Nucleoprotein (NP) and matrix protein 1 (M1) are highly conserved among influenza A viruses and have been attractive targets to develop vaccines to elicit cross-reactive cytotoxic T lymphocytes (CTLs). Yet, external antigens are often presented on major histocompatibility complex class II molecules and elicit humoral immune responses. In this study, we present a physical radiofrequency adjuvant (RFA) to assist recombinant NP and M1 to elicit potent CTL responses. We found recombinant NP/M1 immunization in the presence of RFA could elicit potent anti-NP CTLs and confer significant protection against homologous viral challenges, while NP/M1 immunization alone failed to elicit significant CTL responses or confer significant protection. Interestingly, RFA failed to elicit potent anti-M1 CTL responses or anti-NP or anti-M1 antibody responses. Different from RFA, AddaVax adjuvant was found to significantly increase NP-specific antibody responses but not CTLs. NP/M1 immunization in the presence of RFA or AddaVax similarly reduced body weight loss, while only the former significantly increased the survival. We further found NP/M1 immunization in the presence of RFA did not significantly increase serum IL-6 release (a systemic inflammatory mediator) and rather reduced serum IL-6 release after boost immunization. NP/M1 immunization in the presence of RFA did not induce significant local reactions or increase body temperature of mice. The high potency and safety strongly support further development of RFA-based recombinant NP/M1 vaccine to elicit cross-protective immunity. Vaccine remains the most effective and cost-effective means to control influenza . Current+ and CD8+ T cells against H5N1 virus in human individuals after seasonal influenza A virus infection [Nucleoprotein (NP) and Matrix 1 (M1) are attractive targets for universal influenza vaccine development . Studiesnfection . NP has nfection  and chimnfection . Viral vnfection ,8,9. NP-nfection ,11. In fnfection . In anotnfection . In thisnfection .Besides the novel types of viral vector and mRNA-based vaccines, incorporation of vaccine adjuvants into traditional protein-based vaccines may also elicit vaccine-specific CTL responses and confer cross-protection. Considering protein-based vaccines are mainly presented on MHC class II molecules and elicit humoral immune responses , the canOur recent studies found non-invasive RF treatment of the mouse skin followed by intradermal (ID) delivery of model antigen ovalbumin (OVA) or influenza pandemic 2009 H1N1 vaccine could elicit potent humoral immune responses and at the same time induce OVA and recombinant HA (rHA)-specific CTL responses via induction of cross-presentation of protein antigens . FurtherRecombinant NP (11675-V08B) and M1 (40010-V07E) of influenza A/Puerto Rico/8/34/Mount Sinai (H1N1) expressed by the baculovirus-insect cell expression system were purchased from Sino Biological US Inc. . AddaVax  was purchased from InvivoGen . Fluorescence-conjugated antibodies were purchased from BioLegend .C57BL/6 mice  were purchased from Charles River Laboratories . Animals were housed in animal facilities of University of Rhode Island (URI) and anesthetized for hair removal, RF treatment, and immunization. Animal experiments involving influenza viruses were conducted in animal biosafety level 2 (ABSL2) facility of URI. All animal procedures were approved by the Institutional Animal Care and Use Committee of URI.2 area was used as in our previous report [A cosmetic fractional bipolar RF device equipped with 12 × 12 array of microelectrodes in 2 × 2 cms report . This deHair on the lateral dorsal skin of mice was shaved and completely removed with the help of a hair removal lotion (Nair) as shown in our previous report . Next da2SO4. Optical absorbance (OD450nm) was read in a microplate reader (Molecular Devices). Serum antibody titer was defined as the reciprocal dilution factor that resulted in OD450nm that was ~3 times higher than the background values. For detection of subtype antibody titer, HRP-conjugated anti-mouse IgG1  and IgG2c  secondary antibodies were used.Serum antibody titer was measured by enzyme-linked immunosorbent assay (ELISA) as in our previous report . In deta+ and CD8+ T cells in peripheral blood mononuclear cells (PBMCs), a small volume of blood (~50 μL) was collected into heparinized tubes followed by red blood cell (RBC) lysis. PBMCs were then stimulated with 1 μg/mL NP or M1 in the presence of 4 μg/mL anti-CD28 antibodies overnight. Next day, Brefeldin A  was added 5 h before cell harvest. PBMCs were then stained with fluorescence-conjugated anti-CD4 (RM4–5) and anti-CD8 (53–6.7) antibodies, fixed and permeabilized, and then stained with fluorescence-conjugated anti-IFNγ (XMG1.2) and anti-IL4 antibodies (11B11). Cells were then subjected to flow cytometry analysis in BD FACSVerse.To measure vaccine-specific CD4n = 5) were infected with 100, 101, 102, 103, 104, and 105 TCID50 influenza viruses. Survival and body weight were monitored daily for 14 days. LD50 was calculated by the method of Reed and Muench [Mouse-adapted influenza A/Puerto Rico/8/1934 (H1N1) viruses  were obtained from BEI Resources. LD50 of PR8 viruses was first determined . In bried Muench . For letSerum IL-6 levels were measured by mouse IL-6 ELISA Ready-SET-Go kit . Values were expressed as mean ± SEM (standard error of the mean). One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test was used to compare differences for more than two groups, except otherwise specified. P-value was calculated by PRISM software  and considered significant if it was <0.05.+ and CD8+ T cells. Mice were subjected to prime/boost immunizations of ID NP/M1 alone or in the presence of RFA, or IM NP/M1 in the presence of AddaVax, or ID PBS . Cellula+CD4+ T cells as compared to ID NP/M1 immunization alone. Percentage of NP-specific IFNγ+CD4+ T cells in NP/M1/RFA group was increased by ~3.6 folds as compared to that in NP/M1 group. IM NP/M1 immunization in the presence of AddaVax also significantly increased percentage of NP-specific IFNγ+CD4+ T cells to a level similar to that in NP/M1/RFA group. We further found ID NP/M1 immunization in the presence of RFA also significantly increased NP-specific IFNγ+CD8+ T cells as compared to ID NP/M1 immunization alone  are commonly used as systemic inflammatory mediators and their serum levels are highly associated with systemic reactogenicity of vaccines in humans . Due to Mice were then challenged with 4 × LD50 of PR8 viruses and body weight was monitored daily for 14 days. As shown in Survival of mice after viral challenges was also explored. All mice in PBS control group died or reached humane endpoint of euthanasia within 9 days. Two out of 5 mice in NP/M1 group and 3 out of 5 mice in NP/M1/AddaVax group survived the lethal viral challenge, while 4 out of 5 mice in NP/M1/RFA group survived the challenge . ID NP/M+CD4+ T cells. The potential role of NP-specific IFNγ+CD4+ T cells in induction of potent IFNγ+CD8+ T cells and overall protection remains to be explored. This study indicated RFA was effective to boost recombinant NP/M1 vaccination. RFA was found to significantly increase NP-specific CTL responses and NP/M1-induced protection against homologous viral challenges. External proteins are mainly presented on MHC class II molecules and elicit humoral immune responses. The induction of significant CTL responses against recombinant NP hinted RFA enabled cross-presentation of external antigens. In our previous report, we found RFA also enabled cross-presentation of OVA and rHA to induce potent CTL responses . Interes+CD8+ T cells will be also assessed to explore the ability of NP/M1 immunization in the presence of RFA to elicit durable CTL responses and long-term protection.Our study compared relative immunogenicity and protective efficacy of ID NP/M1 immunization in the presence of RFA to IM NP/M1 immunization in the presence of AddaVax. Due to the high risk of AddaVax adjuvant to induce significant local reactions following ID delivery , IM routRFA was safe to boost vaccination. Serum IL-6 levels were used in our study as a systemic inflammatory mediator due to its close association with systemic adverse reactions of vaccines . RFA faiOther strategies have been explored to elicit anti-NP and anti-M1 CTL responses and confer protection against influenza viral infection. These strategies include the development of DNA and viral vector vaccines and through virus-like particle (VLP) platforms ,26,27,28Conserved internal antigen-based universal T-cell vaccines are under development to induce cross-reactive CTL responses and confer cross-protection against influenza A viruses. Vaccine adjuvants hold a great promise to induce cross-presentation of influenza internal antigens and elicit potent CTL responses. Yet, the majority of approved adjuvants mainly enhance humoral immune responses. We took advantage of our recently developed physical RFA capable of elicitation of potent CTL responses against protein antigens to develop recombinant NP/M1-based universal T-cell vaccines. Results from the current study support the potency and safety of RFA to aid recombinant NP/M1 to induce potent NP-specific CTL responses and protection against homologous viral challenges in murine models. Our data support further development of the physical RFA and recombinant NP/M1-based universal T-cell vaccine.


Particularly, a novel reassortant influenza A(H7N4) virus was first identified in a woman and her backyard poultry flock in Jiangsu Province, China, posing a serious threat to public health. Here, we describe the genetic characterization and pathogenicity of the nine influenza A(H7N4) isolates. Phylogenetic analysis indicated that complex viral gene flow occurred among Asian countries. We also demonstrated a similar evolutionary trajectory of the surface genes of the A(H7N4) isolates and Jiangsu human-related A(H7N4) viruses. Our A(H7N4) isolates exhibited differing degrees of virulence in mice, suggesting a potential risk to other mammalian species, including humans. We revealed multiple mutations that might affect viral virulence in mice. Our report highlights the importance and need for the long-term surveillance of avian influenza virus in migratory birds combined with domestic poultry surveillance along migratory routes and flyways and, thereby, the development of measures to manage potential health threats.A 2-year surveillance study of influenza A viruses in migratory birds was conducted to understand the subsequent risk during the migratory seasons in Dandong Yalu River Estuary Coastal Wetland National Nature Reserve, Liaoning Province, China, a major stopover site on the East Asian-Australasian flyway. Overall, we isolated 27 influenza A viruses with multiple subtypes, including H3N8  viruses, showing a similar evolutionary trajectory with Jiangsu human A(H7N4) viruses in HA and NA genes. Moreover, our isolates exhibited variable virulence (including moderate virulence) in mice, suggesting a potential risk to other mammalian species, including humans. Similar to the H5 low-pathogenicity avian influenza viruses (LPAIVs), the H7 LPAIVs are capable of mutating into highly pathogenic avian influenza (HPAI) variants that can cause high fatality in poultry and pose a serious threat to public health . Since tA novel reassortant AH7N4) virus was first identified in a woman and her backyard poultry flock in Jiangsu Province, China, in January 2018 . PreviouN4 virus n = 2), H4N6 (n = 2), H4N7 (n = 2), H7N4 (n = 9), H7N7 (n = 1), H10N7 (n = 7), and H13N6 (n = 4)  . Here, w (n = 4) .The whole genomes of Liaoning A(H7N4) (Ln/H7N4) isolates were sequenced and compared with each other. Most gene segments among Ln/H7N4 isolates shared a relatively high nucleotide identity (99.7 to 100%), except for the polymerase acidic (PA), nucleoprotein (NP), and nonstructural (NS) genes, which showed 93.6 to 100%, 93.5 to 100%, and 96.4 to 100% nucleotide identity, respectively. As such, the nine isolates were grouped into four genotypes as follows: genotype 1 , genotype 2 [isolate A/eastern curlew/Liaoning/dandong688/2019(H7N4) (abbreviated as D688)], genotype 3 [isolate A/eastern curlew/Liaoning/dandong1144/2019(H7N4) (abbreviated as D1144)], and genotype 4 [isolate A/little curlew/Liaoning/dandong142/2019(H7N4) (abbreviated as D142)].In the overall genetic structure of the global H7 hemagglutinin (HA) tree, two major geographically dependent genetic lineages, American and Eurasian, were represented. Ln/H7N4 and Js/H7N4 were clustered into the Eurasian lineage; however, they were not genetically related to the enzootic A(H7N9) viruses in China . The maxThe timing of evolution events was determined by Bayesian phylogenetic reconstruction and demonstrated by maximum clade credibility trees . The timUsing phylogeographic analysis, we sought to understand further the most likely source of A(H7N4) viruses. Potential events of cross-regional gene flow to the origin of A(H7N4) viruses were identified . For Ln/The amino acid sequence PELPKGR↓GLFGAI was found within the cleavage site of the HA of Ln/H7N4, suggesting that all were LPAIVs . The recn = 2), PA (n = 3), PA-X (n = 6), NP (n = 5), NA (n = 1), M1 (n = 1), NS1 (n = 4), and NS2 (n = 2). D688 also showed high polymorphic amino acid sites, however, with no single unique site. Virus evolution is characterized by adaptations that are specific to the host. Genetic analysis has shown that Ln/H7N4 and Js/H7N4 shared a common ancestor in HA and NA gene segments; therefore, we next compared their amino acid sequences , Ln/H7N4, especially D1144 and D142, displayed polymorphic amino acid sites in PB2 (6 50% egg infective doses (EID50) (50) of D1144 was 5.43 EID50; thus, D1144 exhibits moderate virulence in mice without prior adaption. D142 could be detected in the infected mice's lungs, nasal turbinate, and kidneys  . The 50% kidneys . All D14 kidneys , althougy day 12 . Thus, Dy day 12 . MutatioLive poultry markets have played pivotal roles in the genesis of novel AIVs. In the spring of 2013, a novel A(H7N9) virus emerged in China and infected people exposed to live-bird markets. Phylogenic analysis indicated that the virus emerged through multiple reassortment events in wild birds and obtained internal genes from H9N2 viruses circulating in poultry. H7N9 AIVs were primarily isolated from poultry while occasionally detected in forest bird species. One reasonable explanation for the notable spread of the A(H7N9) virus only in poultry and its geographical limitation to China is that it could not replicate in the waterfowl host, thereby losing the opportunity to infect the migratory birds distributed broadly . In thisb sequences as described previously (https://www.gisaid.org/) EpiFlu database (accession numbers EPI_ISL_445001 to EPI_ISL_445093).After the first case of human infection with the A(H7N4) virus was confirmed in January 2018, to trace whether wild birds carry the A(H7N4) virus, active surveillance of AIV was undertaken during the bird migratory seasons of 2018 to 2019 in the Dandong Yalu River Estuary Coastal Wetland National Nature Reserve, Liaoning Province, China. The samples were oscillated and then centrifuged, and the collected supernatant of all samples was inoculated into 9-day-old specific-pathogen-free (SPF) chicken embryos. Then, 72 h after incubation, the allantoic fluid was harvested, and the hemagglutinin (HA) activity was assayed. The negative allantoic fluid was passaged twice again in embryonated eggs. An HA inhibition assay was performed preliminarily to determine the HA subtype of the isolated virus by using chicken anti-HA serum for each subtype. Viral RNA was extracted from HA-positive samples from the incubated allantoic fluid using a QIAamp Viral RNA minikit  and reverse transcribed using the primer Uni12. Viral cDNAs were amplified by PCR with primers designed for each gene segment . The PCReviously . The whohttps://www.evolgenius.info/evolview-v2).The variation of amino acid sequences among our Liaoning A(H7N4) isolates (abbreviated as Ln/H7N4) and Jiangsu human-related A(H7N4) viruses (abbreviated as Js/H7N4) were visualized by SeqLogo implemented in TBtools to analyze the potential host adaptation molecular characters . The seqhttp://beast.community/logcombiner). Tracer v1.7.1 (http://beast.community/tracer) was used for obtaining an adequate sample size of ≥200. Maximum clade credibility (MCC) trees were combined after removing initial 10% burn-in and then reconstructed using TreeAnnotator v1.10.4 (http://beast.community/treeannotator). MCC trees were edited by FigTree v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree). The time to the most common ancestor was also estimated from the MCC trees. All BEAST runs were run on the cyberinfrastructure for phylogenetic research (CIPRES) science gateway (https://www.phylo.org).Before the analysis, the temporal signal of each data set was investigated using a root-to-tip regression of genetic distances against sampling time in the program TempEst v1.5 . To clarThis study was performed according to the recommendations detailed in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People's Republic of China. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.Studies with the A(H7N4) viruses were conducted in enhanced animal biosafety level 3 (ABSL3+) facility approved for such use by the Ministry of Agriculture and Rural Affairs of China and China National Accreditation Service for Conformity Assessment. All animal studies were approved by the Review Board of the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The facility details and biosafety and biosecurity measures used have been previously reported .50) testing of the three viruses, groups of five BALB/c mice were anesthetized with CO2 and inoculated intranasally with 10-fold serial dilutions 106 to 101 50% egg infectious doses (EID50) of A(H7N4) viruses in a volume of 50 μl. A control group (n = 5) was intranasally inoculated with 50 μl of phosphate-buffered saline (PBS). The mice were monitored for 14 days to assess mortality. The MLD50 was determined using the Reed-Muench method  viruses. Three mice were euthanized at 3 days postinfection (dpi), and different tissues, including the brain, spleen, kidney, lungs, and nasal turbinate were collected. The tissue samples were homogenized and centrifuged at 8,000 rpm. Then, the supernatants were collected and inoculated into three 9-day-old eggs. At 72 h after incubation and 37°C, the activity of HA was tested, and the EID50 was determined using the Reed-Muench method .Three representative A(H7N4) viruses for genotypes 1, 3, and 4 were used for mouse studies, A/shorebird/Liaoning/dandong603/2019(H7N4), abbreviated as D603; A/eastern curlew/Liaoning/dandong1144/2019(H7N4), abbreviated as D1144; and A/little curlew/Liaoning/dandong142/2019(H7N4), abbreviated as D142. As there is no unique molecular feature, the virus of genotype 2  was not selected. Six-week-old female BALB/c mice were obtained from Beijing Vital River Laboratories. For the 50% minimum lethal dose (MLDh method . Three mh method . Statisthttps://www.gisaid.org/) EpiFlu database (accession numbers EPI_ISL_445001 to EPI_ISL_445093).The whole-genome sequences of A(H7N4) isolates in this study were submitted to the Global Initiative on Sharing All Influenza Data (GISAID,


Magnaporthe oryzae. We have previously reported that there are eight RGS and RGS-like proteins named MoRgs1 to MoRgs8 playing distinct and shared regulatory functions in M. oryzae and that MoRgs1 has a more prominent role compared to others in the fungus. To further explore the unique regulatory mechanism of MoRgs1, we screened a M. oryzae cDNA library for genes encoding MoRgs1-interacting proteins and identified MoCkb2, one of the two regulatory subunits of the casein kinase (CK) 2 MoCk2. We found that MoCkb2 and the sole catalytic subunit MoCka1 are required for the phosphorylation of MoRgs1 at the plasma membrane (PM) and late endosome (LE). We further found that an endoplasmic reticulum (ER) membrane protein complex (EMC) subunit, MoEmc2, modulates the phosphorylation of MoRgs1 by MoCk2. Interestingly, this phosphorylation is also essential for the GTPase-activating protein (GAP) function of MoRgs1. The balance among MoRgs1, MoCk2, and MoEmc2 ensures normal operation of the G-protein MoMagA-cAMP signaling required for appressorium formation and pathogenicity of the fungus. This has been the first report that an EMC subunit is directly linked to G-protein signaling through modulation of an RGS-casein kinase interaction.GTP-binding protein (G-protein) and regulator of G-protein signaling (RGS) mediated signal transduction are critical in the growth and virulence of the rice blast pathogen  M. oryzae, previous studies demonstrated that G-protein/cAMP signaling are important for appressorium formation and pathogenicity. One of the eight regulator of G-protein signaling (RGS) and RGS-like proteins, MoRgs1, targets G-protein MoMagA to regulate cAMP levels and growth and virulence of the fungus; however, how MoRgs1 exhibits this function and its own regulation indifferent from other RGS and RGS-like proteins are not clear. We here demonstrated that MoRgs1 is subject to regulation by the casein kinase 2 MoCk2 through protein phosphorylation, and this regulation is also essential for the GTPase-activating protein (GAP) function of MoRgs1. We also showed that the endoplasmic reticulum (ER) membrane complex (EMC) subunit MoEmc2 modulates MoCk2-mediated MoRgs1 phosphorylation. Balanced interactions among MoRgs1, MoEmc2, and MoCk2 ensure normal appressorium formation and pathogenicity of M. oryzae.G-proteins play a significant role in signal perception and transduction during pathogen and host interactions. In the rice blast fungus  Heterotrimeric G-protein signaling plays a fundamental role in regulating various cellular developmental processes in eukaryotic organisms. The largest family of membrane receptors, G-protein-coupled receptors (GPCRs), sense extracellular stimuli by activating G-proteins, consisting of three canonical subunits Gα, Gβ, and Gγ, and downstream effector molecules that modify cellular behaviors –3. In a Magnaporthe oryzae during infection. With its robust turgor pressure, mature appressoria effectively penetrate the host cell allowing rapid invasion and colocalization ) with 2 mM PMSF and proteinase inhibitor cocktail . The lysates were collected into 2.0 ml EP tubes and placed onto the ice for 30 min and shaken every 10 min. Protein was extracted by centrifugation at 15,000 rpm for 10 min at 4°C and collecting the supernatant lysates as total proteins. For GFP-tagged protein detection, samples were analyzed by 12% SDS-PAGE gel with running immunoblotting and combining anti-GFP antibody  and the anti-mouse secondary antibody , detected by ODYSSEY infrared imaging system (software Version 2.1). For S-tagged protein detection, samples were analyzed with anti-S antibody  and the anti-rabbit secondary antibody . For RFP-tagged protein detection, samples were analyzed with anti-RFP antibody  and the anti-mouse secondary antibody . For detecting phosphorylated Pmk1 level, we used anti-phospho-p44/42 MAPK (Erk1/2) antibody  with anti-p44/42 MAP kinase antibody  as control.Tagging at the C terminal or the N terminal of MoMagA is known to jeopardize the Gα function, but a previous report indicated that introducing a tag peptides coding sequence in-frame into the αB-αC loop of MoMagA would effectively address this issue . We therBait constructs were conducted by cloning full-length cDNAs of target genes into pGADT7 and pGBKT7 . Prey constructs and bait constructs were confirmed by DNA sequencing and co-transformed into the yeast strain AH109  following the recommended protocol (BD Biosciences Clontech). Transformants screened by synthetic dextrose medium minus leucine, tryptophan, adenine, and histidine (SD-Leu-Trp-Ade-His) were selected. Yeast strains for positive and negative controls were provided by the BD library construction and screening kit.in vivo, full-length gDNA with 1500 bp native promoter region of MoCka1, MoCKB1, and MoCKB2 were cloned into pXY203, a vector with a S tag  was read. We calculated the change in absorbance values (DA620) for the samples by subtracting the A620 of the control well (A620) control from the A620 of the sample well (A620) sample. The concentration (mM) of free phosphate [Pi] was computed in the sample from the standard curve. The formula was: Enzyme activity (units/l) = [Pi] (mM) × 40 ml ÷ [10 μl × reaction time (min)]. One unit is the amount of enzyme that catalyzes the production of 1 mmol of free phosphate per minute under the assay conditions. The relevant experimental procedures were assessed, as described previously [The fusion constructs His-MoRgs1, His-MoRgs1eviously .P < 0.01 as described previously [Each experiment was performed with three replicates and represented as the mean ± standard deviation (SD). The significant differences between treatments were statistically determined by one-way analysis of variance (ANOVA) comparison and followed by Duncan’s new multiple range tests if the ANOVA analysis is significant at eviously .Genes reported in this article can be found in the GenBank database under the following accession numbers: MoRgs1 (MGG_14517), MoEmc2 (MGG_07480), MoCka1 (MGG_03696), MoCkb1 (MGG_00446), MoCkb2 (MGG_05651), MoMagA (MGG_01818), MoMagB (MGG_00365), MoMagC (MGG_04204), MoMgb1 (MGG_05201), MoMgg1 (MGG_10193), MoRab5 (MGG_06241), MoRab7 (MGG_08144), Mst7 (MGG_00800), MoMac1 (MGG_09898).S1 PRIMERS Checklist(DOCX)Click here for additional data file.S1 Fig2+-Phos-tag SDS-PAGE and normal SDS-PAGE were used to conduct Western blot analysis with the anti-GFP antibody. The extent of MoRgs1 phosphorylation was estimated by the mobility shift assay.MoRgs1-GFP proteins were extracted from transformants at mycelia, conidia, and appressoria stages then treated with phosphatase and phosphatase inhibitors. Mn(TIF)Click here for additional data file.S2 FigC, MoCkb1-YFPC, and MoCkb2-YFPC) with MoRgs1-YFPN were conducted by BiFC. Empty YFPC and empty YFPN constructs were used as a negative control. The co-transformants were observed at the germ tube hooking stage (3 h) with laser scanning microscopy . The mean and standard deviation of fluorescence intensity were denoted over 50 germinated conidia that were randomly chosen.(A) The interaction among all three subunits of MoCk2 holoenzyme  with His-MoRgs1 were conducted by GST pull-down assays. GST-MoCka1, GST-MoCkb1, GST-MoCkb2, His-MoRgs1, and GST were expressed and purified by affinity chromatography. Bound proteins were separated by SDS-PAGE in duplicate and analyzed by Western blot with the anti-His  and anti-GST antibodies . (B) The interaction among all three subunits of MoCk2 holoenzyme (MoCka1-YFP(TIF)Click here for additional data file.S3 Figin vivo. Total proteins treated with phosphatase and phosphatase inhibitors were detected by the GFP antibody. Bands were shifted by Mn2+-Phos-tag SDS-PAGE and normal SDS-PAGE, respectively. (B) Phosphorylation analysis in vitro by the fluorescence detection in tube (FDIT) method. Purified proteins of GST-MoCka1, GST-MoCkb1, GST-MoCkb2, and His-MoRgs1 were constructed for the protein kinase reaction in the presence of ATP. Fluorescence was measured in a microplate reader .(A) Phosphorylation analysis of MoRgs1 (TIF)Click here for additional data file.S4 FigMockb2 strains by LC-MS-MS (Q-E). The five phosphorylation sites  were identified in the wild type strain. Phosphorylation site sand sequences are annotated in the upper panel. (F) A model of five serine sites located at the MoRgs1 domains. (G) Covered peptides and phosphorylated sites in the wild type, ΔMockb2, and ΔMoemc2 strains. Colorful letters represent the amino acid sequences covered by mass spectrometry. Red letters represent phosphorylation sites newly identified.(A-E) Identified differentiated phosphorylation sites of MoRgs1 between the WT and Δ(TIF)Click here for additional data file.S5 Fig5A, and MoRgs15D, respectively. Total proteins were extracted and incubated with the anti-GFP agarose and then eluted for Western blot analysis using anti-RFP or anti-GFP antibodies.Co-IP analysis for the interaction between MoMagA and MoRgs1, MoRgs1(TIF)Click here for additional data file.S6 FigMagnaporthe oryzae MoEmc2), NP_012621.1 (Saccharomyces cerevisiae ScEmc2), KUI71153.1 , PTD09165.1 (Fusarium culmorum TPR repeat protein), KZL69988.1 (Colletotrichum tofieldiae TPR repeat protein), XP_009648592.1 (Verticillium dahliae TPR repeat protein), OQE20945.1 (Penicillium flavigenum TPR repeat protein), TBU37051.1 , NP_850995.1 , and NP_055488.1 (Homo sapiens HsEmc2). (B) MoEMC2 suppressed the heat sensitivity of the yeast Δemc2 strain. 10-fold serial dilutions of BY4741, ΔScemc2, and ΔScemc2 transformed with pYES2-MoEMC2 constructs were grown on SD-Met-Leu-His-Ura  plates at 30°C and 37°C for 4 days and then photographed.(A) The amino acid sequences of diverse Emc2 proteins from corresponding organisms were aligned using the CLUSTAL_W. The neighbor-joining tree was constructed by MEGA 7.0 with 1000 bootstrap replicates. GenBank accession numbers and the corresponding species names are as listed: XP_003711387.1 ((TIF)Click here for additional data file.S7 Fighttp://smart.embl-heidelberg.de/). The positions of the domains within the proteins were indicated by amino acid numbers. The full length of MoEMC2 was divided into NTD, TPR, and CTD domains before being ligated in pGBKT7. (B) MoRgs1 has two DEP domains at the N-terminus and one RGS domain at the C-terminus [(A) Structure and domain prediction of MoEmc2 using SMART Click here for additional data file.S8 FigMoEMC2 gene deletion by homologous recombination in M. oryzae. (B) Gene-specific probe (probe 1) and hygromycin phosphotransferase (HPH) probe (probe2) were used in Southern hybridization. Thick square frames indicate the sites of MoEMC2 and HPH genes. Thin lines below the square frames indicate sequence-specific gene probes.(A) A model of the (TIF)Click here for additional data file.S9 FigMoemc2 strains at the germ tube hooking stage (3 hpi). Insets highlight areas analyzed by line-scan. Bar = 10 μm. Percentage of a pattern showed in image was calculated by observation for 50 germinated conidia that were randomly chosen, and observation was conducted for 3 times. (C) Co-IP assays for the interaction between MoRgs1-GFP with MoCkb1-S in the WT and ΔMoemc2 strains. Total proteins were extracted and eluted from the anti-GFP agarose beads before being analyzed by immunoblotting with corresponding antibodies. T: Total protein E: Elution.(A and B) Fluorescence GFP labeled MoCkb1-GFP, and MoRgs1-GFP fusion constructs were introduced into the WT and Δ(TIF)Click here for additional data file.S10 Fig4 spores/ml) were sprayed onto 2 week-old rice seedlings (CO-39). Diseased rice leaves were photographed and percentages of per 5 cm length leaf lesion area were analyzed by ImageJ after 7 days of inoculation. Values are means of three replications and SD . (C and D) Rice sheath injecting assays in vivo and lesion area statistics. Conidial suspensions (2 × 105 spores/ml) were sprayed onto 4 week-old rice seedlings (CO-39). Diseased rice leaves were photographed and percentages per 5 cm length leaf lesion area were analyzed by ImageJ after 5 days of inoculation. Values are means of three replications and SD . White triangles point out the injection sites. (E and F) Rice sheath injecting assays in vitro and classification statistics. Invasive hyphae  in rice cells were observed at 36 hpi and 4 types of were quantified and statistically analyzed. Error bars represent SD from three independent replicates. (G and H) Appressorium formation assays and statistics analysis. Conidia of the WT, ΔMoemc2 and complemented ΔMoemc2 (ΔMoemc2/MoEMC2) strains were dropped on hydrophobic surfaces and the dynamics of appressorium formation were photographed at various times . Bar = 10 μm. (I) Appressorium formation was assayed on hydrophobic (the upper panel) and hydrophilic (the upper panel) surfaces for 24 hpi. Percentages of Mean and SD were shown at the lower panel. (J) Intracellular cAMP levels in the mycelia of the indicated strains cultured for 2 d in CM were quantified by HPLC . (K) Morphological characteristics of the WT and ΔMoemc2 strains. Percentages of Mean and SD were depicted at the lower panel . Bar = 10 μm.(A and B) Rice spraying assays and lesion area statistics. Conidial suspensions (5 × 10(TIF)Click here for additional data file.S11 Fig(A and D) MoEmc2-GFP transformants were stained by endoplasmic reticulum dye ER-Tracker at conidia and germ tube hook stages. (B and E) MoEmc2-GFP transformants were stained by FM4-64 at the conidia and germ tube hooking stages (3 h). (C and F) Late endosome marker GFP-MoRab7 was co-transformed with MoEmc2-RFP in the WT strain and observation was made at the conidia and germ tube hooking stages. All assays were observed 100 samples with three replicates and insets highlight areas analyzed by line-scan. Bar = 10 μm.(TIF)Click here for additional data file.S12 Fig(A and B) ER-Golgi trafficking inhibitor BFA was used in conidium assays with DMSO solvent as control. Insets highlight areas analyzed by line-scan. Bars = 10 μm. (C and D) Early to late endosome inhibitor EGA assay was conducted in GFP-MoRab5 and MoEmc2-RFP co-transformants . Percentages of the pattern (shown in the images) were calculated by the observation of 100 randomly chosen germinated conidia, and the observation was conducted 3 times. The extent of fluorescence overlap was estimated with the Pearson correlation coefficient calculated by ImageJ coloc2. Mean and standard deviation were indicated in the right column (n = 30). Bars = 10 μm.(TIF)Click here for additional data file.S1 TextThe bait construct AD-MoRgs1 was used to screen a yeast two-hybrid cDNA library constructed with an RNA pool from various stages, including conidia and infectious hyphae .(DOCX)Click here for additional data file.S2 TextP < 0.01). All experiments were conducted with three biological repetitions and three replicates, mean and standard deviations were calculated. 5A: S396A S399A S585A S696A S700A 5D: S396D S399D S585D S696D S700D.a. Colony diameter of the indicated strains on CM and SDC media after 7 days incubation at 28°C. b. Dry weight of hyphal at 2 days after incubation in liquid complete medium at room temperature by shaken at 160 rpm. c. Quantification of the conidial production of the indicated strains formed on SDC cultures in the dark for 7 d, followed by incubation under constant illumination for 3 d at room temperature. d. Percentage of appressoria penetrating rice sheath epidermal cells at 24 h post-inoculation. All different capital letters in column show significant difference Click here for additional data file.S3 TextP < 0.01). All experiments were conducted with three biological repetitions and three replicates, mean and standard deviations were calculated.a. Colony diameter of the indicated strains on CM, OM, MM and SDC agar plates after 7 dark days incubation at 28°C. b. Dry weight of hyphae 2 days after incubation in liquid complete medium at 28°C with 160 rpm. c. Statistics of conidial production of the indicated strains growing 7 dark days on SDC agar plates followed by constant illumination (wavelength of 365 nm) for 3 d at room temperature. d. Percentage of appressorium formation on hydrophobic surface with 24 h post-inoculation. e. Percentage of collapsed appressorium treated with 1 M, 2 M, 3 M and 4 M glycerol solution for 5 min after appressorium formation with 24 hpi. All different capital letters in column show significant difference Click here for additional data file.


Background: Protein arginine methyltransferase 4 (PRMT4) has been reported to play a role in several common cancers; however, the function and mechanism of PRMT4 in hepatocellular carcinoma (HCC) are not fully understood. This study aimed to investigate the role and mechanism of PRMT4 in the progression of HCC.Methods: PRMT4 expression and clinicopathological characteristics were investigated using an HCC tissue microarray (TMA) consisting of 140 patient samples analyzed by immunohistochemistry. CCK-8, crystal violet and Transwell assays were used to determine cell proliferation, colony formation, migration, and invasion of HCC cell lines in which PRMT4 was overexpressed or downregulated. The underlying mechanism of PRMT4 function was explored by Western blot assays.Results: PRMT4 was highly expressed in HCC tumor tissues compared to adjacent nontumor tissues. PRMT4 expression was significantly associated with alpha-fetoprotein levels, tumor size, satellite nodules, and microvascular invasion. Patients with higher PRMT4 expression had a shorter survival time and higher recurrence rate. Functional studies demonstrated that PRMT4 overexpression promoted HCC cell proliferation, migration, and invasion in vitro, while knocking down PRMT4 inhibited these malignant behaviors. Additional results revealed that PRMT4 promoted the progression of HCC cells via activation of the AKT/mTOR signaling pathway. Furthermore, inhibition of the AKT/mTOR signaling by MK2206 or rapamycin significantly attenuated PRMT4-mediated malignant phenotypes.Conclusions: This study suggests that PRMT4 may promote the progression of HCC cells by activating the AKT/mTOR signaling pathway, which may be a valuable biomarker and potential target for HCC. Hepatocellular carcinoma (HCC) is one of the most common malignant tumors with high morbidity and mortality. It is the sixth most common cancer and the third leading cause of cancer-related death worldwide Protein arginine methylation is a widespread posttranslational modification that affects many cellular processes, including transcription, RNA splicing, DNA repair, cell signaling and cell fate determination in vitro. Additionally, we determined the underlying molecular mechanism by which PRMT4 promoted the progression of HCC cells by activating the AKT/mTOR pathway.This study focused on the function of PRMT4 in the progression of HCC. We demonstrated that PRMT4 was upregulated in human HCC tissues and promoted the proliferation, migration, and invasion of HCC cell lines Thirty pairs of HCC tissues and the corresponding nontumor liver tissue were obtained from patients who underwent liver resection at the First Affiliated Hospital of Nanchang University and the samples were subjected to quantitative real-time PCR analysis. To evaluate the expression pattern of PRMT4, we used a tissue microarray (TMA) containing 140 pairs of paraffin-embedded primary HCC tissues and adjacent nontumor liver tissues collected from the First Affiliated Hospital of Nanchang University between January 2012 and December 2016 and analyzed the relationship between the PRMT4 expression pattern and clinical features. Patients in the TMA cohort were followed up regularly, and the clinicopathological characteristics and survival information were recorded. This research was approved by the Ethics Review Committee of The First Affiliated Hospital of Nanchang University and was carried out in compliance with the Declaration of Helsinki. All participants signed an informed consent document before enrollment.2O2 (0.003%) in Tris-HCl , the extent of staining and the staining intensity were examined automatically by the Vectra 2 system  and they were calculated using the H-score as previously described Paraffin-embedded tissue sections were deparaffinized and rehydrated before being subjected to endogenous peroxidase inhibition. The sections were rinsed three times in PBS (0.01 mol/l) and blocked with PBS (0.01 mol/l) containing 5% BSA and 0.3% Triton X-100 for 1 h. After incubation with anti-PRMT4 antibody  at 4°C overnight, the samples were incubated again with the secondary antibody at room temperature for 2 h. After being developed with 3,3'-diaminobenzidine (0.03%) and H-ΔΔCt method Total RNA was isolated from the clinical samples using TRIzol reagent . Then, cDNA was synthesized from 1 μg of total RNA using the PrimeScript RT reagent Kit . Real-time PCR was conducted using SYBR Premix Ex Taq  on a LightCycler . The PRMT4-specific primers were 5'-TCGCCACACCCAACGATTT-3′ (forward) and 5'-GTACTGCACGGCAGAAGACT-3′ (reverse), and the GAPDH primers were 5'-ATGACCCCTTCATTGACCTCA-3' (forward) and 5'-GAGATGATCACCCTTTTGGCT-3' (reverse). The relative expression level of PRMT4 was normalized to GAPDH using the 22.Human HCC cell lines  and the liver cell line THLE2 were purchased from the cell bank of the Shanghai Biology Institute, Chinese Academy of Science . All cells were grown in DMEM (Gibco) containing 1% penicillin/streptomycin (Sangong Biotech) and 10% FBS (Anlite) and maintained at 37 ℃ in a humidified incubator with 5% COThe coding sequence for the full-length PRMT4 was cloned into the p23-ZsGreen plasmid  to generate the PRMT4 expression vector. Lentiviral short hairpin RNA (shRNA) targeting PRMT4  was inserted into the pLKO.1-TRC vector. Cell transfection were performed with Lipofectamine 2000 Reagent  according to the manufacturer's instruction. YY-8103 cells were infected with p23-ZsGreen-PRMT4 lentivirus or negative control vector, and Huh7 cells were infected with pLKO.1-shPRMT4 or negative control duplex with a scramble sequence. Overexpressed and silenced cells were sorted using flow cytometry or selected by puromycin (4 µg/ml) for 4 days. Sequences of the interference targets are the followings: shPRMT4-1: 5'-GATCCCCCATGATGCAGGACTACGTGTTCAAGAGACACGTAGTCCTGCATCATGTTTTTC-3′ (forward) and 5'-TCGAGAAAAACATGATGCAGGACTACGTGTCTCTTGAACACGTAGTCCTGCATCATGGGG-3′ (reverse); shPRMT4-2: 5'-GATCCCCGGACATGTCTGCTTATTGCTTCAAGAGAGCAATAAGCAGACATGTCCTTTTTC-3′ (forward) and 5'-TCGAGAAAAAGGACATGTCTGCTTATTGCTCTCTTGAAGCAATAAGCAGACATGTCCGGG-3′ (reverse); Scramble shRNA: 5′-GATCCCCAATTGCCACAACAGGGTCGTGTTCAAGAGACACGACCCTGCCGTGGCAATTTTTTTC-3′ (forward) and 5′-TCGAGAAAAAAATTGCCACAACAGGGTCGTGTCTCTTGAACACGACCCTGCCGTGGCAATTGGG-3′(reverse).Cells were lysed in RIPA buffer  containing protease inhibitors on ice for 30 min. After centrifugation , the concentrations of protein samples were assessed using Bradford reagent (Sigma). Then, equal amounts of protein were separated by 10% SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% skim milk for 2 h at room temperature, the membranes were incubated with the indicated primary antibodies overnight at 4°C. The primary antibodies used were as follows: PRMT4 , AKT, p-AKT , mTOR, p-mTOR , 4E-BP1, p-4E-BP1, RPS6 , p-RPS6  and GAPDH . Then, the membranes were incubated with corresponding horseradish peroxidase-conjugated secondary antibody  for 2 h at room temperature. The immunoreactive protein bands were visualized with an ECL chemiluminescence system .4 cells per well. After that, 10 μl of CCK-8 solution  was added to the medium of each well at 0, 24, 48, and 72 h before the optical density (OD) was measured at 450 nm using a microplate reader .Transfected cells were seeded into a 96-well plate at a density of 1x10The cells  were grown in 6-well dishes containing culture medium and 10% FBS. The culture medium was changed every 3 days. Following 14 days of incubation, the cells were subjected to crystal violet staining and then photographed. Finally, OD values were measured at 600 nm using a microplate reader.5 cells) was positioned into the top chamber with FBS-free medium, and 500 μL medium with 10% FBS was positioned into the bottom chamber. For the invasion analysis, Matrigel  was used to precoat the membrane surface. After incubation at 37°C for 24 h, the invading cells were fixed with methanol and then stained with crystal violet (0.1%) for 10 min at room temperature. The cells in 5 randomly chosen fields were counted under an optical microscope (Olympus Corp.) at ×200 magnification.HCC cell invasion and migration analyses were carried out using a 24-well Transwell chamber . Briefly, a cell suspension  were subjected to multivariate analysis using the Cox proportional hazards regression model. Differences between groups were compared using Student's t-test or ANOVA followed by Tukey's post hoc test. Differences were considered to be significant at P<0.05.All statistical analyses were processed using the SPSS 22.0 software package (SPSS Inc.) and GraphPad Prism 8.0 (GraphPad Software Inc.). All experimental results were independently repeated at least three times. Data are expressed as the mean ± standard deviation (SD). Categorical data were compared using Chi-square test. The OS and DFS rates of patients with HCC were estimated using Kaplan-Meier analysis. Independent predictors of OS and DFS were determined using univariate analysis and multivariate Cox regression analysis with stepwise selection. The significant prognostic factors upon univariate analysis (P = 0.040), tumor size (P = 0.006), satellite nodules (P = 0.024), and microvascular invasion (P<0.001) were significant parameters that correlated with the expression of PRMT4. Cox's multivariate proportional hazards model identified some risk factors for overall survival (Table P = 0.008); tumor size ≥ 5 cm ; microvascular invasion ; and high PRMT4 expression . The risk factors for disease-free survival were as follows (Table P = 0.012); microvascular invasion ; and high PRMT4 expression . Taken together, these results revealed that PRMT4 was upregulated in clinical HCC tissues and cell lines, and its overexpression correlated with the poor prognosis of HCC patients.First, we collected 30 pairs of HCC tissues and their matched nontumor counterparts and analyzed the expression level of PRMT4 at the mRNA level using qRT-PCR, and the results showed upregulation of PRMT4 in 83.3% 25 of 30) of the HCC specimens compared with their nontumor counterparts Figure A. Furthe5 of 30 oDue to the close correlation between PRMT4 upregulation and tumor size and microvascular invasion, the aforementioned clinical observations encouraged us to investigate the biological function of PRMT4 in HCC cells, including its effects on proliferation, migration, and invasion. First, we exogenously overexpressed PRMT4 in YY-8103 cells, which showed relatively low expression levels of exogenous PRMT4 Figure A. Then, We successfully downregulated the expression of PRMT4 by two targeted shRNAs in Huh7 cell lines, which showed relatively high expression levels of exogenous PRMT4 Figure A. SimilaConstitutive AKT/mTOR signaling pathway activation is known to play a critical role in the development of HCC in vitro. Third, we determined that the PRMT4-related tumor-promoting effect in HCC may depend on the activation of the AKT/mTOR pathway.In this study, we first identified a novel oncogene, PRMT4, whose overexpression predicts poor prognosis for HCC patients. Second, we observed that PRTM4 overexpression could promote the proliferation, migration, and invasion of HCC cells in vitro, while knocking down PRMT4 inhibited these tumor characteristics. These findings revealed that PRMT4 may be a reliable biomarker to predict prognosis for HCC patients.PRMT4 was first identified as a protein with arginine-specific histone methyltransferase activity To explore the underlying mechanism of PRMT4 in HCC, we evaluated the effect of PRMT4 on the AKT/mTOR signaling pathway. The AKT/mTOR signaling pathway is a major signaling pathway that is activated in human malignancies and regulates multiple cellular processes, such as cell proliferation, apoptosis and migration In summary, this study demonstrated that PRMT4 overexpression was associated with aggressive tumor behaviors and a poor prognosis in patients with HCC. PRMT4 promotes HCC progression by activating the AKT/mTOR signaling pathway. Thus, PRMT4 may serve as a valuable biomarker and potential target for HCC.


Remarkably, some of these domains respond to the JNK activity, but not all. Altogether, these analyses show that puc expression regulation is very complex and that JNK activities participate in non-previously known processes during the development of Drosophila.For developmental processes, we know most of the gene networks controlling specific cell responses. We still have to determine how these networks cooperate and how signals become integrated. The JNK pathway is one of the key elements modulating cellular responses during development. Yet, we still know little about how the core components of the pathway interact with additional regulators or how this network modulates cellular responses in the whole organism in homeostasis or during tissue morphogenesis. We have performed a promoter analysis, searching for potential regulatory sequences of  Developmental biology focuses on the analysis of the events occurring during the development of the organism. It aims to understand the mechanisms directing how, from a single cell, multicellular organisms, with their extraordinary variety of shapes and forms, are built through complex morphogenetic processes.Signaling pathways are key for morphogenesis as they transduce extracellular information regulating cell proliferation, determining developmental axis, specifying compartment domains, or modulating cell shape or apoptosis. Remarkably, the number of these pathways is finite. A few signaling modules are activated once and again by divergent stimuli to elicit context-specific cellular responses. To understand how the same transducing cascades regulate so many different events is fundamental to gain insight into how both their activity and their target genes are controlled. The recent literature points to new emerging ideas, considering these pathways more a subject of threshold events and regulatory feedback loops than of on/off signaling switches . It has,C. elegans and Drosophila . These domains covered: upstream sequences and the 5′ untranslated domain (PG1); the first and second intron (PG2); the 5′ half of the third intron (PG3); the 3′ half of the third intron (PG4); and the 3′ untranslated domain and downstream sequences (PG5). The candidate regulatory domains (PG1 to PG5) were cloned in the PPTGal4 vector and transgenic lines were created to study their regulatory effects in vivo . The comparison was performed with three species chosen based on their phylogenetic distance to D. melanogaster   .D. melanogaster non-redundant transcription factor (TF) binding profiles database (see Methods). This survey provided information on potential binding motifs for the 4 RS domains, beyond confirming the presence of c-Jun and Creb sites in RS2  were scanned with the Position-Weight Matrices (PWM) of the TRANSFAC database in order to associate them to known transcription factors (Match Suite) (see Methods). RS2 was the only conserved region that got a statistically accepted score. The matrices that theoretically bind this region are those involving c-Jun, Creb and CRE-BP1/c-Jun heterodimer. The rest of the conserved regions did not get any matrix associated. We also performed a less stringent scan analysis (at 85% relative profile score threshold) employing JASPAR s in RS2 . A numbes in RS2 .puc, partially or completely. For each of the available Gal4 lines, expression was evaluated in embryonic tissues, in first instar larval brains and in third instar full larvae.To test the different Gal4 transgenic lines generated (PG1–PG5 and RS1–RS4), they were crossed with a multimerized UAS-GFP stock . Followipuc transcription start site plus its 5’ untranslated domain is not carrying relevant transcriptional regulatory elements for embryonic development. None of the different insertions of the PG1 construct show detectable expression, neither in embryonic tissues nor in first instar larvae  (The 4kb region comprising the iracles) A. Yet, Giracles) B. This eiracles) C.puc, as well as its second exon, are represented by the PG2 Gal4 line. This region appears to enclose different regulatory elements active in different tissues and periods. PG2-driven expression was first detected in the CNS of stage 15 embryos and patchy at the whole epidermis , in the salivary glands, the pharynx/ring gland and weakly in the midgut and the anal pads. Yet, its most characteristic feature is to be expressed in all body wall muscles and the nervous system A–C. Its In the third instar larvae, the expression directed by the PG3 domain in muscles recapitulates that observed in the embryo E. Furthepuc sequences (RS2–4) map in the PG3 intronic fragment. The complex embryonic expression pattern observed for the PG3 domain is only in part reproduced by the RS2 to RS4 conserved sequences. The RS2 motif, residing at the 5′ end of the PG3 sequence, is activated at the end of embryogenesis (late stage 17) in the salivary glands and in a subset of the lateral body wall muscles, in the posterior spiracles and in the CNS   A,B. Furtpattern) C,D. No epattern) . In thirpattern) F,G.puc 3’ non-coding sequences examined  includes its last exon, 3′UTR and sequences downstream. The PG5 sequence is activated during the last embryonic stage (stage 17) in the salivary glands and in the embryonic maxilla at the anterior tip of the embryo. Expression is also detected in segmentally iterated groups of cells, positioned dorsally and slightly posteriorly with respect to the brain lobes occupying symmetrical positions at the branching points of the tracheal dorsal trunk  has been thoroughly employed as a readout of JNK signaling activity. Yet, puc has, in places, been found to be expressed in the absence of JNK. We tested a subset of the new enhancers generated in our screening for dependence on JNK activity and evaluated the expression of these new lines in a hep minus maternal and zygotic background. A stock carrying the 1hep mutation and a multimerized UAS-GFP inducible marker was built. Homozygous, maternally rescued, 1hep females, of the aforementioned stock, were crossed to males of the Gal4 lines PG2 to PG4 and RS1 to RS3. Hemizygous 1hep male embryos not maternally rescued were identified in the progeny by their characteristic dorsal open phenotype. GFP expression was monitored in these living embryos. Remarkably, much of the expression directed by the new enhancers is not JNK dependent.As mentioned above, puc, the 1hep embryos display GFP expression in the remnants of the salivary glands and the trunks of the tracheal system. The epidermal expression seems to be reduced but some signal still remains  are unaffected in 1hep embryos .A251.1puc and E69puc enhancer trap expression both in 1hep and 2bsk allelic conditions .RS2-directed expression recapitulates in part the PG3 muscle pattern but not its tracheal expression. It also shows a quite distinct expression in the CNS, mostly in glial cells. In the absence of JNK activity, the muscle and CNS expression (as it happens for PG3) are maintained D. Intere1hep mutants, remnants of GFP expression in the salivary glands and scattered cells that seem to be of tracheal origin are found. The epidermal and terminal tracheal cells expression seems to be abolished in the loss of JNK activity    , or to a1hep maternal and zygotic condition). Red boxes point to patterns of expression that are eliminated in 1hep mutants.The patterns of expression directed by each Gal4 line (PGs and RSs) in embryos and third instar larvae, and on the embryonic and first instar larvae CNS, when relevant, are summarized. Grey boxes relate to the non-specific expression detected in salivary glands and posterior spiracles . Green boxes highlight expression patterns not observed in the wild-type condition, that are switched on in the absence of JNK activity , Caupolican (caup), or Mirror  show high relative scores. Their expression patterns, however, seem unrelated to the expression of the RS1 element [dl) binding site . Dl acts together with Dif downstream of the Toll pathway, positively regulating hemocyte proliferation. Interestingly, an adjacent Deaf-1 binding site (MYND-type zinc finger factor), showing a very high relative binding score, was also found. Deaf-1 acts downstream of Cactus (IκB homolog) and Dif and Dorsal (NF-κB homologs) regulating the immune response and the expression of Drosomycin, a well-known target of the JNK signaling cascade [pan) . Wingless and JNK signaling have been found to cooperate on dorsal closure, ventral patterning and compensatory cell proliferation amongst other events [Within the RS1 domain, binding sites for members of the Iroquois (Iro) family, such as Araucan , body wall muscles, heart, allary muscles and anal pad . The mos closure ,57.bsk expression is almost ubiquitous and it regulates cell shape and stress responses amongst other processes. In the context of the RS2, we have shown that the absence of Hep (JNKK) triggers a specific response switching on the RS2 enhancer in epithelial cells at the edge of the open epidermis (egr) leading to cell death [puc in the VNC and its morphogenesis [puc CNS expression remains to be evaluated.pidermis . It has ll death . It is tll death  and regull death . It alsoogenesis . The potpuc expression is dynamically regulated in a subset of eve expressing neurons during VNC morphogenesis [Besides the Creb/Jun bipartite site, RS2 carries a relevant binding site for Dbx (NK-like homeobox transcription factor), which is expressed in neuroblasts of the VNC, and it is involved in axon guidance and neuron fate commitment. Dbx affects the differentiation of neurons via cross-repressive interactions with the products of Exex and Eve transcription factors . Interesogenesis  pointingdpp (TGF) signaling pathway, Mad  and Brinker , were identified. The dpp and JNK signaling pathways have been shown to cooperate in multiple developmental processes [ara and caup, on one side, and mad and brinker, on the other are all expressed in the trachea primordia and the developing tracheal system , as well as on the embryonic epithelia. All the above factors could certainly be linked to puc expression in these tissues mediated by RS3.RS3 specifically drives reporter expression to the tracheal system and the epidermis. This expression, in embryos, is reduced in response to a loss of JNK activity . No regurocesses ,64. In tdll) (NKL homeobox transcription factor), well known for specifying limb  versus body wall  fates during embryogenesis, but that it is also expressed in the head epidermis, labial and maxillary sensory complexes [CG4328 is expressed in the embryonic brain  and the larval ventral nerve cord and is predicted to be involved in neuron differentiation [Lim3 is expressed in embryonic/larval neurons and regulates neuronal sub-type identity, including motor neuron identity. exex is expressed in and regulates the differentiation of motor neurons that project axons to ventral body wall muscles [exex negatively interacts with the products of Lim3 and eve (see above) to govern neuronal specification and differentiation. Lim3 and exex are expressed in near-identical patterns though neither is required for the activation of expression of the other, although they show a complex epistatic relationship [exex causes a thickening of ventral motor projections , defects in longitudinal connectives and collapse of commissures [Last, RS4 directed activity is limited to a very weak expression at the VNC in embryos and at the maxillary primordia and anal pads and scattered in the tracheal system in larvae. Relevant in this context could be Distal-less  we designed five pairs of primers that were employed to amplify fragments of genomic DNA. As the expected fragments are around 4 to 6 Kb long, we used a long template PCR system from Roche. These PCR fragments were first cloned in a PGEM T easy vector. Secondly, we subcloned these fragments in a Gal4 expression vector, the pPTGal [white + gene marker which will lead to a red eye color on a white-background. RSs were directly cloned (after amplification employing appropriate primers) in the Bgl2 site of the pPTGal Vector.In order to amplify the upstream, intronic and downstream potential regulatory sequences of e pPTGal . pPTGal y- w- embryos with a helper plasmid that contains a transposase gene. The hatched flies were crossed individually with y- w- flies. Generally, one female or male potential transgenics with three other y- w- flies. To map these insertions in the second or third chromosome, the flies were crossed with a double balancer stock .Transgenic flies were generated by injections in puc Genomic (PGs) domains and the Regulatory Sequences (RSs) are presented below and referred to the D. melanogaster r6.40 Genome database (version FB2021_05) [The coordinates of the 2021_05) . 3R refepuc Genomic 1 (PG1): 3R: 8,099,435–8,105,718; Includes the puc promoter and upstream sequences.puc Genomic 2 (PG2): 3R: 8,105,378–8,110,997; Includes the puc exons 1, 2 and, partially, 3 and introns 1 and 2.puc Genomic 3 (PG3): 3R: 8,111,181–8,116,293; Includes the puc exon 3, partially, and the 5’ half of intron 3.puc Genomic 4 (PG4): 3R: 8,115,938–8,121,127; Includes the puc 3’ half of intron 3 and, partially, exon 4.puc Genomic 5 (PG5): 3R: 8,120,573–8,122,620; Includes the puc exon 4 and downstream sequences.Regulatory Sequence 1 (RS1) (94 bp): 3R: 8,106,539–8,106,632AAAAAATGTAAAGCAAAGCAGGTTTCTCAAGCGGCCTGGCAACGCTGAAAAACCCGCTTTGAAACGACCTTTCGAGTACGAATTCATCGGCACARegulatory Sequence 2 (RS2) (127 bp): 3R: 8,112,003–8,112,128GAGACGCAAAAAAGGGGGCACGAGGTGGGAACGAGGAGAGAACTTTAGCCGTGGAATATAATGCTGACGTCAATCGCATTTTTCCATTTCCATTTTCTATGTCCAAAGCTGTTCATCAAGTATTTTTRegulatory Sequence 3 (RS3) (58 bp): 3R: 8,112,946–8,113,003TTGTTGTTGCTGTTGCGTGTTGGCGTTGGCAGACAGCATGGCGGCAGCGGCGTCGTCGRegulatory Sequence 4 (RS4) (92 bp): 3R: 8,115,816–8,115,911GGTGACATTTTTGTTGGCCCCCGTGCGCATAATTTCAATATTCGTCAGCCGGCTATTTTTAAAATGCAAAAATCGACCAAGCGAAAAAAAATpuc in different Drosophila species was done using the Vista Point tool  on the D. melanogaster Apr. 2004 Genome Release [The whole genome comparative analysis to identify conserved regions in or around  Release .in silico: first, position-weight matrices of the TRANSFAC database [https://genexplain.com/services/%20match%20tools, accessed on 1 July 2021); second, the JASPAR collection of transcription factor DNA-binding matrices  was employed [D. melanogaster database (153 entries) complemented with a subset of manually selected general eukaryotic transcription factors up to a collection of 229 entries  1; 10XUAS-IVS-Syn21-GFP-p10hep (this work)w; If/CyO; MKRS/TM6BAll crosses were performed at room temperature.Embryos were collected at 25 °C and aged at 18 °C for 17–19 h, then dechorionated in 100% bleach for 90s. They were washed thoroughly with water and processed. Whole mount embryos were fixed and de-vitellinized according to .Drosophila embryo dissections for generating flat preparations were performed according to [rding to . BrieflyDrosophila embryos was performed using the following primary antibodies: mouse anti-Fas2 , rabbit anti-GFP tag polyclonal , mouse anti-Repo  and rat anti-Elav .Immunostaining of whole mount or flat-dissected stage 16–17 The secondary antibodies used for detection were: Goat anti-Rabbit IgG (H + L) Alexa Fluor 488 conjugate (A-11008), Goat anti-Mouse IgG (H + L) Alexa Fluor 555 conjugate (A-21422) and Donkey anti-Rat IgG (H = L) Alexa Fluor 555 conjugate (A-48270). All secondary antibodies were used in a dilution of 1:600 and were from Invitrogen.Immunostained embryos were oriented on Poly-L-Lysine coated slides and mounted in Vectashield anti-fading medium  using a 22 mm × 6 mm glass coverslip (thickness 1) as a spacer between the slide and the overlaying coverslip. Flat-prepped immunostained embryos were mounted in the same way.Image acquisition was performed on a Zeiss LSM 700 inverted confocal microscope, using a 25× oil immersion lens (1.3 NA) for whole mount embryos and a 40× oil immersion lens (1.3 NA) for flat-dissected embryos respectively. Z-stacks spanning the whole embryo thickness were acquired with a step size of 2 µm.Third instar larvae were anesthetized in a chamber saturated with ether fumes for 8 min at room temperature. Next, they were stretched and aligned with forceps on a piece of double-sided tape, adhered to a glass slide and covered with a 3:1 mix of Halocarbon Oil 27/700 with a Refraction Index of 1.47. Two 22 mm × 6 mm glass coverslip (thickness 1) were used as a spacer between the slide and the coverslip (thickness 1.5). Images were acquired on a Superesolution DRAGONFLY 505 (Andor) installed on an inverted confocal microscope (Nikon Eclipse Ti2) using a 10× multiple immersion lens (0.8 NA). 150 slices with a step size of 2 µm were collected.


Rheumatoid arthritis (RA) occurs in about 5 per 1,000 people and can lead to severe joint damage and disability. However, the knowledge of pathogenesis and treatment for RA remains limited. Here, we found that histone demethylase inhibitor GSK-J4 relieved collagen induced arthritis (CIA) symptom in experimental mice model, and the underlying mechanism is related to epigenetic transcriptional regulation in macrophages. The role of epigenetic regulation has been introduced in the process of macrophage polarization and the pathogenesis of inflammatory diseases. As a repressive epigenetic marker, tri-methylation of lysine 27 on histone H3 (H3K27me3) was shown to be important for transcriptional gene expression regulation. Here, we comprehensively analyzed H3K27me3 binding promoter and corresponding genes function by RNA sequencing in two differentially polarized macrophage populations. The results revealed that H3K27me3 binds on the promoter regions of multiple critical cytokine genes and suppressed their transcription, such as IL6, specifically in M-CSF derived macrophages but not GM-CSF derived counterparts. Our results may provide a new approach for the treatment of inflammatory and autoimmune disorders. Rheumatoid arthritis (RA) is an aggressive immune mediated joint disease with synovial inflammation and joint destruction of extracellular matrix, which involves various immune cell populations, such as T cells, B cells and macrophages . In the Leishmania donovani down-regulated iNOS gene expression by influencing H3K27me3 repressive role in LPS and IFNγ stimulated J774 macrophages , a methyltransferase, which is the component of polycomb repressive complex 2 (PRC2) complex, is the “writer” that leads to high level of H3K27me3. In contrast, demethylases such as Ubiquitously Transcribed tetratricopeptide repeat on chromosome X (UTX) and Jumonji domain containing protein 3 (JMJD3), is the “eraser” that specifically decreases di- and trimethylation on H3K27, thus lead to transcriptional activation . RecentlIn the current study, our results revealed that demethylase inhibitor GSK-J4 could relieve the RA symptoms in Collagen Induced Arthritis (CIA) mice by reducing IL6 expression in macrophage populations. We further compared the levels of common epigenetic modifications in M-CSF and GM-CSF derived macrophage populations (M-BMDMs and GM-BMDMs), and identified H3K27me3 as the transcriptional suppressor in M-BMDMs specifically. GSK-J4 was found to repress gene transcription and NFκB binding activities in GM-BMDMs, in which the steady state level of H3K27me3 is quite low. Overall, these findings identify H3K27me3 as a critical regulator of monocyte/macrophage-mediated inflammation during RA progression and demonstrate GSK-J4 is feasible to reduce RA severity, which might shed light on the potential therapeutic application of macrophage epigenetic regulators in RA patients.Eight weeks C57BL/6J and DBA/1J male mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Mice were housed under a 12-hour light/dark cycle in a temperature-controlled room (22°C) with free access to water and food. C57BL/6J mice were used for BMDM isolation, and DBA/1J mice were used for CIA model. All mice were maintained and used with the approval of the Committee of Experimental Animals of the Ocean University of China (OUC-SMP-2018-12-02) and conformed to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health .Human monocytic THP-1 cells were cultured in RPMI 1640, supplemented with 10% low-endotoxin fetal bovine serum , 1% Pen/Strep, and differentiated into macrophage by incubation with 100 nM Phorbol 12-myristate 13-acetate (PMA) for 72 hours.Details of primary BMDM isolation have been described . After cth day of growth factors mediated differentiation at the final concentration of 10 μM or 30 μM, respectively and the cells were further cultured for 48 hours. LPS (100 ng/ml) was then used to stimulate BMDM for indicated time before the sample collections.For the experiments with inhibitors, DZNeP  or GSK-J4  were added into medium on the 5Cells were lysed and RNA was extracted using TRIzol reagent. One microgram total RNA was reverse transcribed into cDNA using One Step PrimeScript™ RT-PCR Kit (Takara) according to the manufacturer’s instructions. The real-time PCR reaction was carried out on StepOne Plus  and the primer sequences were listed in https://www.gsea-msigdb.org/gsea/index.jsp).The mRNA of BMDMs were extracted using TRIzol, then reversed transcribed to make the cDNA library. The cDNA library was then sequenced on an Illumina platform. Genes with corrected p value < 0.05 and |log2 (Fold Change)| >1 were assigned as significant differentially expressed genes (DEGs). Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis of DEGs were conducted by DESeq. Gene set enrichment analysis (GSEA) was performed on GSEA 4.1.0 according to the instructions provided on the GSEA website  against UTX or JMJD3 by Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen) according to the manufacturer’s instructions. Forty-eight hours post transfection, BMDM were used for LPS treatment.Chromatin immunoprecipitation (ChIP) assay was carried out by using a ChIP assay kit , according to the manufacturer instructions and our previous experience . BrieflyCells were lysed in lysis buffer , briefly sonicated, kept on ice for 30 minutes, and centrifuged at 15,000 g for 15 minutes. The protein concentration in supernatant was quantified by BCA assay kit . Loading buffer were then added into 20-30 μg total proteins, heated at 95°C for 5 minutes, then cell lysates were resolved in 8%-12% SDS-PAGE. The proteins were then transferred onto a nitrocellulose membrane (Invitrogen). After overnight incubation with appropriate primary antibody at 4°C, the stripes were incubated with HRP linked second antibody for 1 hour at room temperature, then visualized with ECL western blotting substrate. The primary antibodies used in this study include: anti-EZH2 , UTX , JMJD3 , GAPDH , H3K27Ace , H3K4me , H3K9me , H3K20me , H3K27me3 , and H4K20me3 .The level of IL6 in the supernatants of cultured cells post LPS challenge were measured. For mice serum, peripheral blood was centrifuged at 1,000 g for 15 minutes before serum collection. For mice tissues, 10 mg tissues were isolated and grinded in 1.0 ml lysis buffer. After centrifugation at 1,000 g for 15 min, the supernatants were collected. The levels of IL6 , IL10, TNFα (Cloud-clone) and IL17 (ExCell Bio) were measured by enzyme linked immunosorbent assay (ELISA) according to the manufacturer instructions.Cells in lymph gland were isolated to make single cell suspension. Cells were blocked with blocking buffer  for 20 minutes and then stained with fluorescence conjugated antibodies for 30 minutes at 4°C. The samples were washed with FACS buffer , acquired and analyzed by FACS Arial III (BD Biosciences). The FlowJo software was utilized for data analysis .Arthrosis tissues were fixed in 4% paraformaldehyde and embedded in paraffin and then stained with hematoxylin-eosin (H&E). For immunofluorescence, sections were deparaffinized, inactivated peroxidase, and after antigen retrieval, blocked in PBST containing 10% goat serum and incubated with primary antibodies against IL6 , H3K27me3  and CD11b  overnight at 4°C. Sections were then counterstained with DAPI and examined using a Zeiss microscope. Randomly selected fields of ten views per mice, and the positive cell fluorescence intensity was calculated using Image J software. Six mice per group were analysed.The media of GSK-J4 treated or siRNA transfected BMDMs were collected and centrifuged at 500 g for 10 mins. The lactate dehydrogenase (LDH) concentration in supernatants was detected using a LDH assay kit . Detailed experimental procedures have been described elsewhere .Nuclear extracts from BMDMs were prepared by high salt extraction . ElectroNFκB binding activity was measured using TransAM DNA binding ELISAs (Active Motif) following the manufacturer instructions. Quantitative data were analyzed with indicated statistical tests and visualized in R, Prism, or Excel software.Data is represented graphically as the mean ± SD. Statistical analyses were performed using Prism version 8. Paired or Unpaired t-test, One- or Two- way ANOVA were used for calculating p values as indicated in each figure legend. p < 0.05 was considered statistically significant.Abnormal secretion of IL6 and other cytokines have been found to play a critical role in RA pathology , 40. UsiTo verify whether H3K27me3 in macrophages plays a vital role in GSK-J4 treated CIA mice, the slides of mice hind legs were stained for IL6, H3K27me3 and CD11b by immunofluorescence. There were at least two distinct CD11b positive macrophages, CD11b+H3K27me3+ and CD11b+IL6+, were found between cartilago articularis and synovial membrane of CIA mice. Upon CIA modeling, the level of H3K27me3 significantly reduced, which could be reversed by GSK-J4 administration –44, the Numerous studies have demonstrated that GM-CSF (GM-BMDM) and M-CSF (M-BMDM) derived macrophages exert quite different phenotypes, which are partially represented by their different gene expression patterns , 47–49. To further analyze the mechanism of GSK-J4 regulation of IL6 expression in macrophages, a variety of common epigenetic modifications that have either enhanced or inhibited roles on transcription, including H3K27Ace, H3K4me3, H4K20me, H3K9me3, H4K20me3, H3K27me3 were evaluated in M-BMDMs and GM-BMDMs. Interestingly, the level of H3K27me3 was significantly different in these two populations . To furtvia RNA sequencing analysis. After comparison with the reference genome, all the genes were filtrated and DEGs were identified. GSK-J4 was shown to be able to significantly down-regulate LPS stimulated genes. However, GSK-J4 itself has limited effects on transcriptome  can be found below: The animal study was reviewed and approved by Committee of Experimental Animals of the Ocean University of China.ZZ, CZ, and JY designed research. ZZ, YZZ, and DG performed research. ZZ, WH, and QS contributed reagents/analytic tools. ZZ and XX analyzed data. ZZ, CZ, and JY wrote the paper. All authors contributed to the article and approved the submitted version.This work was supported by Key R&D Program of Shandong Province (2020CXGC010503) and Shandong Provincial Major Science and Technology Innovation Project (2018SDKJ0402).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Prion diseases are associated with conformational conversion of cellular prion protein into a misfolded pathogenic form, which resembles many properties of amyloid fibrils. The same prion protein sequence can misfold into different conformations, which are responsible for variations in prion disease phenotypes (prion strains). In this work, we use atomic force microscopy, FTIR spectroscopy and magic-angle spinning NMR to devise structural models of mouse prion protein fibrils prepared in three different denaturing conditions. We find that the fibril core region as well as the structure of its N- and C-terminal parts is almost identical between the three fibrils. In contrast, the central part differs in length of β-strands and the arrangement of charged residues. We propose that the denaturant ionic strength plays a major role in determining the structure of fibrils obtained in a particular condition by stabilizing fibril core interior-facing glutamic acid residues. Protein aggregation into insoluble, beta-sheet rich fibrillar aggregates  is linkeA prime example of such polymorphic nature is the prion protein , a glycoIt has been observed in a number of studies that the environment, under which protein aggregation occurs, has a profound effect on the resulting structure and morphology of amyloid fibrils. Various factors, including pH , agitatiOne means of combating this problem is to determine the condition–structure relationship between the environment used to generate prion fibrils and their type. However, this is complicated by the fact that such aggregates are insoluble and non-crystalline, thus greatly limiting conventional protein structure assays. Transmission electron  or atomiIn this work, we investigate mouse prion protein folding domain (MoPrP(89-230)), which corresponds to proteinase K-resistant part of pathogenic PrP, and is often used for in vitro studies . We prepEscherichia coli and purified as described in the Materials and Methods section. Since spontaneous aggregation of recombinant prion protein is too slow under ambient conditions, the addition of denaturing agents is required. Commonly used prion protein aggregation conditions include either 2 or 4 M guanidinium hydrochloride (GdnHCl) in phosphate buffer solution [Recombinant MoPrP(89-230) was expressed in solution ,44,45 assolution ,47,48. Bsolution ,47,48 an−1 (main minimum in the second derivative spectrum), indicating one dominant type of hydrogen bonding in the beta-sheet structure [−1 and two clearly expressed minima (1628 and 1614 cm−1) in the second derivative spectrum, indicating the presence of two types of hydrogen bonding. When the aggregates were reseeded, the main maximum shifted to 1614 cm−1 and the second derivative main minimum at 1614 cm−1 only had a shoulder at 1628 cm−1. Such a change is associated with the decreased proportion of weaker hydrogen bonds in the beta-sheet structure. Reseeding also caused the fibrils to become highly elongated  [Similarly to G2, the U3G1 condition fibril FTIR spectra did not have any notable changes upon reseeding I,J, withht 5 nm, M and, opht 5 nm, K–N. The  nm, −1) , however13C,15N-labeled MoPrP(89-230) fibrils spontaneously aggregated in U3G1 condition (without reseeding) showed poor resolution -solution  similar solution .13C CP, 15N CP, 13C INEPT and 2D DARR and NCA spectra of U-13C,15N-labeled, Thr-, Arg-unlabeled MoPrP(89-230) fibrils produced at the three above-mentioned aggregation conditions. Their side-by-side comparison suggests a higher similarity between fibrils produced in presence of GdnHCl only (G2 and G4 conditions), whereas the fingerprints of fibrils obtained in U3G1 condition indicate a significantly altered fibril structure. The most striking difference is a low-field-shifted 13Cα resonance that was assigned to Val209. These results suggest that the denaturing agent type is more important than concentration in determining the final fibril structure. 13C INEPT spectra show many peaks from mobile polypeptide regions, indicating that the fibrillar core is constituted of only a portion of the amino acid sequence. However, almost no differences between the 13C INEPT spectra of the three samples were observed, suggesting that the fibrillar cores are confined to approximately the same protein region.13C and 15N backbone chemical shift assignment of the MoPrP(89-230) fibrils, we recorded four sets of 3D NCOCX, NCACX, CANCO and CONCA spectra on U-13C,15N-labeled fibrils prepared in U3G1 condition and on U-13C,15N-labeled, Thr-, Arg-unlabeled samples prepared in all three conditions  is a characteristic of the amino acid sequence and does not depend on the particular aggregation condition, implying physiological significance of the identified fibril core region. The identified structured core (residues Pro165–Ala224) of our prepared MoPrP(89-230) fibrils fits very well with the hydrogen-exchange mass spectrometry (HX-MS) data on MoPrP fibrils [Site-specific assignments of the protein resonances are requisite for atomic- and residue-level structural studies by NMR spectroscopy. In order to perform residue-specific nditions . Analysinditions . Figure  fibrils , but doe fibrils ,40,41. T fibrils . In fact fibrils . Therefo13Cα,β chemical shifts from random-coil values were calculated  G2 fibril in the central region. The HuPrP D178N mutant has an additional beta strand at the N-terminus starting at Asn159 and lacks the C-terminal helical turn and beta strand, while in the central part secondary structure motifs fit well to those found in the MoPrP(89-230) G2 and G4 fibrils.Despite the differences between regions forming the fibril core, the locations of secondary structures partly coincide between the mouse and human prion protein fibrils . The wt 13C and 15N nuclei. Given the high similarity of secondary structures between our MoPrP(89-230) U3G1 and full-length HuPrP fibrils, we created a structural model for the U3G1 fibrils based on the HuPrP cryo-EM structure. Thus, modifications were introduced only in the N- and C-termini, where the first beta strand was extended by 6 residues and the C-terminal helical turn and last beta strand was shifted by 2 residues to match the above-identified MoPrP(89-230) secondary structures. Next, to identify regions exhibiting tertiary structural differences between the MoPrP(89-230) fibrils formed in the three different conditions, we assigned protein side chain resonances based on 2D RFDR and DARR spectra  and calcTo gain atomic-level insight into the structural differences of these regions displaying the largest chemical shift variation, we analyzed dipolar recoupling C(HH)C spectra, where several well-resolved cross-peaks in the methyl-group region report on the conformation of Ile182, Ile184, Val209, Val210, Met205, Met206, Glu207 and Glu211 side chains . For U3GLocation of amino acid side chains in amyloid fibril structures is usually determined by the rule of thumb that hydrophobic groups are oriented towards the fibril core, while charged groups are facing the solvent. Our data indicate that the structure of MoPrP(89-230) G2 and G4 fibrils does not follow this rule, since either one (G2) or two (G4) negatively charged glutamic acid side chains are oriented towards the interior of the fibril core. The MoPrP(89-230) assembly into such an unfavorable arrangement seems to be driven by formation of extended beta sheets  and avoidance of a helical turn motif. Furthermore, the fact that this destabilizing conformation forms only in presence of high concentration GdnHCl (2 M and 4 M) further suggests that the denaturant is involved in its stabilization. The most obvious difference between our studied aggregation conditions is the ionic strength of the denaturant and, thus, the negative charges in the fibril interior most likely are compensated by ion pair formation with guanidinium. Notably, these tertiary structural differences between the fibrils formed in the three conditions may have further effects on the quaternary structure . Although the G2 and G4 conditions also featured a reduced pH (6.0 compared to 7.4 for U3G1 condition), it most likely did not contribute to the structural differences. The only residue types that could change protonation state in this pH range are histidines (His177 and His187), which are located in the N-terminal region showing identical secondary structures between the three fibrils.DPGQGGGTHN QWNKPSKPKT NLKHVAGAAA AGAVVGGLGG YMLGSAMSRP MIHFGNDWED RYYRENMYRY PNQVYYRPVD QYSNQNNFVH DCVNITIKQH TVTTTTKGEN FTETDVKMME RVVEQMCVTQ YQKESQAYYD GRRS), was a generous gift of Prof. Witold K. Surewicz. MoPrP(89-230) expression was done by growing BL21(DE) One Star E. coli strain transformed with pRSETB vector in minimal nutrition M9 medium composed of: 6.0 g/L Na2HPO4, 3.0 g/L KH2PO4, 1.0 g NaCl, trace metals, 0.10 mM CaCl2, 2.0 mM MgSO4, 1.0 g/L 15N labeled ammonium chloride, 2.0 g/L 13C glucose, 1×concentrate Gibco MEM vitamin solution , and 0.10 g/L ampicillin. One colony from LB agar plate was inoculated into 20 mL LB medium and grown for 14 h at 37 °C under 180 RPM agitation. Afterwards, 10 mL of this culture was centrifuged at 3000× g for 5 min and resuspended into 1 L of M9 medium. The culture was grown for approximately 8 h at 37 °C under 220 RPM agitation until the optical density at 600 nm reached 0.6–0.9. The expression was induced with 0.50 mM of IPTG, and the culture was grown for an additional 16 h at 20 °C under 220 RPM agitation.The expression vector pRSETB harboring nucleic acid sequence encoding N-terminally truncated mouse prion protein (MoPrP(89-230), bold text) fused to an N-terminal linker containing 6×His tail and a thrombin cleavage site  was expressed as described above, but the culture harvested from 10 mL LB medium was suspended and grown in M9 medium with 0.20 g/L of each Arg and Thr amino acids. The selective unlabeling efficiency was determined by solution NMR to be above 90% by comparing peak intensities in 1H-15N HSQC spectra of Thr-, Arg-unlabeled and fully labeled samples .The prion protein fibrils were prepared in three different conditions. For two conditions, lyophilized MoPrP(89-230) was dissolved in 50 mM sodium phosphate buffer (pH 6.0), containing either 2 M or 4 M GdnHCl, while the third condition the sample was dissolved in PBS buffer (pH 7.4) containing 3 M Urea and 1 M GdnHCl. In all cases the final protein concentration was 0.5 mg/mL (~27 µM). The solutions were then distributed into 1.5 mL test-tubes  and incubated at 37 °C and 220 RPM agitation  for three (2 M GdnHCl and 1 M GdnHCl with 3 M Urea samples) or seven (4 M GdnHCl samples) days. The formed aggregates were sonicated for 10 min on ice using a Sonopuls 3100 ultrasonic homogenizer . 1 mL of each sonicated aggregate solution was mixed with 1 mL of fresh, non-aggregated protein solutions  and incubated at 60 °C under quiescent conditions for 2 h. The aggregate sonication and reseeding steps were repeated 5 times, each time mixing the aggregate solution with fresh protein solution in equal volumes, resulting in a final volume of 32 mL. Subsequently, the aggregate solution was distributed into 16 × 1.5 mL test-tubes and centrifuged at 20,000× g, 20 °C for 30 min and resuspended into 1 mL of D2O. This centrifugation and resuspension procedure was repeated 4 times, with the final suspension volume of 0.20 mL. The samples were then sonicated for 20 s using a MS-72 tip . FTIR spectra were recorded using 256 interferograms per spectrum with 2 cm−1 resolution and analyzed using GRAMS software .FTIR measurements were performed as described previously ,52. In bAFM measurements were performed as described previously . Briefly13C cross-polarization (CP), 15N CP, 13C INEPT, 2D NCA, NCO, DARR, RFDR and C(HH)C, 3D NCACX, NCOCX, CANCO, and CONCA spectra were acquired. The MAS frequency was 17 kHz during acquisition of 2D DARR, RFDR and C(HH)C spectra, while for other spectra it was 12.5 kHz. Temperature regulation was set to 273 K. Spectra acquisition and processing parameters  are summarized in 13Cα,β chemical shifts from random coil values [i = (ΔδCαi − ΔδCβi).All spectra were recorded on an 800 MHz Bruker Avance III HD spectrometer  equipped with a 3.2 mm H/C/N E-free MAS probe. For all the samples in this project 1D l values  using th13C/15N labels is required, which is beyond the scope of this study.We have prepared MoPrP(89-230) fibrils in three different conditions widely used for studies of prion proteins and have structurally characterized them using AFM, FTIR and MAS NMR spectroscopy. The backbone and side chain resonances have been assigned for most residues through analysis of 2D and 3D MAS NMR spectra and locations of secondary structures have been identified though secondary chemical shift analysis. The results show that regardless of the aggregation condition the MoPrP(89-230) fibrillar core is comprised by residues Pro165 to Ala224, which highlights the physiological significance of this protein region and agrees well with earlier results obtained by HX-MS. However, it does not match with the fibrillar core of human or Syrian hamster prion proteins, which could be due to differences in their amino acid sequence. Analysis of chemical shift differences between the MoPrP(89-230) fibrils obtained in the three conditions points to a nearly identical structure for the N- and C-terminal parts of the fibril core, while the largest structural differences are found in the central region near residues Asp178–Asn181 and Lys204–Met213. Further analysis of dipolar recoupling ssNMR spectra indicates that the U3G1 fibrils have a structure that closely resembles the wt HuPrP fibrils prepared in presence of 2 M GdnHCl. The spectra for G2 and G4 fibrils reveal an altered structure with one or two glutamic acid residues facing inwards, which results in formation of longer beta strands and absence of a helical turn motif. Altogether, these findings suggest that the role of the denaturant in driving MoPrP(89-230) aggregation into a particular fibrillar structure is related to its ability to stabilize fibril core interior-facing glutamic acid residues though charge screening. Therefore, the fibrils obtained in presence of high concentrations of the charged GdnHCl denaturant may be less biologically relevant due to significant deviation from physiological ionic strength. The aggregation conditions could have further effects on the quaternary structure as the protofilament interactions often involve inter-chain salt bridges. To study these effects, preparation of new fibril samples with mixed