790M, the IC50 values of osimertinib and nazartinib were around 100 nM, which was 10- to 100-fold higher than those for classic+T790M mutations. On the contrary, osimertinib and nazartinib showed similar efficacies in cells expressing EGFR exon 20 insertions. The findings highlight the diverse mutation-related sensitivity pattern of EGFR-TKIs. These data may help in the selection of EGFR-TKIs for non-small cell lung cancer patients harboring EGFR mutations.Third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) were developed to overcome  EGFR) gene mutations ; PC-9ER [EGFR exon 19 deletion (delE746-A750) + T790M]; BID007 [EGFR exon 20 insertion (A763_Y764insFQEA)]; and H1975 [EGFR L858R + T790M]. PC9 and BID007 cells were a kind gift from Dr. Susumu Kobayashi . H1975 cells were purchased from the American Type Culture Collection . PC-9ER cells became resistant to erlotinib after chronic exposure to this molecule and acquisition of the EGFR T790M mutation. Cell authentication for H1975 was performed in June 2015.Four human NSCLC cell lines were used: PC-9 [Erlotinib and afatinib were purchased from LC Laboratories . Osimertinib was purchased from Selleck Chemicals . Nazartinib (#B5889) was purchased from Apexbio. Antibodies recognizing total EGFR (#2232), total AKT (#9272), phospho-AKT , total p44/42 MAPK (#9102S), and phospho-p44/42 MAPK  were purchased from Cell Signaling Technology . The phospho-EGFR (Y1068) antibody (44788G) was purchased from Invitrogen/Life Technologies , and the actin antibody was purchased from Sigma-Aldrich .EGFR mutations were cultured in RPMI-1640 growth medium, supplemented with 10% fetal bovine serum, at 37°C in a humidified 5% CO2 incubator. Ba/F3 cells expressing wild type EGFR were cultured in RPMI-1640 growth medium, supplemented with 10% fetal bovine serum, at 37°C in a humidified 5% CO2 incubator with EGF (10 ng/mL). The EGFR mutations examined in this study included: delL747_P753insS (exon 19del); L858R; delL747_P753insS + T790M (exon 19del + T790M); L858R + T790M; A763_Y764insFQEA; Y764_V765insHH; A767_V769dupASV; D770_N771insNPG; G719S; L861Q; G719S + T790M; L861Q + T790M; delL747_P753insS + T790M + C797S (exon 19del + T790M + C797S); and L858R + T790M + C797S.Ba/F3 cells stably expressing either wild type or mutated EGFR were created as previously described . Ba/F3 cThe MTS -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay was performed as previously described . PC-9, PCells were treated with EGFR-TKI at concentrations of 0.01–1 μmol/L for 4 h. Cells were lysed in Cell Lysis Buffer . Equal amounts of protein were loaded per lane on sodium dodecyl sulfate-polyacrylamide gels. Separated proteins were transferred to polyvinylidene fluoride membranes. The membranes were incubated overnight with primary antibodies at 4°C and then incubated with secondary antibodies for 1 h. For the detection of proteins, the membranes were incubated with agitation in LumiGLO reagent and peroxide , and then exposed to X-ray film.EGFR, exon 19del + T790M, Y764_V765insHH, A767_V769dupASV, G719S + T790M, or L861Q + T790M were seeded in 6-well plates. The cells were treated with EGFR-TKIs (0.1 μM) for 48 h. Control cells were treated with the same concentration of the vehicle, DMSO. We analyzed the apoptotic status of cells using the Annexin V Apoptosis Detection Kit APC , according to the manufacturer's protocol. The proportion of apoptotic cells was evaluated by flow cytometric analysis, using the Gallios flow cytometer system .Ba/F3 cells harboring wild type 50 values were calculated using GraphPad Prism software.Statistical analysis was performed using the GraphPad Prism software, version 4.0 . The ICEGFR mutation analyses were performed on genomic DNA extracted from tumor samples by using the PNA-LNA PCR clamp method.This study included NSCLC patient data. The study was reviewed and approved by the Institutional Review Board of Keio University School of Medicine . Tumor samples were obtained with written informed consent.


Saccharomyces cerevisiae upon the loss of the M-2 dsRNA alone and the M-2 along with the L-A-lus dsRNAs. We provide a comprehensive study based on the high-throughput RNA-Seq data, Gene Ontology and the analysis of the interaction networks. We identified 486 genes differentially expressed after curing yeast cells of the M-2 dsRNA and 715 genes affected by the elimination of both M-2 and L-A-lus dsRNAs. We report that most of the transcriptional responses induced by viral dsRNAs are moderate. Differently expressed genes are related to ribosome biogenesis, mitochondrial functions, stress response, biosynthesis of lipids and amino acids. Our study also provided insight into the virus–host and virus–virus interplays.Competitive and naturally occurring yeast killer phenotype is governed by coinfection with dsRNA viruses. Long-term relationship between the host cell and viruses appear to be beneficial and co-adaptive; however, the impact of viral dsRNA on the host gene expression has barely been investigated. Here, we determined the transcriptomic profiles of the host  Totiviridae and Partitiviridae) have widespread homologs in the nuclear genomes of eukaryotic organisms, such as plants, arthropods, fungi, nematodes, and protozoa, suggesting that viral genes might have been transferred horizontally from viral to eukaryotic genomes ) were generated. The absence of corresponding dsRNA(s) in particular cells was confirmed by functional tests, dsRNA gel electrophoresis and RT-PCR . S. cerevisiae M437 [L+M+] K2-killer strain, which naturally possesses L-A-lus and M-2 dsRNAs. Hereafter, upregulation indicates higher and downregulation lower transcription levels in dsRNA(s)-free cells compared to M437 [L+M+].To elucidate changes in gene transcription of the yeast strain induced by different sets of dsRNA viruses, whole transcriptome profiling was performed. Transcriptional changes induced by the absence of viral dsRNA(s) in M437 [L+M−] and M437 [L−M−] cells were compared to the wild type p < 0.05 significance level) after eliminating the M-2 dsRNA  were moderate and did not exceed the limit of four-fold change . Only 19 genes in M437 [L+M−] and 22 genes in M437 [L−M−] cells were upregulated four-fold or more, while downregulation for more than four-fold was documented for 39 genes in M437 [L+M−] and 66 genes in M437 [L−M−] cells in comparison to the reference strain. Representative examples of the up- and down-regulated genes were evaluated by qRT-PCR confirming the data obtained in whole-transcriptome profiling experiments . To characterize the cellular activities connected with the viral dsRNA elimination, we calculated the enrichment of “biological process”, “cellular component” and “function” gene ontology (GO) terms associated with genes of altered transcription. We identified 363 and 231 statistically enriched GO terms of genes altered by the elimination of M-2 and both M-2 and L-A-lus, respectively . In transcriptional response to the M-2 dsRNA elimination, RNA-related processes such as ribosome biogenesis and assembly (Fold enrichment (F.E.) of 7.5 and 8.4, respectively); metabolic processes of rRNA, ncRNA, and tRNA ; and RNA 5′-end and rRNA processing  were positively stimulated A. Nucleoside biosynthetic and metabolic processes were highly enriched , including GMP biosynthesis and nucleoside salvage . Genes involved in RNA secondary structure unwinding, ribosomal subunit and protein export from the nucleus  were upregulated in response to the M-2 virus loss. Functional allocation of the upregulated genes fell into the activities of RNA methyltransferase (F.E. of 10.8), RNA-dependent ATPase and RNA helicase (F.E of 7 of each), and RNA polymerase I and III (F.E of 12.5 of each) . GO termElimination of the M-2 dsRNA resulted in downregulation of genes involved in distinct biological processes . Genes iWhile genes upregulated in the M-2-free cells positioned within highly enriched and quite specific categories, elimination of the L-A-lus and M-2 dsRNAs triggered fewer number of broader processes in terms of upregulation A. More tDownregulated genes are involved in highly similar processes in response to elimination of the M-2 dsRNA alone or both L-A-lus and M-2 viruses A. M437  cells, three interconnected groups related to oxidation-reduction, amino acid biosynthesis and transmembrane transport were built . ProductPRX1-encoded mitochondrial peroxiredoxin with thioredoxin peroxidase activity has most interactions in the group and is known to be induced during oxidative stress , dehydrogenases  and peroxidases . e stress A. Importantly, elimination of either M-2 or M-2 and L-A-lus dsRNA altered expression of several genes, related to oxidation-reduction processes. However, these genes do not overlap and were shown to be involved in distinct processes: in M-2 dsRNA-free cells they are associated with energy generation and in M-2 and L-A-lus free cells they are related to stress response. ARG1, -7 and -5,6) were the most downregulated (more than five-fold) in this group.Interactions among gene products of downregulated genes related to amino acid biosynthesis were represented by the highest confidence level B. CertaiInteractions of negatively regulated genes in transmembrane transport group were represented at medium confidence level B. Most oLTV1, BUD23, NSR1, FYV5, FYV7, LRP1, MRT4 and HGH1). The majority of those upregulated genes are related to ribosomes and RNA processing. In line with previous observation, part of genes conferring increased resistance of target-cell to K2 toxin in corresponding knockout strains were downregulated upon M-2 dsRNA elimination . These negatively regulated genes are involved in several processes . Genes, whose deletions were shown to confer either increased susceptibility  or resistance  to K2 toxin, were preferentially downregulated upon elimination of both L-A-lus and M-2 viruses. We compared the list of differently expressed genes of virus hosting cells, with the genes conferring increased resistance or sensitivity of target cell to the K2 killer toxin . There wIn this study, we addressed the impact of dsRNA viruses on transcriptional status of native strain M437 [L+M+]. Prolonged infection of yeast with L-A-lus and M-2 viruses resulted in versatile coadaptation of viruses and host, thus the strains representing virus-naive conditions—either M-2 free or L-A-lus and M-2 free—were prepared manually. In such a way, transcriptional alterations of wild type strain M437 were described from the perspective of dsRNA-cured cells. We measured transcript levels using RNA-Seq, a robust and an extremely sensitive standard for analysis of global gene expression ,42,43,44The Venn diagram represents numbers of differently expressed genes identified in M437 [L+M−] and M437 [L−M−] cells A.Elimination of M-2 dsRNA alone led to exclusive upregulation and downregulation of 189 and 104 genes, respectively. Combined removal of the L-A-lus and M-2 dsRNAs resulted in unique upregulation and downregulation of 246 and 276 genes, respectively, suggesting that L-A-lus virus has a more profound impact on host gene expression. M437 [L+M−] cells share number of downregulated and upregulated genes  with M437 [L−M−] cells. It might be the consequence of co-adaptation and additive effects of small transcriptional changes induced by the elimination of both dsRNA viruses. There was also a small number of differently expressed genes that were oppositely regulated in M437 [L+M−] and M437 [L−M−] cells, further confirming high compatibility of L-A-lus and M-2 dsRNA viruses.M-2 dsRNA is responsible for toxin and immunity-ensuring component production. Maintenance of K2 toxin demands higher cellular energy production; thus, M-2 removal should be associated with a relief for a host cell by lowering energy costs for synthesis of viral RNA and proteins. Propagation of L-A-lus dsRNA and capsid proteins in host cells also requires more resources than completely cured, dsRNA-free cells. Our study uncovers the processes related to amino acid and lipid biosynthesis, transport and energy production to be positively regulated upon infection with L-A-lus and M-2 dsRNAs, whereas ribosome biogenesis and stress responses are downregulated following the infection. This observation is consistent with the notion that killer cells could be more competitive and less stress-aware due to the presence of the host protection system. It is also possible that dsRNA viruses might have yet unrevealed roles in controlling of cellular RNA metabolism and other processes, not linked directly to the maintenance of a virus in the cell.mak mutations affect M-1 virus propagation by targeting the supply of proteins from the L-A virus and that the translation of the L-A mRNA depends critically on the amount of free 60S ribosomal subunits ) ) and M437 [L−M−] , cured of the M-2 dsRNA and both L-A-lus and M-2 viruses, respectively, were used for gene expression profiling. S. cerevisiae yeast strain į’1 (MATį leu2-2 [kil-0]) was used as the sensitive strain for testing of killing phenotype [kil-K2]) , harborihenotype .Yeast cells were grown in standard YPD medium . To test the K2 activity, MBA medium , adjusted to pH 4 with 75 mM phosphate-citrate buffer and supplemented with 0.002% methylene blue dye, was used.S. cerevisiae strain į’1 and grown for 2 days at 25 °C. Colonies lacking killer phenotype (not forming lysis zones) were selected for dsRNA isolation and visual inspection by gel electrophoresis. Absence of M-2 dsRNA was also confirmed by 2-step RT-PCR. M-2 dsRNA was eliminated from the M437 cells by means of moderate heat treatment. Yeast cells were spread onto YPD-agar plates and incubated at 37 °C for 4–5 days. The surviving colonies were replica-plated on MB-agar plates overlaid with sensitive to K2 toxin S. cerevisiae strain M437 was transformed with pYAK-G-LA-1gag  and yeast colonies after the dsRNA curing process were spotted onto MBA medium seeded with sensitive yeast strain į’1 (1 × 10henotype .8 cells) were washed with 1 mL 50 mM EDTA solution, resuspended in 1 mL 50 mM TrisCl pH 8.8 containing 1% ā-mercaptoethanol and incubated at room temperature (RT) for 15 min. Cells were precipitated and mixed with 800 ģL TES  solution, then 600 ģL phenol added and incubated for 30 min by using moderate shaking. The upper aqueous phase, separated by centrifugation at 15,000 g for 30 min, was treated with 60 ģL 3M NaAc pH 5.2 and 660 ģL 2-propanol. The pellet was collected by centrifugation at 15,000 g for 10 min, washed with cold 75% ethanol and resuspended in 16 ģL of nuclease-free water. For DNA elimination, additional incubation with DNAse I at 37 °C for 30 min was performed. DNAse I was inactivated by adding EDTA to final concentration of 5 mM and incubating at 65 °C for 10 min. Total RNA was used for cDNA synthesis and subsequent PCR reaction for checking M-2 and L-A-lus dsRNA presence. After total RNA isolation, rRNA and other single-stranded RNAs were subsequently removed by precipitation with 2.8 M LiCl for 16 h at 4 °C and centrifugation at 15,000 g for 45 min at 4 °C. Double-stranded RNR was precipitated from the aqueous phase by ethanol [Yeast cells were grown in liquid YPD medium at 25 °C for 16 h with shaking at 250 rpm. Collected cells , according to the manufacturer’s instructions. L-A-lus-specific primers (5′-CATGCATTTAAAAAGTTCTGGAC and 5′-GTGCTAACTAGAGCATGTGTAAGG) and M-2-specific primers (5′-GGGAAAAAATGAAAGAGACTACCACCAG and 5′-GGGCTAGCCGCTGTCACATTC) accordingly were used in cDNA synthesis. The same primers were used to detect L-A-lus and M-2 virus specific cDNAs conducting PCRs with DreamTaq DNA Polymerase . PCR cycling parameters for L-A-lus detection consisted of an initial denaturation at 95 °C for 3 min; following as 30 cycles of 95 °C for 30 s, 54 °C for 30 s and 72 °C for 2 min; and a final extension step at 72 °C for 5 min. The following thermocycler conditions were used for M-2 dsRNA detection: 95 °C initial denaturation for 3 min, followed by 30 cycles of 95 °C for 30 s, 72 °C for 80 s; and a final extension step at 72 °C for 5 min. PCR products analyzed by electrophoresis on 1% agarose gel.https://dna.macrogen.com/) using the Agilent Technologies 2100 Bioanalyzer prior proceeding to RNA-Seq. The RNA integrity number (RIN) of total RNA was greater than 8.0.The total RNA was isolated using GeneJET RNA Purification Kit , according to the Yeast Total RNA Purification Protocol. DNA from total RNA samples was eliminated by DNAse I  treatment at 37 °C for 30 min. RNA quantity and integrity were primarily evaluated by agarose gel electrophoresis see . Additiohttps://dna.macrogen.com/). The cDNA libraries were constructed and sequenced for three independent biological replicates with 100 bp paired-end reads on the Illumina HiSeq 2000 platform . All resulting RNA-Seq data have been made available in GEO, with accession number GSE100290. cDNA library construction, quality control and sequencing were performed by Macrogene Inc., South Korea (S. cerevisiae S288C reference genome (NCBI reference sequence: GCF_000146045.2_R64_genomic_20170309) with the following alignment parameters: maximum number of mismatches 2; minimum length fraction 0.9; minimum identity within the mapped sequence 0.95; maximum number of best-scoring hits for a read 30. The expression values for each transcript were calculated as Reads Per Kilobase of transcript per Million mapped reads (RPKM). The “Exact Test” for two-group comparisons was implemented and the Empirical analysis of DGE tool was applied. Transcripts with expression fold change ≥1.5 and an FDR-corrected p-value ≤ 0.05  were chosen for further analyses.Prior to assembly, reads were subjected to trimming and filtering using CLC Genomics Workbench v. 8.5 . Low quality reads were discarded . We also removed: ambiguous nucleotides (ambiguities 1), adapter sequences and sequences less than 50 nucleotides in length. Reads were mapped to the http://www.yeastgenome.org). Differentially expressed transcripts were subjected to term enrichment analysis using GOTermFinder (http://go.princeton.edu/cgi-bin/GOTermFinder) [p values were calculated with the hypergeometric test, using the Benjamini and Hochberg false discovery rate (FDR) correction for the enrichment of each GO term. Fold enrichment (F.E.) was determined by dividing the frequency of specific gene cluster to the total frequency for each GO term.Specific gene functions and biological pathways were annotated according to SGD  . SignifiThe protein network was created by STRING v. 10  and impoFYV7, ARO10 and TAF10 genes were performed using Luminaris HiGreen qPCR Master Mix , and the protocol according to the manufacturer’s instructions. The following primer pairs were used: TAF10 (5′-CCTATCATTCCCGATGCAGT-3′ and 5′-AGCTCTCGCCTGACTGTTGT-3′), ARO10 (5′-CCTGGTGATGTTGTCGTTTG-3′ and 5′-TGAGCGTTTGAGTGGTCTTG-3′), FYV7 (5′-GGGTACAGCCAAGCAAAATC-3′ and 5′-ATTGCCCTGGCTTCCTTAAT-3′). All measurements were taken in triplicates. The expression ratios were calculated using the 2−ÄÄCT method [Quantitative PCRs for T method .


Acute respiratory distress syndrome (ARDS) is characterized by inflammatory injury to the alveolar and capillary barriers that results in impaired gas exchange and severe acute respiratory failure. Nuclear orphan receptor Nur77 has emerged as a regulator of gene expression in inflammation, and its role in the pathogenesis of ARDS is not clear. The objective of this study is to investigate the potential role of Nur77 and its underlying mechanism in the regulation of endothelin-1 (ET-1) expression in lipopolysaccharide (LPS)-induced A549 cells and an ARDS rat model. We demonstrate that LPS induced Nur77 expression and nuclear export in A549 cells. Overexpression of Nur77 markedly decreased basal and LPS-induced ET-1 expression in A549 cells, whereas knockdown of Nur77 increased the ET-1 expression. LPS-induced phosphorylation and nuclear translocation of NF-κB and p38 MAPK were blocked by Nur77 overexpression and augmented by Nur77 knockdown in A549 cells. In vivo, LPS induced Nur77 expression in lung in ARDS rats. Pharmacological activation of Nur77 by cytosporone B (CsnB) inhibited ET-1 expression in ARDS rats, decreased LPS-induced phosphorylation of NF-κB and p38 MAPK, and relieved lung, liver, and kidney injury. Pharmacological deactivation of Nur77 by 1,1-bis-(3′-indolyl)-1-(p-hydroxyphenyl)methane  had no effect on ET-1 expression and lung injury. These results indicated that Nur77 decreases ET-1 expression by suppressing NF-κB and p38 MAPK in LPS-stimulated A549 cells in vitro, and, in an LPS-induced ARDS rat model, CsnB reduced ET-1 expression and lung injury in ARDS rats. By  to ARDS . It is k to ARDS , 58, 62. to ARDS , 26, 28, to ARDS , 98. Elu to ARDS . Compare to ARDS .Nur77, a member of the nuclear hormone receptor 4A (NR4A) subgroup of the nuclear receptor superfamily, is encoded by an immediate early gene and expressed in multiple tissues in response to diverse stimuli . Origina2 incubator. All studies on these cells were performed with subcultures after three to five passages.A549 cells were obtained from American Type Culture Collection . Cells were cultured in RPMI-1640 medium containing 10% (vol/vol) fetal bovine serum (GIBCO) at 37°C in a 5% COGCCAAACTGGACTACTCCAAGTTTCAAGAGAACTTGGAGTAGTCCAGTTTGGTT-3′ ] was constructed in pGPU6/Neo plasmid vector, and a plasmid carrying a nontargeting control sequence served as a negative control . All of the plasmids mentioned above were synthesized by GenePharma . A549 cells at ∼60–80% confluence were transfected using Lipofectamine 2000 Reagent  according to the manufacturer's instructions, and stable cell lines were obtained by geneticin 418  screening. Overexpression and knockdown of Nur77 were verified by real-time PCR (RT-PCR).Human full-length Nur77 (exNur77) was constructed in pcDNA3.1 plasmid vector, and the empty vector served as a negative control (exNC). Short-hairpin RNA targeting human Nur77 ; LPS and SB-203580 group  were designed using primer premier 5 software (Premier Biosoft) and synthesized by Shenggong . The primer sequences were as follows: Nur77 (human) sense: 5′-ATACACCCGTGACCTCAACCA-3′, antisense: 5′-TTCTGCACTGTGCGCTTGAA-3′; Nur77 (rat) sense: 5′-CGTGCCTTTAAGCCCATAGC-3′, antisense: 5′-TCTGGAATGAGGAGATACATCAGTCT-3′; ET-1 (human) sense: 5′-AAACCCACTCCCAGTCCACC-3′, antisense: 5′-CCAAGTCCATACGGAACAACG-3′; ET-1 (rat) sense: 5′-CTGGACATCATCTGGGTCAACA-3′, antisense: 5′-GGCTCGGAGTTCTTTGTCTGC-3′; GAPDH (human) sense: 5′-ccacccatggcaaattccatggca-3′, antisense: 5′-tctagacggcaggtcaggtccacc-3′; and GAPDH (rat): sense: 5′-CGTATCGGACGCCTGGTTA-3′, antisense: 5′-GACTGTGCCGTTGAACTTGC-3′.The expression of ET-1 and Nur77 mRNA of A549 cells and rat lung tissue was analyzed by RT-PCR. Strand DNA was synthesized with the RevertAid First Strand cDNA Synthesis Kit  as per the manufacturer's instructions. The quantitative RT-PCR was performed on an iQ5 multicolor RT-PCR detection system (Bio-Rad) by using FastStart Universal SYBR Green Master  according to the manufacturer's instructions. The primers for the amplification of Nur77, ET-1, and GAPDH [GenBank accession no. t) value was measured, and the comparative gene expression was calculated by the 2t−ΔΔC method as described previously.After 5 min of initial denaturation at 95°C, PCR was carried out for 40 cycles at 95°C for 10 s and 60°C for 30 s. GAPDH was performed simultaneously and used as the housekeeping gene. The threshold cycle , following the manufacturer's protocol. MPO concentrations in the BAL and lung tissue were measured with a rat MPO ELISA kit  according to the manufacturer's instructions.2HPO4, 2.7 mM KCl, and 1.5 mM KH2PO4) containing 0.1% Tween 20. The membrane was washed three times with PBS-Tween buffer, incubated with primary antibodies against phospho-NF-κB p65 , phospho-p38 MAPK , Nur77 , and β-actin  in PBS-Tween buffer at 4°C overnight, and rinsed three times with PBS-Tween buffer, and then the membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG  for 60 min at room temperature. The blots were subsequently washed three times in PBS-Tween buffer followed by visualization with enhanced chemiluminescence reagents  and exposed to an X-ray film . The scanned images were imported into an image analyzer . Scanning densitometry was used for semiquantitative analysis of the data.Protein concentrations of rat lung tissue homogenate and cell lysates were measured using the bicinchoninic acid protein assay kit (Beyotime) with bovine serum albumin (BSA) as the standard. Extracts containing equal amounts of total protein (20 μg) were loaded on SDS-PAGE (4–15% polyacrylamide), subjected to electrophoresis, and electrophoretically transferred to a polyvinylidene difluoride filter membrane that was then blocked for 2.5 h at room temperature with 5% nonfat milk in PBS  as per the manufacturer's instructions. In brief, cytoplasmic extracts (500 μg) were incubated with anti-Nur77  or negative control IgG  at 4°C on a rotator for 4 h. After a short centrifugation, protein complexes were incubated with 25 μl protein G magnetic beads for 1 h at 4°C on a rotator. Subsequently, the protein beads were precipitated using a magnet. Immune complexes were collected in 20 μl 2× reducing loading buffer  after four washes with Co-IP/wash buffer and then subjected to Western blotting with anti-Nur77 , anti-NF-κB p65 , and anti-p38 MAPK .4/ml) on glass bottom cell culture dishes , incubated overnight, and then treated as described previously. Cells were rinsed two times with 1× PBS (pH 7.4) and fixed in 4% paraformaldehyde at room temperature for 30 min. Fixed cells were washed three times and then permeabilized with 1 ml of 0.3% Triton X-100 for 20 min. After blocking with PBS containing 4% BSA for 30 min, cells were incubated with primary antibody  in 4% BSA/PBS overnight at 4°C and then washed and incubated with FITC-conjugated anti-rabbit IgG antibody (1:150 dilution) for 1 h at room temperature. Cells were washed, dried, and mounted in medium containing DAPI  and imaged on an Olympus Fluoview 500 IX71 Confocal Laser Scanning Microscope.A549 cells were seeded (5 × 101) neutrophils in the alveolar space, 2) neutrophils in the interstitial space, 3) number of hyaline membranes, 4) amount of protein-associated debris, and 5) extent of alveolar septal thickening. The total injury score was derived from the following calculation: score = [20 ×  for HE staining. Six slices were selected from each group, and six fields of each slice were visualized by an Olympus BX53F microscope  with an Olympus DP73 digital camera (Olympus). Pathologists unaware and blinded to the nature and characteristics of the sample examined the HE-stained slides. The degree of pathological damage was evaluated based on lung the injury scoring system  or Nur77  overnight at 4°C. After three washes of 5 min with PBS, sections were incubated with peroxidase-conjugated IgG antibody. Following three rinses of 5 min with PBS, the slides were then stained using the biotin-avidin peroxidase method. After development, slides were counterstained with hematoxylin.Immunohistochemistry was performed after blocking endogenous peroxidase activity with 3% Ht-test for comparison between two groups or one-way analysis of variance followed by Bonferroni's post hoc test for multiple comparisons. Histological scores were analyzed with the Kruskal-Wallis nonparametric test followed by Dunn's pairwise comparison. Significance was defined as P values <0.05. All tests were performed using SPSS version 21.0 software .Data normality was determined by Shapiro-Wilk and Kolmogorov-Smirnov tests (α = 0.05). The data with normality distribution are presented as means ± SE and were analyzed using the two-tailed Student's A and B), and Nur77 was shown to be transferred from nuclei to cytoplasm . Overexpression of Nur77 decreased basal and LPS-induced ET-1 mRNA and protein expression (C and D). Both SC-514 and SB-203580 inhibited LPS-induced ET-1 mRNA and protein expression (E and F).ET-1 mRNA and protein expression was augmented in Nur77 knockdown A549 cells at both basal and LPS-stimulated conditions , A and Bpression , C and Dpression , E and FLPS-induced phosphorylation of NF-κB p65 and p38 MAPK was enhanced by knockdown of Nur77 G and wasI and J, top). Nur77 overexpression blocked this nuclear translocation, and Nur77 knockdown promoted it . The LPS-induced redistribution of phospho-NF-κB p65 and phospho-p38 MAPK was partially inhibited by pretreatment with SC-514 and SB-203580, respectively .LPS triggered phospho-NF-κB p65 and phospho-p38 MAPK to transfer from cytoplasm to nuclei (Co-IP was performed to test the interactions between NF-κB p65/p38 MAPK and Nur77 with cytoplasmic extracts of A549 cells with or without LPS treatment. As shown in A and B), whereas C-DIM8 did not change Nur77 expression in LPS-induced ARDS rats (A and B). CsnB as well as SC-514 and SB-203580 inhibited ET-1 mRNA expression in lung (D and E) in ARDS rats.CsnB, an agonist for Nur77, increased Nur77 mRNA and protein expression in LPS-induced ARDS rats , A and BRDS rats , A and B in lung C and ET- in lung , D and EA and B). SC-514 and SB-203580 pretreatment also exhibited similar effect on LPS-induced phosphorylation of NF-κB p65 and p38 MAPK, respectively (A and B).LPS activated NF-κB and p38 MAPK signaling by increased phosphorylation of NF-κB p65 and p38 MAPK, whereas pretreatment with CsnB partly blocked LPS-induced phosphorylation of NF-κB p65 and p38 MAPK , A and Bectively , A and BImmunohistochemistry of lung tissue sections shows that Nur77 is expressed in alveolar epithelial cells, airway epithelial cells, alveolar macrophages, and bronchial smooth muscle A and exhA–H). Pretreatment with SC-514 and SB-203580 also exhibited lung-protective effects with decreases of lung injury score from 0.78 ± 0.06 to 0.55 ± 0.05 and from 0.78 ± 0.06 to 0.44 ± 0.06, respectively .Rats administered with LPS exhibited altered alveolar capillary barrier with increased total BAL protein concentration A and incA and B). LPS administration caused significantly increased creatinine levels that were partially reversed by pretreatment with CsnB (P < 0.05) (A–E).Liver pathology showed that treatment of LPS caused hemorrhage, focal necrotic and hepatocyte balloon and inflammation, and an increased liver injury score, which were partially alleviated by pretreatment with CsnB , A and Bith CsnB C. Renal ith CsnB D. In LPSith CsnB D. Pretreith CsnB D and red < 0.05) E. C-DIM8 < 0.05) , A–E.ARDS is a clinical syndrome that is characterized by pulmonary edema, refractory hypoxemia, and multiple organ failure resulting from excessive inflammatory response to diverse insults . Nur77 hThe present study shows that LPS induced Nur77 expression and nuclear export both in A549 cells and ARDS rats. In addition, immunohistochemistry of lung tissue sections show that Nur77 is expressed in alveolar epithelial cells, airway epithelial cells, alveolar macrophages, and bronchial smooth muscle cells in the lungs. Nur77, coded by the immediate-early gene NR4A1, is synthesized immediately after the resting cells are stimulated by extracellular signals . The abiNur77 functions through both genomic and nongenomic actions, depending on the stimuli and its subcellular localization . Nur77 pLPS-induced Nur77 nuclear export was detected in the present study, indicating that cytoplasmic Nur77 may predominantly contribute to the mechanism of ARDS. However, our results are different from those where incubation of primary human monocytes with LPS increased Nur77 nuclear translocation . There aBy interacting with NF-κB p65 and p38 MAPK, Nur77 inhibited LPS-induced NF-κB and p38 MAPK activation, resulting in downregulation of ET-1 in LPS-stimulated A549 cells and in the ARDS rat model. These findings give further evidence that nongenomic function of Nur77, such as protein-protein interactions, may contribute to the downregulation of LPS-induced ET-1 expression. Activation of NF-κB p65 and p38 MAPK is involved in the pathogenesis of ARDS through inducing proinflammatory mediators expression , 47, 100This finding is consistent with the fact that Nur77 acted as a negative regulator of ET-1 in thrombin-stimulated vascular endothelial cells through an inhibitory interaction with the c-Jun/AP-1 pathway . AnotherWe demonstrated that CsnB, an agonist of Nur77, decreased pulmonary neutrophilic inflammation and ET-1 expression and attenuated alveolar capillary barrier dysfunction, resulting in alleviation of hypoxemia and improvement of lung, liver, and kidney injury in LPS-induced ARDS rats, whereas, C-DIM8, a pharmacological inactivator of Nur77, exhibited neither influence on ET-1 regulation nor therapeutic effects in ARDS rats.Neutrophils play a key role in the development of ARDS . ExcessiET-1 is one of the inflammatory mediators in the pathogenesis of ARDS , 77, 81.CsnB, a pharmacological activator of Nur77 , 97, hasThe treatment of ARDS predominantly depends on lung-protective ventilation and judicious fluid balance, and no pharmacological therapies have demonstrated robust effectiveness , 72. CsnC-DIM8, an Nur77 antagonist, mimics the effects of short-interfering RNA for Nur77 and deactivates Nur77 , 39, 99.Although the A549 cell line showed a functional resemblance as a type II alveolar epithelial cell, further validation using primary cells is needed.In summary, Nur77 decreases ET-1 expression by suppressing NF-κB and p38 MAPK in LPS-stimulated A549 cells in vitro and in an LPS-induced ARDS rat model. The Nur77 agonist CsnB reduces ET-1 expression and exerts a systemic protective effect in ARDS rats.81160013, 81060007, and 81560321 from the National Natural Science Foundation of China and the 139 Training Plan of Guangxi Medical High Level Backbone Talents and Project of Guangxi Colleges and Universities Key Laboratory of Intensive Care Medicine and Molecular Immunology.This study was supported by Grant Nos. No conflicts of interest, financial or otherwise, are declared by the authors.Y.J., Y.Z., X.H., and W.L. performed experiments; Y.J., Y.Z., X.H., Y.Q., W.L., and S.X. analyzed data; Y.J., Y.Z., X.H., Y.Q., S.X., and L.P. interpreted results of experiments; Y.J., Y.Z., S.R.S., and L.P. drafted manuscript; Y.J., Y.Z., X.H., Y.Q., W.L., S.X., S.R.S., and L.P. approved final version of manuscript; X.H. and S.X. prepared figures; Y.Q. and L.P. conception and design of research; S.R.S. and L.P. edited and revised manuscript.


Mst77F is a Drosophila SNBP with unique DNA condensation properties in vitro, but its role during spermiogenesis remains unclear. Here, we show that Mst77F is required for the compaction of sperm DNA and the production of mature sperm, through its cooperation with protamine-like proteins Mst35Ba/b. We demonstrate that Mst77F is incorporated in spermatid chromatin as a precursor protein, which is subsequently processed through the proteolysis of its N-terminus. The cleavage of Mst77F is very similar to the processing of protamine P2 during human spermiogenesis and notably leaves the cysteine residues in the mature protein intact, suggesting that they participate in the formation of disulfide cross-links. Despite the rapid evolution of SNBPs, sperm chromatin condensation thus involves remarkably convergent mechanisms in distantly related animals.In most animals, the bulk of sperm DNA is packaged with sperm nuclear basic proteins (SNBPs), a diverse group of highly basic chromosomal proteins notably comprising mammalian protamines. The replacement of histones with SNBPs during spermiogenesis allows sperm DNA to reach an extreme level of compaction, but little is known about how SNBPs actually function  We concluded that these new alleles, named Δ1Mst77F, Δ2Mst77 and Δ3Mst77F, are null or at least strong loss-of-function alleles. All three alleles induce complete male sterility at the homozygous state. We noted, however, that trans-heterozygous combinations of these mutant alleles occasionally produced rare progeny (about 0.5% of the control)   gene targeting system  to obtaif Mst77F a in ordegermline . The tarromosome b. Among op codon c. Westernt males d. We concontrol) . AlthougDf(3L)BSC452, a deficiency that uncovers Mst77F , did not complement Mst77F mutant alleles. Surprisingly, however, Df(3L)ri-79c ri-79c does not uncover Mst77F and does not affect its normal expression . Similarly, we showed that the c06969Mst77F Piggybac insertion complemented Δ1Mst77F sterility (b). Unfortunately, we have not been able to obtain the 1Mst77F allele, which is probably no longer available.As expected, henotype ) fully cterility . Consistterility  and did 1/DfMst77F(3L)ri-79c males is probably unrelated to Mst77F function. Analysis of the new ΔMst77F alleles however establishes that Mst77F is importantly required for male fertility.We conclude that the previously reported sterility of 2.2.Mst77F mutant males revealed the absence of mature sperm in seminal vesicles, thus explaining the sterility phenotype (a). As expected, the initiation of spermiogenesis (the differentiation of post-meiotic spermatids) was not affected by the absence of Mst77F. Groups of 64 elongating spermatid nuclei were indistinguishable in wild-type and Mst77F mutant males until the late canoe stage, shortly before individualization (b). Following this stage, bundles of mutant spermatids appeared severely disorganized compared with the tightly clustered control spermatids (b). We then stained testes with fluorescently labelled phalloidin to reveal the actin-based individualization complexes (IC) (c). Individualization occurs with the progression of these structures from nuclei toward the distal tip of flagella  antibody additionally detected an abundant, specific band of approximately 17 kDa . Moreover, only this smaller Mst77F band was detected in extracts prepared from seminal vesicles, which only contain mature sperm . Taken together, these results suggest that Mst77F is synthesized as a precursor that undergoes proteolytic maturation, with only the shorter form persisting in mature sperm.We also used an anti-Mst77F [171–184] antibody that was raised against a peptide from the C-terminus of Mst77F protein . Interes5r panel) . In prota, lower panel) is in apparent support of the processing of Mst77F from its N-terminus. However, the Mst77F-EGFP transgene failed to rescue the fertility and the spermiogenesis defects of Mst77F mutant males, despite the apparent incorporation of this recombinant protein in spermatid nuclei (P{mRFP1-gMst77F}) (a). In contrast with Mst77F-EGFP, the mRFP1-Mst77F transgene rescued the fertility of Mst77F mutant males . Strikingly however, the mRFP1-Mst77F nuclear fluorescence completely disappeared at the onset of individualization , thus recapitulating the immunostainings obtained with the anti-Mst77F [FL] antibody. To confirm that N-terminal processing of the recombinant protein induced the loss of mRFP1 fluorescence, we analysed protein extracts from testes and seminal vesicles of rescued males in WB. In testicular extracts stained with the anti-Mst77F [171–184] antibody, the 25–37 kDa Mst77F band (L) shifted to an apparent size of about 55 kDa , as expected from the presence of the mRFP1 tag (expected size of the recombinant protein: 60 – 65 kDa). However, the size of the small band (S) remained unchanged . In seminal vesicles of the same males, the full-length recombinant protein was not detected but the short isoform was still detected . Finally, we also analysed protein extracts from testes and seminal vesicles of Mst77F-EGFP males (in a Mst77F wild-type background). Interestingly, a specific band migrating at an intermediary position (about 37 kDa) was detected with anti-Mst77F [171–184] antibody in both testes and seminal vesicles of Mst77F-EGFP males , thus indicating that the recombinant protein is processed like the endogenous Mst77F. By contrast, the S-GFP band was not detected using the anti-Mst77F [FL] antibody . Thus, both immunofluorescence and western blot analyses indicate that the anti-Mst77F [FL] antibody recognizes an epitope in the N-terminus and can thus be considered specific to pre-Mst77F (figure 6c).The fact that the Mst77F-EGFP protein (with the EGFP fused to the C-terminus of Mst77F) remains present in mature sperm nuclei in a wild-type background figure 6Mst77F}) a. In connt males . We thenMst77F} aOur results thus demonstrate that Mst77F is first synthetized as a precursor protein (pre-Mst77F), which is processed by proteolysis of its N-terminus to generate the mature form of Mst77F present in sperm chromatin.3.Mst77F demonstrates that this SNBP is required for male fertility by allowing the proper organization of sperm chromatin following the histone-to-protamine transition. We observed that, in the absence of Mst77F, spermatid nuclei appeared normal up until the passage of the IC. The observed disruption of Mst77F mutant spermatid nuclei during individualization suggests that incomplete nuclear compaction physically disturbs the passage of IC around nuclei. However, we cannot exclude the possibility that, incomplete DNA compaction indirectly triggers the arrest of spermiogenesis through a putative checkpoint of spermatid chromatin state.The analysis of loss-of-function alleles of Mst35Y copies are also present on the Y chromosome, but they so far appear non-functional  antibody. Among the individual 19 lines, 15 lines showed no bands corresponding to Mst77F protein (data not shown). Three individual lines  were randomly selected and the genomic DNA lesion was precisely determined by PCR amplification with the primer set 5′-ATATGGCGCCGATCTGCG-3′ and 5′-TGGTTCCTGCGGAAGTGC-3′ and sequencing.Mst77F mutants were generated as previously described . The 20 n system . gRNA-Ms4.3.4.3.1.Mst77F gene locus  to 17-bp downstream of the end of the 3′UTR) was amplified from single 1118w female genomic DNA using the following primers: 5′-ATCGAATTCAGTGGTTGAAACCCCGG-3′ and 5′-ATCGCGGCCGCAATATGGGTAGAAATTTGATCAGAC-3′. The PCR fragment was cloned into the EcoRI and NotI of the pBluescript SK+ vector (pBS-gMst77) and then transferred into the pW8 vector. Transgenic flies were generated by standard P element-mediated germline transformation.The genomic region covering the 4.3.2.mRFP1 coding sequence was inserted into the pBluescript SK+ vector with SalI and EcoRI sites (pBS-mRFP1). PCR fragment amplified from the pBS-gMst77F vector with 5′-ATCGAATTCATGAGTAATCTGAAACAAAAGGA-3′ and 5′-ATCGCGGCCGCAATATGGGTAGAAATTTGATCAGAC-3′ primers was inserted into the pBS-mRFP1 vector with EcoRI and NotI sites (pBS-mRFP1-g2Mst77F). Next, PCR fragment amplified from the pBS-gMst77F vector with 5′-ATGCGGTACCAGTGGTTGAAACCCCGG-3′ and 5′-ATCGTCGACTTTGCAACCAATTCTTGCTCG-3′ primers was inserted into the pBS-mRFP1-g2Mst77F vector with KpnI and SalI sites. To delete 6 bp SalI site before mRFP1 start codon, mutagenesis was performed using 5′-ATGGCCTCCTCCGAGGACG-3′ and 5′-TTTGCAACCAATTCTTGCTCG-3′ primers. Finally, this construct was subcloned into pW8 with KpnI and NotI sites.To insert the mRFP1 tag into the N-terminal of the Mst77F protein, first, the 4.3.3.eGFP cDNA without stop codon was cloned into the pBluescript SK+ vector in SalI and EcoRI site, and sequentially 6xHis tag with stop codon was inserted in EcoRI and SpeI site. A downstream of 985 bp from just after stop codon, which included the Tpl94D 3′UTR was amplified from the y w genomic DNA with 5′-ATGACTAGTTCATCATGTCACCCACTTCAC-3′ and 5′-ATGCGGCCGCTTTCATCCAGCTGAAATCGC-3′ primers and cloned into the pBS-eGFP-6xHis vector in SpeI and NotI site. Next, 956 bp upstream from TSS of Tpl94D and open reading frame (ORF) without stop codon was amplified from the y w genomic DNA with 5′-ATGCGGTACCGTTATACTAAGGGCTACC-3′ and 5′-ATGCGTCGACTAAGTCTGATATGAAAATGC-3′ primers, and cloned in KpnI and SalI site. This construct was subcloned into the pW8 vector with KpnI and NotI sites.4.4.4.4.1.Mst77F was obtained from the Drosophila Gene Collection (DGC) (Clone ID #RH09844). Mst77F cDNA was amplified using a primer set , and cloned into the EcoRI and XhoI sites of the pGEX-4T-1 plasmid , and into EcoRI and SalI sites of the pMAL-cRI plasmid (New England Biolabs). These plasmids were both transformed into E. coli BL21-CodonPlus (DE3)-RIL (Agilent Technologie). First, E. coli was incubated at 37°C for 3 h, and after adding 1 mM isopropyl beta-d-thiogalactoside (IPTG), incubated at 30°C for more 3 h. Cells were harvested, and the lysis buffer  was added, and briefly sonicated. After centrifugation, the supernatant was collected as a soluble fraction. For glutathione S-transferase (GST) fused Mst77F recombinant protein, the soluble fraction was incubated with glutathione sepharose 4B  and eluted by glutathione, and dialysed with 1× PBS buffer, and used as an antigen for the rabbit polyclonal antibody. For maltose-binding protein (MBP) fused Mst77F recombinant protein, the soluble fraction was incubated with amylose resin (New England Biolabs) and eluted by maltose, and dialysed with 1× PBS buffer, and used for the affinity purification.Full-length cDNA of 4.4.2.Mst35Bb was obtained by PCR from cDNA library of y w testicular total RNAs. Mst35Bb cDNA with EcoRI and XhoI restriction site was amplified using a primer set , and cloned into the pGEX-4T-1 plasmid  in EcoRI and XhoI site, and the pMAL-cRI plasmid (New England Biolabs) in EcoRI and SalI site. The following steps were performed as anti-Mst77F [FL] antibody generation.Full-length cDNA of 4.4.3.Tpl94D was amplified by RT-PCR from y w testicular total RNAs using a primer set , and cloned into the BamHI and XhoI sites of the pET-21a (+) plasmid (Novagen) in BamHI and XhoI site. This plasmid was transformed into E. coli BL21-CodonPlus(DE3)-RIL cells (Stratagene). Inclusion body from this E. coli culture was suspended in 8 M urea and applied to two sequential columns, HiTrap Q and HiTrap SP  that were linked in tandem, as previously described for a recombinant histone purification [The full-length cDNA of fication . The unf4.5.Testicular protein extract and WB were performed as previously described . Fifteen4.6.1118w females each. After 7 days, parents were discarded, and the total number of adult progenies was determined at 18th day. Finally, the average number of the adult progenies in all vials was calculated.Ten single 0–1-day-old males were crossed with two virgin 4.7.For each testis, the number of normal (bundled) and abnormal (disrupted) post-IC spermatid nuclear bundles was counted to determine the following ratio: number of normal bundles/total number of bundles. Twenty testes were analysed for each genotype and the ratio average was calculated.4.8.mageJ and Adobe Photoshop software.For each analysed genotypes, at least five pairs of testes with seminal vesicles from 0–1-day-old males were dissected, stained and observed as previously described . Primary