In current seasonal influenza vaccines, neutralizing antibody titers directed against the hemagglutinin surface protein are the primary correlate of protection. These vaccines are, therefore, quantitated in terms of their hemagglutinin content. Adding other influenza surface proteins, such as neuraminidase and M2e, to current quadrivalent influenza vaccines would likely enhance vaccine efficacy. However, this would come with increased manufacturing complexity and cost. To address this issue, as a proof of principle, we have designed genetic fusions of hemagglutinin ectodomains from H3 and H1 influenza A subtypes. These recombinant H1-H3 hemagglutinin ectodomain fusions could be transiently expressed at high yield in mammalian cell culture using Expi293F suspension cells. Fusions were trimeric, and as stable in solution as their individual trimeric counterparts. Furthermore, the H1-H3 fusion constructs were antigenically intact based on their reactivity with a set of conformation-specific monoclonal antibodies. H1-H3 hemagglutinin ectodomain fusion immunogens, when formulated with the MF59 equivalent adjuvant squalene-in-water emulsion (SWE), induced H1 and H3-specific humoral immune responses equivalent to those induced with an equimolar mixture of individually expressed H1 and H3 ectodomains. Mice immunized with these ectodomain fusions were protected against challenge with heterologous H1N1 (Bel/09) and H3N2 (X-31) mouse-adapted viruses with higher neutralizing antibody titers against the H1N1 virus. Use of such ectodomain-fused immunogens would reduce the number of components in a vaccine formulation and allow for the inclusion of other protective antigens to increase influenza vaccine efficacy. Orthomyxoviridae family [Influenza is a major global pathogen that causes significant morbidity and 290,000–650,000 human deaths annually, with a potential 10–100-fold higher toll in a pandemic [e family . Influene family . Current0) chain that associates non-covalently and folds to form homotrimers [Influenza vaccination is the preferred and most cost-effective intervention tool currently available to prevent influenza virus infection and disease. Licensed influenza vaccines include inactivated whole or split viruses, (recombinant) viral subunit, and live attenuated vaccines . Seasonaotrimers . Each heotrimers . Hemagglotrimers ,9. Neutrotrimers . Both HAotrimers . Due to otrimers ,12,13. HFurthermore, vaccine strains need to be updated every year . Thus, tOne of the primary drawbacks of including other antigenic components in the vaccine formulations is the technical difficulty of adding additional elements to an already quadrivalent HA vaccine formulation. Genetic fusions of HA ectodomains from influenza A virus subtypes are a potential approach to overcome this issue. However, it was unclear if this could be done without severely compromising antigen conformational integrity and yield. To study these issues, we designed recombinant hemagglutinin ectofusion constructs, where an H3 hemagglutinin ectodomain was genetically fused with an H1 ectodomain with the help of a flexible linker and heterologous trimerization sequences. We show that the resulting designed, recombinantly expressed, H1–H3 HA ectodomain-fused (hereafter abbreviated to ectofusion) immunogens elicit both H1 and H3 specific humoral immune responses in mice and protect against heterologous challenge with both H1 and H3 influenza viruses, comparable to an equimolar mixture of separately expressed and purified individual H1 and H3 ectodomains. These recombinant HA ectofusion based immunogens can potentially help minimize the total number of HA constructs that need to be produced under Good Manufacturing Practices (GMP), allowing for additional non-HA components to be included, thus boosting vaccine efficacy. Relative to conventional inactivated virus, use of recombinant protein-based immunogens allows for improved characterization of individual components, adjustment of their relative stoichiometry, avoids both the presence of egg-adapted mutations and the requirement for large numbers of pathogen free eggs, and allows for increased scalability and rapidity of manufacture.The Expi293 transient expression system was used for rapid and high-yield production of designed immunogens and antibodies from mammalian cells. Expi293 suspension-adapted cells, derived from the human 293F cell line, were maintained in Expi293™ Expression Media . In our hands, these gave considerably higher (~3–10 fold) yields than adherent 293 or suspension 293F cells. Use of mammalian cells enabled testing of multiple constructs in a short period of time and also ensured native glycosylation. Light and heavy chain genes of the CR9114, C05 and MA2077 antibodies were synthesized and cloned into the pCDNA3.4 by GenScript ,26,27. AThe amino acid sequences of H1, H3 hemagglutinin ectodomains were derived from A/Hawaii/70/2019 (GenBank Protein Accession: QGW43678.1) and A/Hong Kong/45/2019 (GISAID Accession: EPI1691930) influenza strains, respectively. Genes for H1, H3 ectodomains  were human codon-optimized and cloned under control of the cytomegalovirus (CMV) promoter by GenScript Inc. . The HA ectofusion construct genes (mMH3H1F_02TE and mMH3FH1F_02TE) were cloned under control of the CMV promoter using three fragment Gibson recombination . The cod6 cells/mL). Cells were then incubated in a 37 °C incubator with a humified atmosphere of 8% CO2 on an orbital shaker. Post 18–22 h of transfection, ExpiFectamine™ 293 Transfection Enhancer 1 and Enhancer 2  were added. Culture was harvested five days post transfection, and proteins were purified from culture supernatant by nickel affinity chromatography. The two-fold diluted supernatant was incubated with phosphate buffer saline (PBS) (pH 7.4) equilibrated Ni-Sepharose 6 Fast Flow resin  for 4–5 h at 4 °C under mild-mixing conditions to facilitate binding. Unbound and non-specific proteins were removed by passing ten column volumes of wash buffer . Bound proteins were eluted from the column using an imidazole gradient . Eluted fractions containing the protein of interest were pooled and dialyzed against PBS (pH 7.4) using a 6–8 kDa (MWCO) dialysis membrane (Spectrum Labs). Protein purity was analyzed on SDS-PAGE followed by Coomassie staining.Polyhistidine-tagged recombinant proteins mMH1_02TE, mMH3_02TE, mMH3H1F_02TE, and mMH3FH1F_02TE were individually transiently transfected and expressed, extracellularly, using Expi293F cells as described previously . BrieflyThe thermal stability of the HA ectodomains and ectofusion immunogens were determined using nano-DSF  on a Prometheus NT.48 instrument (Nano Temper) . ThermalPurified HA ectofusion complexes  were analyzed for the nature of particle distribution and overall homogeneity using NS-TEM. Carbon coated copper TEM grids were glow discharged in a GloQube glow discharge system for 30 s prior to sample addition. Purified samples (3.5 μL of 0.1 mg/mL) were applied onto the Cu grids and incubated at room temperature for 1 min. Excess sample was carefully blotted off with a Whatman filter paper. This was followed by negative staining using a 1% solution of freshly prepared uranyl acetate. Data were acquired on a 120 kV Talos L120C room temperature electron microscope, equipped with a bottom mounted Ceta camera (4k × 4k) at a calibrated pixel size of 2.42 Å/pixel at specimen level.Two sets of raw micrographs for mMH3H1F_02TE and mMH3FH1F_02TE were imported into EMAN 2.2 for further assessment of the protein complexes . Approxi−1). ASTRA™ software (Wyatt Technology) was used for data analysis. The theoretical molecular weight of proteins was calculated using the ExPASy-ProtParam tool. An addition of 1.5 kDa per glycosylation site was added to the overall molecular weight.Size exclusion chromatography was performed using a Superdex-200 10/300 analytical gel filtration column . Purified protein samples (75 μg) were injected into the column, equilibrated with 1X PBS buffer (pH 7.4), and eluted at room temperature at a 0.5 mL/min flow rate. In-line UV detector , refractive index detector (WATERS 2414), and triple angle MALS scattering  detectors were used for molar mass determination . First, the GLM sensor chip was activated using EDC and sulfo-NHS , followed by Protein G (10 μg/mL) (Sigma) immobilization in various channels for 300 s (30 μL/min) in the presence of 10 mM sodium acetate buffer (pH 4.0) and then using 1 M ethanolamine excess sulfo-NHS esters quenched. Nearly 1000 response units (RU) of monoclonal antibodies MAb2077, C05, and CR9114 were immobilized at a flow rate of 5 μg/mL for 100 s. Five different concentrations  of HA ectofusion immunogens were passed at a flow rate of 30 μL/min for 100 s over the chip surface, followed by a dissociation step of 200 s. After each kinetic assay, the chip was regenerated in 0.1 M Glycine-HCl (pH 2.7), and kinetic parameters were obtained by fitting the data to a simple 1:1 Langmuir interaction model using Proteon Manager.v/v antigen: SWE ratio per animal per dose, i.e., 20 μg of antigen in 50 μL of PBS and 50 μL of SWE) at day 0 (prime) and day 21 (boost). Squalene-in water emulsion (SWE) adjuvant composition is similar to MF59 adjuvant and is free of intellectual property rights facilitating its open access for use. Moreover, it was shown to be safe and effective in preclinical studies for various vaccine candidates [50 mouse-adapted Bel/09 (H1N1) or X-31 (H3N2) virus in 20μL of PBS. X-31 (H3N2) is engineered to express surface HA glycoprotein genes of A/Aichi/2/68 (H3N2) and the remaining genes are derived from A/Puerto Rico/8/34 virus [t-test with Bonferroni Dunn’s correction method.All immunization experiments were carried out at the Central Animal Facility, Indian Institute of Science. Groups of 5 BALB/c mice (6–8 weeks old) were immunized intramuscularly with 20 μg of test ectofusion immunogen along with SWE adjuvant (1:1 ndidates . Adjuvan34 virus . Survivat-test was performed for pairwise ELISA endpoint titer comparisons.Enzyme linked immunosorbent assay (ELISA) was performed to determine serum antibody titers against test immunogens. Briefly, 4 μg/mL of test immunogens (50 μL/well) were coated on 96 well Nunc plates  for 2 h at room temperature, under constant shaking at 300 rpm on a MixMate thermomixer . After washing with 1X PBS containing 0.05% Tween 20 (PBST) four times, plates were blocked with 3% skimmed milk in PBST (blocking buffer) for 1 h at room temperature. Antisera raised against test immunogens were serially diluted four-fold in blocking buffer and were added to wells. Plates were then incubated for 1 h, 300 rpm at room temperature, followed by three washes with PBST, after which 50 μL of ALP-conjugated goat anti-mouse IgG secondary antibody (1:5000) diluted in blocking buffer was added (50 μL/well) and incubated at room temperature for 1 h. Plates were washed with PBST (four times) followed by the addition of pNPP liquid (SIGMA-ALDRICH) substrate (50 μL) to each well and then incubated at 37 °C for 30 min. Optical density at 405 nm was measured. The highest serum dilution possessing signal above 0.2 O. D at 405 nm was considered the endpoint titer for ELISA. Data were plotted using Prism v8.4.3 . Two-tailed Student’s t-test was performed for pairwise HI titer comparisons.HI titers to vaccine-matched and challenge-matched viruses were tested with mice sera. Immunized mice sera were heat-inactivated and treated with receptor destroying enzyme  before use. Sera were two-fold serially diluted with cold PBS buffer and incubated with the indicated viruses (4 HAU/well), and then incubated with 1% Guinea pig red blood cells (RBC) for 30 min at room temperature. Hemagglutinin inhibition (HI) titers were recorded as the highest serum dilution at which no agglutination was observed. Two-tailed Student’s 2 with 50 TCID50 viruses. Serum-virus mixture was then transferred to 96 well plates, and 1.5 × 105 MDCK-London cells/mL were added to each well. Plates were then incubated for 48 h at 37 °C in 5% CO2, and cytopathic effects were observed. MN assay for matched virus was only performed with group I, III, V (H1) and group II, IV and VI (H3) as groups (I and II), (III and IV), (V and VI) were identical in terms of their immunogens, only the challenge virus was different. The neutralization titer in the assay is the highest serum dilution at which no cytopathic effect was observed. Two-tailed Student’s t-test was performed for pairwise MN titer comparisons.Viruses were grown in Madin-Darby Canine Kidney (MDCK) cells in the presence of TPCK-treated trypsin (1 µg/mL) and stored at −70 °C. Immune mice sera samples were heat-inactivated and treated with receptor destroying enzyme  before use. Immunized sera samples were two-fold serially diluted and incubated for 1 h at 37 °C in 5% COHemagglutinin ectodomains from influenza A subtypes H1 and H3 were genetically fused. Two ectofusion immunogens were designed, containing either a single foldon trimerization motif at the C- terminus of the H1 hemagglutinin ectodomain (designated as mMH3H1F_02TE), or two foldons, one foldon at the C-terminus of the H3 hemagglutinin ectodomain and the other at the C-terminus of the H1 hemagglutinin ectodomain (designated as mMH3FH1F_02TE) A.In the mMH3H1F_02TE ectofusion, the C-terminus of the H3 HA ectodomain (residues 17-518) was connected to the N-terminus of the H1 HA ectodomain (18-515) with a soluble GSA linker. The length of the GSA linker for connecting H3 to the H1 hemagglutinin ectodomain was calculated using UCSF Chimera Visualization software. The three C-termini of H3 HA are situated at the vertices of an equilateral triangle with an edge of length 14.3Å. The corresponding distance between N- termini of H1 HA is 36.5 Å. The H3 and H1 HA ectodomains were aligned along their three-fold symmetry axes. To avoid a steric clash between the H1 and H3 hemagglutinins, planes defined by the three N and three C-termini, respectively, were separated by a perpendicular distance of 19.2Å. The resultant shortest distance was determined between the C-termini of H3 and the N-termini of H1 HA to be 21.8Å. A ten-residue flexible GSA linker (L10) accommodates this distance, enabling the genetic fusion of the H3 ectodomain with the H1 ectodomain B. Similam~57.3 °C) than mMH3FH1F_02TE (Tm~54.4 °C).The designed hemagglutinin ectofusion immunogens, and individual H1, H3 HA ectodomains were transiently expressed in Expi293F cells and purified from Expi293F culture supernatant using single-step nickel affinity chromatography. Coomassie-stained non-reducing SDS-PAGE was used to assess the purity of fusion construct designs, which was approximately 90–95% A. The yiThe oligomeric state of the designed HA ectofusion immunogens and individual HA ectodomains was probed by size exclusion chromatography—multi-angle light scattering experiments (SEC-MALS). mMH1_02TE, mMH3_02TE, mMH3H1F_02TE and mMH3FH1F_02TE exist as trimers in solution as the calculated molecular weights (mMH1_02TE: 214.5 (±0.4%) kDa; mMH3_02TE: 284.1 (±0.4%) kDa; mMH3H1F_02TE: 402.1 (±0.7%) kDa; and mMH3FH1F_02TE: 426.0 (±0.2%) kDa) were in good agreement with the expected theoretical molecular weight for the corresponding trimers  .Further characterization of the HA ectofusion immunogens was performed using room temperature negative staining transmission electron microscopy (NS-TEM). TEM revealed monodispersed immunogen particles in several orientations A,B. The Surface plasmon resonance was performed to assess the antigenicity and proper folding of designed hemagglutinin ectofusion immunogens. The binding of the designed H1H3 HA ectofusion immunogens to a panel of bnAbs  was determined. CR9114 is a HA stem-directed broadly neutralizing antibody (bnAb) while C05 is an H3 head-directed bnAb ,27. MA20We further evaluated the immunogenicity of the hemagglutinin ectofusion immunogens: mMH3H1F_02TE, mMH3FH1F_02TE relative to the individual H1 and H3 ectodomains mixed in equimolar amounts (mMH1_02TE + mMH3_02TE) in mice. Animals were intramuscularly immunized with 20 µg of SWE adjuvanted immunogen in a prime-boost regimen with a three-week interval. Adjuvant-treated mice were used as controls. H1 and H3 hemagglutinin-specific antibodies in sera samples were measured 14 days post-boost using ELISA, HI, and MN assays. Interestingly, ectofusion immunogens elicited both H1 and H3 specific humoral immune responses, comparable to individual H1 and H3 hemagglutinin ectodomains mixed in an equimolar ratio (mMH1_02TE + mMH3_02TE). The ELISA endpoint titers range from 25,600–409,600 against untagged H1 and H3 proteins. H1 HI titers against both homologous (A/Guangdong-Maonan/SWL1536/2019) and heterologous (A/Belgium/145-MA/2009 (Bel/09)) viruses were high (160–1280) for all three groups. However, H3 HI titers were low in all cases. Similarly, microneutralization titers were high against H1 virus (160–1280) and low for H3 virus (10–40) for all groups for both homologous and heterologous virus . Since t50 of Bel/09 (H1N1) or X-31 (H3N2) virus. mMH3H1F_02TE and mMH3FH1F_02TE immunogens both conferred 100% protection against the Bel/09 H1N1 virus  in terms of immunogenicity and protective efficacy, although in the latter case, the immunogen concentration was double of that in the ectofusion groups. Both ectofusion immunogens elicited H1 and H3 specific humoral immune responses in mice and protected against Bel/09 (H1N1) and X-31 (H3N2) virus challenge. We evaluated the immunogenicity of the designed ectofusion immunogens using hemagglutination inhibition (HI) and virus microneutralization (MN) assays. The hemagglutination inhibition assay detects HA-specific antibodies that inhibit agglutination and are a known correlate of protection .The HI titers were high against both H1 viruses . However, HI titers against H3 were low for both H3 viruses. It is known that the HI assay works less well for recent human H3N2 than for H1N1 viruses ,38. The There was complete protection in all groups except for 80% protection observed in the mMH3FH1F_02TE group against H3 X-31 challenge. Higher morbidity was observed against the X-31 challenge relative to the Bel/09 challenge, consistent with the lower homology of immunogen with H3 virus (85%) than H1 virus (96%) and the presence of several mutations in known neutralizing epitopes in the H3 challenge virus. As a consequence of these mutations, antibodies generated against the antigen will poorly neutralize the heterologous challenge virus, thereby lowering protective immunity.Overall, our data demonstrate that ectodomain fusion can be tolerated without loss of immunogenicity. This is an important result because it shows proof of principle that such ectofusions are viable immunogens, and this strategy offers a path to including more antigens in the vaccine formulation. However, there is some reduction in antigen yield. Future studies will focus on how other modifications, such as a change in linker length or the use of alternate signal peptides, can be used to address this issue. When only a single HA in the vaccine changes, it is possible that characterizing new HA ectodomain fusion proteins might be more complicated than replacing a single component. The current recombinant Flublok vaccine includes four HA components. If one could successfully make two ectodomain fusion of, for example, H1-H3 influenza A and HY-HV influenza B components, the number of components could be reduced from four to two. It could similarly be applied to additional NA components. It remains an open question as to whether this apparent simplification would come with reduced yield and other issues. The present study is a proof of principle that ectodomain fusion is possible without loss of conformational integrity. Much further work needs to be done to examine if this process is robust to the precise identity of the ectodomains and whether this approach will actually find utility in real world influenza vaccines.Recombinant ectofusion immunogen designs would potentially be advantageous for vaccine manufacturers since they reduce the number of components that need to be GMP manufactured. However, as with seasonal vaccines, these HA ectofusion immunogens would need to be changed annually to accommodate recommended strain changes in annual influenza vaccine formulations. This is certainly an inconvenience if only a single strain changes in the formulation, and only a single ectofusion is used. However, if a similar approach also works for the two influenza B components and the ectofusion approach is robust to minor changes in individual HA sequences, then this would facilitate incorporation of other protective antigens in the vaccine formulation as discussed below. These HA ectofusion molecules will likely provide only sub-type or strain-specific protection . Given tRecently broadly protective anti-NA antibodies have been identified against influenza A and B viruses ,19,46. A


RNF6 is a RING finger protein with oncogenic potential. In this study, we established colon-specific RNF6 transgenic (tg) mice, and demonstrated that RNF6 overexpression accelerated colorectal carcinogenesis compared to wild-type littermates in a chemically induced colorectal cancer (CRC) model. To understand whether transcriptional activity of RNF6 underlies its oncogenic effect, we performed integrated chromatin immunoprecipitation (ChIP)-sequencing and RNA-sequencing analysis to identify splicing factor 3b subunit 2 (SF3B2) as a potential downstream target of RNF6. RNF6 binds to the SF3B2 promoter and the overexpression of RNF6 activates SF3B2 expression in CRC cells, primary CRC organoids, and RNF6 tg mice. SF3B2 knockout abrogated the tumor promoting effect of RNF6 overexpression, whereas the reexpression of SF3B2 recused cell growth and migration/invasion in RNF6 knockout cells, indicating that SF3B2 is a functional downstream target of RNF6 in CRC. Targeting of RNF6-SF3B2 axis with SF3B2 inhibitor with pladienolide B suppressed the growth of CRC cells with RNF6 overexpression in vitro and in vivo. Moreover, the combination of 5-fluorouracil (5-FU) plus pladienolide B exerted synergistic effects in CRC with high RNF6 expression, leading to tumor regression in xenograft models. These findings indicate that tumor promoting effect of RNF6 is achieved mainly via transcriptional upregulation of SF3B2, and that RNF6-SF3B2 axis is a promising target for CRC therapy. Colorectal cancer (CRC) is one of the most common malignancies worldwide and the third leading cause of cancer deaths . IncidenCRC is a heterogeneous disease with multiple pathogenic mechanisms, which involves genetic and epigenetic alterations –5. Gene Rnf6 transgenic (tg) mice to assess the role of RNF6 in CRC development. Integrative chromatin immunoprecipitation (ChIP)-sequencing and RNA-sequencing unraveled splicing factor 3b subunit 2 (SF3B2) as a downstream transcriptional target of RNF6. Functional validation showed that RNF6 promotes CRC cell proliferation and invasion in a SF3B2-dependent manner. We also evaluated the therapeutic efficacy of SF3B2 inhibitor, pladienolide B, for the treatment of RNF6-expressing CRC in in vitro and in vivo models.In this study, we have constructed colon-specific The human CRC cell lines  were purchased from the American Type Culture Collection  and maintained according to the instructions from ATCC. 293T cell line was purchased from Invitrogen . All cell lines were obtained between 2013 and 2015. Primary CRC organoids (PDO74 and PDO828) were kindly provided by Prof. Catherine O’Brien  and cultured as described previously [® HD (Promega). Viral particles in the cell culture supernatant were filtered with 0.45 µm filters and added to target cells.Single guide RNA (sgRNA) oligonucleotides  targeting RNF6 or SF3B2 (Supplementary Table According to the EZ-ChIP™ protocol (Merck Millipore), SW480 cells were fixed with 1% formaldehyde for cross-linking at room temperature. Glycine was added to the cells to quench unreacted formaldehyde. After sonication, protein–DNA complexes were immunoprecipitated by anti-RNF6 antibody (proteintech) Supplementary Table Nuclear protein was extracted from CRC cells and EMSA was conducted by using a biotin-labeled EMSA kit , according to the manufacturer’s instruction. The sequence of the wild-type probe is 5′-TCAACTCAAAGTTTCCTCTCC TCCAGG-3′. The sequence of the mutant-type probe is 5′-TCAACTCAAAGAGCGTGCTCCTCCAGG-3′. Briefly, 3 µg of nuclear proteins were incubated with 10×binding buffer, 1.0 µg poly (dI-dC), and 0.5 µL biotin-labeled probes for 20 min at room temperature. Specificity of the DNA protein complex was confirmed by competition with unlabeled cold-competitor oligonucleotide or mutant-type oligonucleotide added to the mixture. In the supershift assays, 0.1 or 0.4 μg of RNF6 antibody (proteintech) was added to the respective reactions and incubated for 30 min at room temperature, with normal rabbit IgG as a control. The reaction mixtures were separated by polyacrylamide gel electrophoresis and transferred to a nylon membrane. Finally, the membrane was incubated with chemiluminescence substrate buffer, and the bands were visualized using CoolImager .Sixteen paired primary CRC and adjacent non-tumor specimens were obtained from treatment naïve CRC patients at the Prince of Wales Hospital, the Chinese University of Hong Kong. A cohort of 151 primary colorectal tumors and adjacent normal tissue RNA were obtained from Peking University Cancer Hospital . Samples were confirmed histologically and staged according to TNM staging system. CRC data were also downloaded from the TCGA Colon and Rectal Cancer database (606 patients). The study was approved by the Clinical Research Ethics Committee of The Chinese University of Hong Kong and Peking University Cancer Hospital. All subjects provided informed consent for obtaining the study specimens. This study was carried out in accordance with the Declaration of Helsinki of the World Medical Association.Rnf6 tg mice (pCAG-loxp-stop-loxp-Rosa26-RNF6) were generated by Biocytogen (China). RNF6-IRES -eGFP (enhanced green fluorescent protein) was cloned into the Rosa26 WT allele to generate a gene-targeting vector, which were then transfected into embryonic stem cells with C57BL/6 background. After clone selection and identification by PCR and southern blot, positive clones were injected into mouse blastocysts to generate chimeric mice. Chimeric mice were mated with WT C57BL/6 mice to obtain the Rosa26-RNF6 mice. CDX2-CreERT2 tg mice were purchased from The Jackson Laboratory, which express CreERT2 in adult epithelium of the distal intestinal tract under the CDX2 gene promoter. Rosa26-RNF6 mice were crossed to CDX2-CreERT2 to generate Rnf6 tg/CDX2-Cre mice.Conditional Rnf6 tg/CDX2-Cre mice were intraperitoneally injected with TAM (100 mg/kg in corn oil) for 4 consecutive days. Two weeks after the first dose of TAM, mice were injected intraperitoneally with 10 mg/kg AOM (Sigma-Aldrich) once a week for 6 consecutive weeks [To drive the intestinal-specific overexpression of RNF6, 7-week-old ve weeks . Mice we6 cells/tumor) were injected subcutaneously into the right dorsal flank of 6-week-old male Balb/c nude mice. Six days after implantation, mice were randomly divided into two groups: (1) vehicle and (2) pladienolide B. Pladienolide B (Santa Cruz) (3 mg/kg) was given intraperitoneally to each mouse on day 0, 2, 4, and 6, as described previously [3) = length × (width)2/2. For the combination treatment study, mice injected with RNF6-overexpressing cells were randomly divided into four groups when the tumor volume reached ~50 mm3, and treated with (1) vehicle, (2) 5-FU, (3) Pladienolide B, and (4) 5-FU + Pladienolide B. Pladienolide B was given as above, and 5-FU (30 mg/kg) was given intraperitoneally once every 5 days. All experimental procedures were approved by the Animal Ethics Committee of the Chinese University of Hong Kong.Control vector or RNF6 stably overexpressing HCT116 cells   and burden  compared to wild-type mice  Fig. . H&E sta19) Fig. . Moreoveice Fig. . In addiice Fig. . We nextice Fig. . Compareice Fig. . CollectP value <0.05. Knockdown of RNF6 in SW480 cells was confirmed by qPCR and Western blot analysis  Fig. . ChIP-PCays Fig. , therebyRnf6 tg mice had increased SF3B2 protein and mRNA expression in colon tissues as compared to WT mice , TCGA cohort      (Supplementary Table n = 587). Kaplan–Meier curve showed that SF3B2 mRNA expression was associated with poor patient survival   Fig. . Multivast) Fig. . SF3B2 eTo investigate the functional role of SF3B2 in CRC, we knockout SF3B2 with two sgRNAs in DLD1 and SW480 cells  after the knockdown of RNF6, consistent with impaired RNA splicing . The results indicated that pladienolide B inhibited SF3B2 protein expression but had no significant effect on SF3B2 mRNA expression Fig. . We thenNext, we tested the effect of pladienolide B in primary CRC-derived organoids. Two human CRC organoids, PDO74 and PDO828, cultured in 3D Matrigel were transfected with empty vector or RNF6 plasmid and treated with pladienolide B. Overexpression of RNF6 also activates SF3B2 expression in primary CRC organoids Fig. . We founTo further validate the inhibitory effect of pladienolide B on RNF6-expressing tumors, we administered the drug into nude mice harboring HCT116 xenografts with vector or RNF6 overexpression. In line with our previous data, RNF6 overexpression accelerated tumor growth in the group without pladienolide B treatment Fig. . In cont50 values for doxorubicin and 5-FU were 300 nM and 10 μM, respectively  and volume (P < 0.001). Taken together, these data suggest that the combination of 5-FU and pladienolide B may be a novel strategy in treating CRC with high RNF6 expression.Given that RNF6 confers drug resistance towards 5-FU while concomitantly promoting sensitivity to pladienolide B, we reasoned that the combination of 5-FU plus pladienolide B could be a promising strategy for targeting CRC with high RNF6 expression. We first quantitatively examined synergism between 5-FU and pladienolide in vitro according to the Chou–Talalay Method . We founRNF6, a RING finger E3 ligase, has been shown to be an oncogene by virtue of its E3 ligase activity , 18, 19.Rnf6 tg mice. Transcriptional activity of RNF6 appears to be distinct from its E3 ligase activity, as its ligase targets, such as TLE3 and SHP-1 [The majority of RNF proteins are believed to function as E3 ubiquitin ligases, however, their RING domains can also bind to DNA and mediate transcription. Here, we are the first to demonstrate that RNF6 could also function as a transcription factor in promoting oncogenesis. Our integrative analyses of ChIP-seq and RNA-seq datasets identified SF3B2 as a key downstream target of RNF6 in CRC. We showed that RNF6 binds to the promoter region of SF3B2 to activate its transcription in CRC cells. Moreover, enhanced SF3B2 expression was found in colon tissues of nd SHP-1 , 8, wereCritically, the oncogenic effect of RNF6 is highly dependent on SF3B2, as the deletion of SF3B2 abrogated RNF6-induced cell growth, colony formation, and cell migration and invasion. Moreover, the overexpression of SF3B2 promoted CRC cell growth and restored malignant phenotypes in cells with RNF6 depletion. These results collectively suggested that SF3B2 functions as a downstream factor essential for RNF6-promoted colorectal tumorigenesis. Besides RNF6, another RNF protein, RNF2, is also reported to possess dual roles in the regulation of oncogenic gene networks via transcriptional upregulation of Cyclin D2 in melanoma . Taken tSF3B2 is a component of splicing factor SF3B complex involved in pre-mRNA splicing . RNA splRNA splicing inhibitors are an emerging class of antitumor drugs attempting to reverse the aberrant RNA splicing activities in cancer cells. Pladienolide B, a naturally existing antitumor macrolide, directly binds to spliceosome-associated 130 (SAP130) subunit of the SF3b complex (SF3B1–7), thereby blocking the spliceosome , 23. Not5-FU-based chemotherapy is the mainstays for the treatment of advanced CRC patients, however, its efficacy is frequently compromised by drug resistance. Here, we revealed the novel association between RNF6 expression and chemotherapy resistance, as CRC cells with ectopic RNF6 expression are more resistant to 5-FU treatment. Against this backdrop, we next tested if pladienolide B could be employed to sensitize high RNF6-expressing CRC cells to 5-FU treatment. Indeed, we found a striking synergistic effect between pladienolide B and 5-FU in CRC with RNF6 overexpression in both in vitro and in vivo models. This work collectively highlights the potential of pladienolide B as a drug candidate, and provides a rationale of combination of 5-FU plus pladienolide B for CRC patients with high RNF6 expression. Apart from pladienolide B, several SF3b inhibitors are currently under preclinical and clinical developments. One such example is E7107, a derivative of pladienolides. E7107 showed inhibition of solid tumor growth with response rate of ~30%; however, it is associated with vision loss in a few patients in clinical trials , 26. MorIn conclusion, RNF6 plays a pro-tumorigenic role in CRC via upregulation of SF3B2. RNF6 binds to promoter and drives the transcription of SF3B2, which in turn, mediates tumorigenic phenotypes in CRC Fig. . The tarSupplementary TablesSupplementary MethodsSupplementary Figures


This revealed SF5 as a superior alternative to the CF3 group. The valueof the SF5 bioisostere as a 19F MRI reportergroup within a biological or pharmacological context is by far underexplored.Here, we compared the biological and pharmacological activities ofdifferent TF derivatives and their 19F MR properties . The 19F MR SNR efficiencyof three MRI methods revealed that SF5-substituted TF hasthe highest 19F MR SNR efficiency in combination with anultrashort echo-time (UTE) MRI method. Chemical modifications didnot reduce pharmacological or biological activity as shown in thein vitro dihydroorotate dehydrogenase enzyme and T cell proliferationassays. Instead, SF5-substituted TF showed an improvedcapacity to inhibit T cell proliferation, indicating better anti-inflammatoryactivity and its suitability as a viable bioisostere in this context.This study proposes SF5 as a novel superior 19F MR reporter group for the MS drug teriflunomide.Fluorine ( Thisprospect heralds an age when exact locations and concentrations ofdrugs can be determined in patients to inform drug therapies.8 The signal-to-noise ratio (SNR) achieved with 19F MRIis limited because of the low availability of 19F nucleiin vivo. Additionally, specific 19F magnetic resonance(MR) properties  and spin–spin (T2) relaxationtimes) and pharmacological properties  could influence SNR and 19F MR signal detection.More than one-third of prescribeddrugs contain fluorine  methods.15 Recently,we detected teriflunomide (TF), a dihydroorotate dehydrogenase (DHODH)inhibitor, in an animal model of multiple sclerosis (MS) using nonlocalized 19F MRS.16 TF inhibits the mitochondrialDHODH enzyme that catalyzes the synthesis of the pyrimidine nucleotideprecursor orotate necessary for DNA replication, including CNS-specificT cells that are key players in the MS pathology, thereby exertingits anti-inflammatory action in MS.17 Despiterecent advances to improve radiofrequency coil sensitivities,18 there are still limitations that impede detectionof 19F drugs in vivo with 19F MRI or localized 19F MRS, while these methods will be essential to locate drugswithin specific tissue and study their distribution in vivo. SNR wasa major limitation in our previous study due to the low TF availabilityin vivo, thereby prohibiting imaging or localized MRS to specify itslocation in vivo.16Fluorinated drugs can be detected in vivousing 5) group has gained an immanentrole in organic materials, polymers, and liquid crystals and has alsorecently emerged as a bioisostere for lipophilic groups like CF3, tert-butyl, halogen, or nitro groups inbiologically active compounds.19 SF6 has been studied extensively in the gas phase as a diagnostictool for pulmonary 19F MRI.25 However, surprisingly, the corresponding derived organic -SF5 group has thus far not been investigated as a 19F MRI reporter group.The pentafluorosulfanyl. Derivatives of TF (Alfa Aesar) that is catalyzed by the DHODH enzyme.35 The reaction was performed in duplicate in volumes of 30 μLin a 384-well plate in 50 mM tris–HCl buffer, containing 73nM coenzyme Q10 (Selleckchem), 0.1% Triton X-100 (Sigma-Aldrich),150 mM KCl (Sigma-Aldrich), 211 nM DCIP (Thermo Fisher), and 7.5 nMDHODH (Biozol). The reaction was started by the addition of the substrate l-DHO, performed at 30 °C, and monitored over 60 min usinga Tecan Safire2 Multimode Microplate Reader (Tecan).The inhibitory activity ofTF derivatives on the DHODH enzyme was measured with a colorimetricenzyme activity assay. Drug concentration ranged from 50 μMserially diluted in DMSO to 4.9 nM with a final DMSO concentrationof 1%. DHODH inhibition was studied by monitoring the reduction of2,6-dichloroindophenol (DCIP) (Alfa Aesar) via color change . The colorimetric reactionis associated with the oxidation of 36 Compounds(10 μL) were plated on 96-well plates and stored at 4 °C.The inhibitory activity of TF derivativeson T cell proliferation was studied after isolating peripheral bloodmononuclear cells (PBMCs) from healthy volunteers. Informed, signedconsent was obtained for collecting blood samples. Approval was grantedfrom the ethics commission of the Charité (EA1/023/15). Compoundswere dissolved in DMSO (19 mM) and further diluted to final concentrationsranging from 0.39 to 50 μM.Blood (in EDTA) was withdrawn from two male healthy volunteers(aged 25–35), diluted 1:2 in PBS, and layered on Lymphopure(BioLegend) for separating cell populations using the principle ofFicoll density gradient centrifugation .PBMCs were cautiously collected from the corresponding phase and dilutedin PBS. After two washing steps in PBS, the cell pellet was resuspendedin a HEPES–RPMI 1640 buffer and counted using a Neubauer cell-countingchamber.37 The fluorescent dye CFSE is taken up into the cells. Dilutions ofCFSE in daughter cells (1:2 as a result of cell division) result insequential losses in fluorescence intensities. Labeling was performedaccording to the manufacturer’s descriptions using 5 μMCFSE, and staining was quenched using a medium containing 10% FCS.Cells were recounted and resuspended in a cell culture medium , 10 mM HEPES (Gibco),and 1% penicillin (10 U/μL)/streptomycin (10 μg/μL)(Gibco) in an RPMI 1640 medium (Gibco)) at a concentration of 106 cells/mL. To induce T cell proliferation, isolated PBMCswere incubated with the superantigen-similar polyclonal stimulatingreagent CytoStim (Miltenyi Biotec) following the manufacturer’sinstructions. CytoStim was added to the cell suspensions (0.2%).Isolated PBMCs were labeled with carboxyfluoresceinsuccinimidylester(CFSE) (BioLegend) as described elsewhere.2 for 72 h. Wells without DHODHinhibitors (TF or its derivates) served as positive proliferationcontrols, and wells without T cell stimuli and inhibitors served asnegative controls. After incubation, cells were centrifuged, washedwith PBS, and resuspended in FACS buffer (0.5% BSA and 0.1% NaN3 (Sigma-Aldrich) in PBS). Propidium iodide (PI) (BioLegend)was added to each well approximately 5 min before the measurementto stain the dead cell population. The fluorescence of CFSE labelingand PI staining was analyzed by flow cytometry using a BD LSRFortessaflow cytometer (BD Biosciences).Cells with or without CytoStim were distributed in 96-well plates and keptat 37 °C and 5% COTable S1.MRexperiments were performed on a 9.4 TMR scanner . All DHODH inhibitors(TF or its derivates) were diluted in either 100% DMSO (Roth) or 100%human serum. The human serum was prepared from whole blood withdrawnfrom a male healthy volunteer (aged 25) and allowed to stand for 15–30min at room temperature; following centrifugation , the supernatant (serum) was transferred into a new container.Serum samples were stored at −20 °C. Phantoms were preparedin 2 and 1 mL syringes to characterize the spectra, chemical shifts,relaxation times, and signal-to-noise (SNR) efficiencies. Teriflunomide(Sigma-Aldrich and Genzyme) was used as a reference compound. Concentrationsof the compounds were adjusted as indicated in 19F/1H mouse head RF coil was used.12 MR measurements were performed at room temperature (RT).For phantom experiments, a home-built dual-tunable 19F signal and to make frequencyadjustments. Chemical shifts are referenced to trichlorofluoromethane,CFCl3 (δF = 0 ppm).A global single pulse spectroscopy  = 90°, block pulse, 4096 points, dwell time =0.02 ms, excitation pulse = 10,000 Hz, spectral bandwidth = 25,000Hz) was used to detect the T1 in low concentratedserum samples, global spectroscopy with different TRs (TR = 50–8000ms) was used. For spectroscopically determining T2, a CPMG sequence was used .For determining T1 mapping was performedusing RARE(rapid acquisition with relaxation enhancement): TE = 4.6 ms, echotrain length (ETL) = 4, FOV = [16 × 16] mm2, matrixsize = 64 × 64, with 9 variable repetitions times (TR = 25–8000ms). T2 mapping was performed using amultislice multiecho sequence: TR = 2000 ms, FOV = [16 × 16]mm2, matrix size = 64 × 64, with 25 different TEs(TE = 40–1000 ms in steps of 40 ms for long T2 and TE = 8–200 ms in steps of 8 ms for short T2).19F MRI of TF and its derivatives,we optimized the parameters(Table S3) of three MR sequences: RARE,bSSFP , and UTE (ultrashortecho time). For all methods, the image geometry was set to FOV = [28× 28] mm2, matrix size = 96 × 96 in DMSO, matrixsize = 64 × 64 in serum, and slice thickness = 5 mm. Pulse sequenceparameters were optimized based on the relaxation times of each compoundin DMSO and serum.39To determine the most SNR efficientMR technique for acquiring T2 conditions, i.e., in DMSO (TEDMSO = 12.12ms), and short T2 conditions, i.e., inserum (TEserum = 2.29 ms). For the latter condition, aRARE sequence with gradient pulses optimized for short echo spacing(RAREst) was used. We used a flip-back module and, based on simulations,chose the highest possible ETL with a full width at half maximum (FWHM)of the point spread function (PSF) below 1.5 pixels. Given these parameters,we optimized the repetition time (TR) based on the steady state signalintensity equation.38For RARE, the receiverbandwidth was set to 10 kHz to maximizeSNR but limit chemical shift artifacts. Centric encoding was used,and the echo spacing was kept minimal for both long bSSFP was adjusted toFor bSSFP,the receiver bandwidth was set to 100 kHz to achievea short TR = 2.6 ms (TE = 1.3 ms) and thus stability regarding bandingartifacts. For each compound and condition, the flip angle αE as the flip angleFor UTE, we used an FID readout (TE = 0.27 ms) and a readbandwidthof 20 kHz. We used TR = 100 ms and the calculated Ernst angle αAnimal experiments were conductedin accordance with the proceduresapproved by the Animal Welfare Department of the State Office forHealth and Social Affairs Berlin (LAGeSo) and conformed to guidelinesto minimize discomfort to animals (86/609/EEC).5-TF (10 mg/mL)suspended in 0.6% carboxymethyl cellulose were administeredorally (bolus volume of 800 μL) to an anesthetized C57BL/6N mouse. After 20 min, the animal was sacrificed by an overdoseof the anesthetic, the esophagus and duodenum were closed with a surgicalsuture, and the stomach was removed. An ex vivo phantom was preparedin a 5 mL tube filled with 4% paraformaldehyde (Santa Cruz Biotechnology).We used RARE for anatomical 1H MRI of the stomach: TR/TE= 2000 ms/10 ms; TA = 1 min, 4 s; FOV = [28 × 28] mm2, and matrix = 256 × 256, slice thickness = 0.5 mm, and an optimizedUTE sequence for 19F MRI of TF and SF5-TF: TR/TE= 100 ms/0.27 ms; TA = 2 h, 30 min, slice thickness = 5 mm, FATF = 25°, FASF5-TF = 42°, FOV =[28 × 28] mm2, and matrix = 32 × 32.The same molarconcentrations of TF (12.15 mg/mL) and SFZ′ factor was calculated40 using an in-house online Z′calculator to assess the quality of the screening method (http://www.screeningunit-fmp.net/tools/z-prime.php). Screening assays with Z′ values between0.5 and 1 are categorized as excellent.40For DHODH activity, time–absorbancecurves reflect the enzymatic activity over time. We calculated theinitial slope of these curves to quantify the initial velocity ofthe enzyme reaction. A Figure S2). The lymphocyte population was selected from theforward (FSC-A)/sideward scatter (SSC-A) and gated for single cells(FSC-A/FSC-H). Dead cells were excluded by gating for the cell populationSSC-A/PI without PI staining. From this population, the CFSE-labeledcells were gated and displayed in a histogram, dividing the differentgenerations of cells from cell divisions. The percentage of proliferatingcells in relation to the originally labeled parental cell populationwas calculated.For T cell proliferation, we analyzed flow cytometry datausing FlowJo  was calculatedfrom the number of proliferating cells in unstimulated negative andstimulated untreated positive controls. Additionally, the stimulationindex (SI) was determined from the proportion of proliferating cellsfrom nonproliferating onesA Data from triplicatesfrom each donor were combined so that eachconcentration point is represented by three measurement points.41 Briefly, we quantifiedthe spectral signal intensity, performed a Fourier transform to obtainmagnitude spectra, and determined the chemical shift. We estimatedthe T1 and T2 time constants by fitting the measured signal intensities in MRmagnitude images to the equations for T1 and T2 relaxations. SNR estimationsfor RARE, bSSFP, and UTE measurements and image preparation of exvivo samples were performed as previously described.42 ImageJ43 was used for furtherimage analysis. We calculated the SNR efficiency (SNR/sqrt(time))per mol to determine the compounds beneficial for in vivo application.MR image processing and spectral analysis and processing were performedin MATLAB R2018a . MR data was analyzed as detailedin our previous work.3O-TF) and pentafluorosulfanyl-substitutedTF (SF5-TF) demonstrated equal or even better pharmacologicaland antiproliferative activities compared to TF (CF3-TF)as shown by the IC50 values for DHODH inhibition in invitro enzyme and proliferation assays . The impact of TF and its derivatesto inhibit T cell proliferation was also determined from the IC50 values from dose–response curves and by fitting adose–response model to the data points . The T cell stimulation index yielded 47.3.Thetrifluoromethoxy-substituted TF . The activity of CF3O-TF to inhibit DHODH was marginally higher than that of TFas shown by the lower IC50, while that of SF5-TF was similar, and that of the difluoromethyl-substituted di-CF3-TF was lower  and IC50 values .For TF and its derivatives, we calculated the IC0 values 1. Compar3 group witha SF5 groupis expected to result in changes in the side chain geometry and electrondensity. The SF5 group is characterized by a higher electronegativityand lipophilicity.19 The increased T cellinhibitory activity by SF5-TF suggests an increased cellularuptake in T cells as a result of more efficient permeation throughcell membranes due to increased lipophilicity, as has been shown intrypanothione reductase inhibitors exhibiting increased cellular activityand membrane permeability following introduction of SF5.44Substitution of the CFToxicology). All compounds showed a similar impact on cancer cell growth andcytotoxicity to that of TF .While T cell inhibitory activity of SF5-TF was enhanced,its impact on cytotoxicity was similar to that of TF. Furthermore,nucleic acid staining using propidium iodide (PI) did not reveal cytotoxiceffects in T cells, even at higher concentrations .We also performed a cell growthinhibition and cytotoxicity testin HepG2 cells . Since the solubility of TF compounds in serumwas lower than in DMSO, lower concentrations of TF compounds wereavailable in serum (Table S1); this resultedin an expected lower SNR . TF,CF3O-TF, and di-CF3-TF exhibita single peak at −58, −55, and −59 ppm, respectively,in DMSO . Due tothe geometric orientation of the five fluorine atoms to the phenylmoiety, one positioned axially and four in equatorial positions, themain peak shows a line splitting (doublet) and the smaller peak showsan apparent 1:4:6:4:1 intensity pentet .46We studied the ower SNR S6. TF,Cower SNR S6. TF,CTable S2).Spectra of the compounds solubilized in serum were similarto thosein DMSO; although we observed a slight change in the chemical shiftand peak broadening  were measured for all TF derivatives  were also measured for all compounds , a reduction in T1 reduces the time required between differentexcitations, thereby enabling more efficient data acquisition. Thereductions in relaxation times could be attributed to dipole–dipoleinteractions and chemical shift anisotropy.48 Besides modifications of side chains, the addition of a paramagneticdopant such as lanthanide chelates, in particular, gadolinium-containingcontrast agents, has been used for shortening the T1. complexes for modulating the 19F signal.50 Increased 19F MR signals from 19F nanoparticles were observed in close proximity to blood–brainbarrier disruptions, indicating a T1 shorteningeffect not only for 1H but also for19F as aresult of the paramagnetic contrast agent Gd–DTPA leakage.51While a reduction in g the T1. For 19F 19F groups. These propertieswere also severely affected by the serum environment. SF5-TF is administered as a suspension, and SF6 is administeredas a gas. All six fluorines on SF6 produce one signal,whereas the SF5 group has an AX4 spin system and producestwo separate signals. Most notably, the 19F T2 relaxation times of TF and its derivatives were drasticallyshortened in serum. The increase in FWHM of the 19F MRspectra in the serum compared to DMSO also suggests a T2* shortening.52 The observed shortening in relaxation is presumably attributed tounspecific binding of the compounds to serum proteins.53 Similar to SF5-substituted TF, thefluorinated gas SF6 exhibits a short transversal relaxationtime.20 SF5 is an organic derivativeof SF6 in which one of the fluorine atoms is replaced byan organic residue. However, there are notable T1 and T2 differences between SF5-TF and SF6.21 At 9.4 T, the T1 of SF6 at room temperature (300K) was reported to be <200 ms21 andis even shorter (1.24 ms at 1.9 T) when administered to rats as agas .20 The T1 of SF5-TF was notstrongly reduced in the presence of serum (T2 of SF5-TF was strongly reduced (by 90%) in thepresence of serum. Differences between SF5-TF and SF6 with respect to T1 and T2 can be attributed to differences in chemicalenvironments.23Differencesin chemical shifts and relaxation times can be attributed to the differentphysicochemical properties of the of serum 2, and we19F MRS.16 The above limitations in relaxation times, particularlywhen protein bound, restrict SNR. Due to the associated rapid lossin 19F signal during T2 relaxation,signal detection with 19F MRI is expected to be more challenging.Recently, we coulddetect TF in vivo when employing 19F relaxation times for TF and its derivativesto calculate the most efficient parameters for three 19F MRI acquisition methods: rapid acquisition with relaxation enhancement(RARE), balanced steady-state free precession (SSFP), and ultrashortecho time (UTE) (Table S3). Selection ofthe appropriate MR acquisition method according to the MR propertiesof the compound within a specific environment is essential to acquiredata with the best SNR efficiency. A thorough characterization of 19F compounds41 is typically necessaryto adapt SNR efficient 19F MR acquisition methods to thespecific MR characteristics of these compounds.38We usedthe determined 19F MR images of all compoundsusing the corresponding optimized sequences  and compared SNR efficiencies (SNR per molecule dividedby the square root of the acquisition time) to identify the most suitedmethod for each compound, in DMSO and in serum  were relapse-free when receiving14 mg of teriflunomide, compared to those receiving 7 mg of TF (39.5%).55 Thus, the higher efficacy of SF5-TF might have downstreambenefits for MS patients, and future in vivo experiments in the animalmodel will be needed to clarify whether SF5-TF administeredat the same dose as TF will be more efficacious at treating neurologicaldiseases. Apart from a ∼3× gain in the 19FSNR efficiency in vitro, we observed an SNR gain of ∼2 forSF5-TF compared to TF in the stomach ex vivo. This variationin the SNR gain could reflect variations in pH in the stomach thatcould affect relaxation times of TF.16 Futurecomparative studies are needed to focus on organ systems more relevantto the pathophysiology of MS such as the CNS.One interpretationof these results is that the increased activityof SF19F MRIacquisition methods when comparing TF with its substitutedderivatives  and SF5-TF = 10 mg/mL (n = 3)) and acquired 19F MR images of the stomachs ex vivo using the optimized parameters for SF5-TF in the stomach, comparedto 5.8 ± 2.9 for TF .We aligned thebiological activity with SNR efficiency of all ivatives 3A. Comparameters3B,C. We 5, to a pharmacologically active compound, to increasedetection in vivo. To our knowledge, this is the first study thathas performed an in-depth investigation of 19F modificationson pharmacological and biological activities, alongside the MR reporterfunction. Here, we addressed SNR by modifying the CF3 sidechain of TF to vary the 19F MR properties and identifypotential SNR-boosting derivatives. Substituting the CF3 side chain by CF3O, SF5, or di-CF3 did not compromise the pharmacological and antiproliferativeactivities. Of note, the superiority of the SF5-substitutedderivative to inhibit T cell proliferation compared to TF indicatesthat some fluorination strategies might even improve the biologicalfunction.Inthis study, we introduced modifications, including those ofSF5 as a potentialtheranostic markerfor detecting and studying the biodistribution of fluorinated drugsnoninvasively, particularly during pathology. However, in vivo studieswill now be necessary to study the versatility of the SF5 bioisostere in combination with the UTE method for in vivo 19F MRI and to determine its superiority over teriflunomideto treat disease in the animal model of multiple sclerosis.Our study indicates SF


For the development of an optimized Egyptian H9N2 vaccine candidate virus for poultry, various recombinant Egyptian H9N2 viruses generated by a PR8-based reverse genetics system were compared in terms of their productivity and biosafety since Egyptian H9N2 avian influenza viruses already possess mammalian pathogenicity-related mutations in the hemagglutinin (HA), neuraminidase (NA), and PB2 genes. The Egyptian HA and NA genes were more compatible with PR8 than with H9N2 AIV (01310) internal genes, and the 01310-derived recombinant H9N2 strains acquired the L226Q reverse mutation in HA after passages in eggs. Additionally, the introduction of a strong promoter at the 3′-ends of PB2 and PB1 genes induced an additional mutation of P221S. When recombinant Egyptian H9N2 viruses with intact or reverse mutated HA (L226Q and P221S) and NA (prototypic 2SBS) were compared, the virus with HA and NA mutations had high productivity in ECES but was lower in antigenicity when used as an inactivated vaccine due to its high binding affinity into non-specific inhibitors in eggs. Finally, we substituted the PB2 gene of PR8 with 01310 to remove the replication ability in mammalian hosts and successfully generated the best recombinant vaccine candidate in terms of immunogenicity, antigenicity, and biosafety. H9N2 avian influenza A viruses (AIVs) have been reported in human cases, and they act as internal gene donors for the generation of new highly pathogenic, reassortant H5N1 AIVs and low pathogenic H7N9 AIVs that cause poultry as well as human infections ,4,5,6. SG1-like H9N2 AIVs may have evolved in chickens, and the genetic constellation supporting efficient replication and transmission may have been selected. In the case of Y439-like H9N2 AIVs, passaging through embryonated chicken eggs (ECEs) resulted in the predominance of the NA stalk-deleted mutant strain and may reflect the importance of balancing hemagglutinin (HA) and neuraminidase (NA) activities. These phenomena have already been observed in natural H5Nx HPAIVs, and HA–NA balancing may be a key factor in viral fitness in chicken infections .HA protein is a transmembrane glycoprotein exposed outside of virus particles, and the receptor binding site (RBS) in the globular head of HA binds to host cell receptors. Avian and human influenza A viruses (IAVs) have different preferences for sialic acids, which are α2,3- and α2,6-linked  to galactose, and mutations in the RBS change receptor binding preferences and host specificity. The E190D and G225D mutations in the H1 subtype and the Q226L and G228S (H3 numbering) mutations in the H2 and H3 subtypes increase α2,6SA preference, and currently isolated H9N2 viruses already have Q226L mutations ,17,18. NThe cooperation of surface HA and NA proteins with internal proteins may also be important for viral fitness, and the appearance and predominance of new reassortant AIVs in nature may reflect the importance of compatibility between surface and internal proteins as well as between internal proteins ,25,26,27The commercial inactivated H9N2 AI vaccine for poultry has been extensively used in Egypt since 2012, but the dissimilarity of the vaccine strain with the field strain led to an antigenic drift in the antigenic site and escaping mutants ,34,35. TIn this study, we generated various recombinant Egyptian H9N2 viruses with different gene constellations to find the optimized recombinant vaccine strains using a 6 + 2 reverse genetics system. The compatibility of the HA and NA of Egyptian H9N2 AIVs with six internal genes of avian and human IAVs was tested, and the effects of mutations observed in the RBS of HA, 2SBS of NA, and PB2 of Egyptian H9N2 AIVs on viral fitness and their acquisition orders were investigated.Recombinant viruses were generated by a pHW2000 plasmid-based reverse genetics system provided by Dr. Robert Webster . Six internal genes  of A/Puerto Rico/8/1934 (H1N1) (PR8) and A/chicken/Korea/01310/2001 (H9N2)  and the 3′ end promoter-mutated PB2 and PB1 genes of 01310 (01310-U4) were used in this study ,39. 293TThe complete HA, NA, and PB2 genes of H9N2 AIVs were all collected from the GISAID Epiflu database, and the nucleotide sequences of HA and NA were aligned with the MEGA program. The nucleotide sequences were translated, and the amino acid mutations at specific residues were compared to calculate frequencies with the BioEdit program (v7.2.5).HA and NA genes were synthesized according to the coding region of A/chicken/Egypt/ME543V/2016(H9N2) , and the common 5′- and 3′-end noncoding region sequences of Egypt wild-type H9N2 isolates were determined from the GISAID database . The prokaryotic promoters and ORFs in the H9 gene sequence were predicted with the Berkeley Drosophila Genome Project (BDGP) program. Site-directed mutagenesis was performed with a Muta-Direct™ site-directed mutagenesis kit (iNtRON) and primer sets according to the manufacturer’s instructions . The HA 2 incubator, 1 mL of Opti-MEM (Life Technologies) and 4 µg/well of L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin  were added and incubated for another 24 h. Two hundred microliters of cell supernatant was inoculated into 10 day old SPF ECEs (VALO biomedia GmbH) and incubated for 3 days. Virus passages through ECEs were repeated 3 or 4 times, and allantoic fluid was harvested and stored at −80 °C until use. The presence of virus was detected with a hemagglutination assay using 1% (v/v) chicken red blood cells (RBCs), and the full genome sequences were confirmed by RT-PCR and sequencing as previously described . Ea. Ean = 55 EID50/0.5 mL of virus diluted in DMEM with 1 µg/mL TPCK-trypsin was inoculated into MDCK and A549 cells (5 × 105 cells/mL) seeded in a 12-well plate for 1 h, and the inoculated virus was eliminated. Infected cells were washed twice with phosphate-buffered saline, and 1 mL of fresh medium was added for 3 days of incubation. Cell supernatant was obtained every 24 h , and the viral titer at each time point was measured at a 50% tissue culture infective dose (TCID50) through 10-fold diluent inoculation into MDCK cells prepared in 96-well plates and calculated with the Spearman–Karber method.The safety of recombinant Egypt H9N2 virus as an inactivated vaccine candidate was indirectly evaluated by infection in mammalian host-originated cell lines, Madin–Darby canine kidney (MDCK) cells, and A549 cells (KCTC). A total of 5 × 10The inactivated recombinant Egyptian H9N2 vaccine efficacy test in chicken was approved and conducted in the facility at KBNP Inc. (IACUC no. KBNP 20-02). Inactivated oil emulsion vaccines were prepared with four recombinant Egypt H9N2 viruses: rEgH9N2(P) (K161ME), rEgH9N2(P)-310PB2 (K162ME), rEgH9N2(P)-P221S-L226Q-av2SBS (K163ME), and rEgH9N2(P)-P221S-L226Q-av2SBS-310PB2 (K164ME). Allantoic fluids containing each virus were inactivated by mixing with 0.2% formaldehyde (Sigma-Aldrich) and incubated at 37 °C for 24 h. The formaldehyde-treated virus was inoculated into 10 day old SPF ECEs to confirm virus inactivation, and confirmed viruses were mixed with ISA70 (SEPPIC) at a ratio of 3:7 to make an oil-emulsion vaccine. Ten one day old SPF chickens of each group were vaccinated with 0.2 mL of the oil-emulsion vaccine via the subcutaneous route. Serum from each group was collected at 0 and 3 weeks postvaccination (wpv) and used to measure serum antibody titers.2) hemagglutination titer (HAT) of four vaccine strain antigens for 40 min. Then, 25 μL of 1% chicken RBCs was added, and serum antibody titer was recorded after 40 min of incubation.Serum samples of each vaccinated group were treated at 56 °C for 30 min prior to serological testing to eliminate nonspecific hemagglutination inhibition reactions caused by proteins present in the serum, and the HI test was performed following the WHO Manual on Animal Influenza Diagnosis and Surveillance. Briefly, each serum sample was serially diluted two-fold with phosphate-buffered saline (PBS) and reacted with the same volume of 4  and centrifuged at 45,000 rpm  for 1 h. The sediment was eluted in 100 μL of sterilized water, and purified viral protein was deglycosylated with PNGase F . Twenty microliters of total viral protein and degycosylated protein was treated with 4X Bolt™ LDS Sample Buffer (Thermo Fisher Scientific) for 5 min at 95 °C and separated with NuPAGE 4–12% Bis-Tris Protein Gels (Life Technologies Co.). The protein band was confirmed with Coomassie Brilliant Blue G-250 staining  for 1 h and destained with a solution consisting of 10% ethanol and 7.5% acetic acid. For IgY detection in purified virus, SDS-PAGE gels were transferred to nitrocellulose membranes. The transferred membrane was incubated with blocking buffer (5% skim milk) for 1 h at room temperature, and HRP-conjugated goat anti-chicken IgG (IgY) antibody (Thermo Fisher Scientific)  was added for 1 h after three washes with TBST. HRP was developed with BioFX TMB One Component HRP Membrane Substrate (SurModics).To compare the antigen quantity of vaccine candidate viruses in the same volume of allantoic fluid, the virus was purified using OptiPrep™ Density Gradient Medium (Sigma-Aldrich) and ultracentrifugation . One hundred EIDp < 0.05 was defined as statistically significant.The significance of the results between experimental groups was evaluated by a one-way analysis of variance  , and E. coli promoter and open reading frames (ORFs) around the toxic regions and found the Shine–Dalgarno sequence  and the start codon (1281–1283). The GAGG sequence was mutated to GAAG, and transformed E. coli was incubated at low temperature (33 °C) to decrease the expression of the putative toxic gene. We successfully cloned the complete HA gene. Interestingly, the putative toxic ORF (144aa) had the same reading frame of HA2 of H9, but it had characteristics of outer-membrane proteins (OMPs)  (ME543) were used. In contrast to the NA gene sequence, the complete HA gene sequence could not be synthesized without a nucleotide (nt) deletion ranging from 1336 to 1365 (H9 numbering), which resulted in stop codons at amino acids 437 and 453 . Therefos (OMPs) . The obs7.58±0.14 to 108.53±0.70 in EID50/mL, The A/Puerto Rico/8/1934 (PR8)-based Hoffmann’s bidirectional pHW2000 plasmid and reverse genetics systems were used for recombinant virus generation . To incrThe frequencies of P221S and L226Q mutations in HA and 2SBS mutations in NA among H9N2 AIVs isolated during 1970–2020 are summarized in 8.07±0.06 EID50/mL) than rEgH9N2(P) (109.42±0.38 EID50/mL). However, rEgH9N2(P)-L226Q-av2SBS with L226Q, av370L, and av400L and rEgH9N2(P)-P221S-L226Q-av2SBS with L226Q, P221S, av370L, and av400L mutations showed higher (1010.03 ± 0.06 EID50/mL) and similar virus titers (109.37 ± 0.12 EID50/mL), respectively -L226Q showed the highest affinity, but the additional P221S mutation (rEgH9N2(P)-P221S-L226Q) decreased the affinity to an avian receptor analog (3′SLN). However, the combination of all the mutations, L226Q, P221S, av370L, and av400L, (rEgH9N2(P)-P221S-L226Q-av2SBS) restored binding affinity A. In con2−3 HAU after heat treatment at 56 °C for 4 h (In contrast to rEgH9N2(P)-P221S, the HA titers of the remaining strains gradually decreased to maintain 2 for 4 h A. The L2 for 4 h B.Egyptian H9N2 AIVs already had I66M, I109V, and I133V mutations (MVVs) in their PB2 genes, which may be a prerequisite for additional MPM acquisition, and they cumulatively acquired additional mutations   31,32].,32.31,32p > 0.05, To date, the 01310 PB2 gene has been successfully used for the development of AI vaccine strains in terms of high virus titers, strong immunogenicity, and mammalian nonpathogenicity ,37,40. TThe potential mammalian pathogenicity of AIVs can be predicted by replication efficiency in MDCK and A549 cell lines. In particular, A549 is an in vitro model that is in good correlation with the BALB/c mouse model . The gro9) at 3 weeks postvaccination (wpv), but K16E had the highest HI titers to homologous and heterologous strains. Interestingly, K163ME and K164ME with HA and NA mutations showed relatively low HI titers to homologous and heterologous strains.Four Egyptian H9N2 strains, rEgH9N2(P) (briefly K161ME), rEgH9N2(P)-310PB2 (K162ME), rEgH9N2(P)-P221S-L226Q-av2SBS (K163ME), and rEgH9N2(P)-P221-L226Q-av2SBS-310PB2 (K164ME), were selected to prepare formalin-inactivated oil emulsion vaccines and compare their immunogenicities and antigenicities in chickens . All of To understand why the four inactivated vaccines showed different immunogenicities and antigenicities, purified viral proteins were compared by SDS-PAGE, and the quantity of separated protein was measured using ImageJ 1.53a. Interestingly, the amounts of NP (PNGase F-treated) and NP + HA1 (PNGase-untreated) were higher in K161ME and K163ME than in K162ME and K164ME A. K164MEE.coli plasmids, and our cloning failure was unexpected. HA gene cloning using E. coli after gene synthesis or amplification is the starting point of reverse genetics, and the problem needs to be solved. To our knowledge, the identification of toxic fragments in HA2 to E. coli has rarely been reported. We analyzed various H9N2 HA genes recently deposited in the database, but we could not find similar putative E. coli ORFs (data not shown). Although this may be an uncommon case, we can solve similar problems when we confront them in the future.To date, various HA genes of AIVs have been cloned into The low compatibility of Egyptian HA and NA genes with six 01310 internal genes, which supported the high growth of 01310E20, resulted in decreased replication efficiency but might have been a good condition for the outgrowth of quasispecies that acquired mutations, improving replication efficiency. The commonly shared reverse mutation L226Q may be the best way to recover the replication efficiency of recombinant strains with incompatible internal genes and the NA gene encoding low affinity to avian receptors ,44,45. CThe strong promoters in PB2 and PB1 genes induced an additional HA mutation, P221S, and it may contribute to the outgrowth of the quasispecies among only the L226Q-bearing viral population in ECEs. The P221S mutation has been reported in H5N1 AIVs to reduce HA trimer integrity to cause low heat and acid stabilities ,50. The In this study, the higher compatibility of six PR8 internal genes with Egyptian than its own HA and NA genes was clearly demonstrated by the growth kinetics study in mammalian cells . AlthougEgyptian H9N2 AIVs have evolved for more than 10 years in chickens, with some occasional infections in pigs and humans, and it is unclear in which host animals they acquire MPMs, especially the E627V mutation ,54. For To date, the gain-of-function of viruses has been a concern for a long time due to the potential risks of laboratory and manufacturing leaks of viruses, but why the generation of PR8-derived vaccine strains against mammalian pathogenic AIVs has not been discussed in biosafety and biosecurity aspects in depth is strange . In our The immunogenicity and antigenicity of the whole virus vaccine depend on the antigen amount and epitope similarity, respectively. The highest HI antibody titers of rEgH9N2(P)-310PB2 against homologous and heterologous antigens need to be addressed . AlthougIn this study, recombinant Egyptian H9N2 vaccine candidate viruses with different genetic characteristics were generated using a plasmid-based reverse genetics system, and we compared the productivity in ECEs, the mammalian pathogenic risk, and the antigenicity as a poultry vaccine. A PR8-derived recombinant vaccine strain possessing parent HA and NA genes and the 01310 PB2 gene was successfully selected as the best vaccine candidate against Egyptian H9N2 AIVs undergoing mammalian adaptation in terms of immunogenicity, antigenicity, and mammalian nonpathogenicity.


Serpina6, which encodes corticosteroid-binding globulin (CBG). Klf15-deficient mice have profoundly low CBG, reduced plasma corticosteroid binding capacity, and heightened mortality during inflammatory stress. These defects are completely rescued by reconstituting CBG, supporting that KLF15 works primarily through CBG to control plasma corticosterone homeostasis. To understand transcriptional mechanisms, we generated the first KLF15 cistromes using newly engineered Klf153xFLAG mice. Unexpectedly, liver KLF15 is predominantly promoter enriched, including Serpina6, where it binds a palindromic GC-rich motif, opens chromatin, and transactivates genes with minimal associated direct gene repression. Overall, we provide critical mechanistic insight into KLF15 function and identify a hepatocyte-intrinsic transcriptional module that potently regulates systemic corticosteroid transport and inflammation.Circulating corticosteroids orchestrate stress adaptation, including inhibition of inflammation. While pathways governing corticosteroid biosynthesis and intracellular signaling are well understood, less is known about mechanisms controlling plasma corticosteroid transport. Here, we show that hepatocyte KLF15 (Kruppel-like factor 15) controls plasma corticosteroid transport and inflammatory responses through direct transcriptional activation of  The hepatocyte KLF15-CBG axis, revealed by KLF15 cistromes, controls systemic corticosteroid transport and inflammation. In mammals, circulating corticosteroid hormones are synthesized by the adrenal gland and orchestrate adaptations to physiological stress across multiple organs (Klf15−/−) are viable (Serpina6 [which encodes corticosteroid-binding globulin (CBG)] in hepatocytes to potently control plasma corticosteroid transport in vivo and demonstrate that this KLF15-Serpina6 transcriptional module plays a critical homeostatic role in the systemic response to inflammatory stress. In the course of these studies, we define the genome-wide binding patterns of KLF15 and provide important mechanistic insight into the function of this widely studied transcription factor.Kruppel-like factor 15 (KLF15) is the most ancient member of the modern-day KLF family of zinc finger transcription factors , the major site of de novo corticosteroid synthesis in mammals. In addition, expression of key enzymes in the corticosteroid biosynthetic pathway was not reduced in adrenal glands of Klf15−/− mice (fig. S1B), and adrenal gland weight did not differ between genotypes (fig. S1C). The principal stimulus for corticosteroid biosynthesis by the adrenal cortex is the peptide hormone ACTH (adrenocorticotropic hormone), which is secreted by the anterior lobe of the pituitary gland. Klf15−/− mice did not have decreased plasma ACTH concentration (fig. S1D), and we did not detect Klf15 expression in the pituitary gland (fig. S1E). Collectively, these observations suggested that the low plasma total corticosterone concentration in Klf15−/− mice could not be attributed to a defect in biosynthesis, prompting us to find another plausible molecular mechanism that could explain this hormone defect.Our study began with the unexpected observation that ntration . We expeKlf15−/− mice , while KLF15 knockdown with a short hairpin RNA (shRNA) decreased Serpina6/CBG mRNA and protein abundance (SERPINA6 (fig. S1J), confirming that this regulatory axis is conserved in human hepatocytes. As circulating cytokines and hormones can influence CBG abundance in vivo (Klf15 by crossing Klf15flox/flox and Albumin1-Cre strains to generate Albumin1-Cre:Klf15flox/flox mice (termed KLF15-LKO). We found that KLF15-LKO mice had decreased CBG in liver . Hence, the genome-wide binding sites of this intensely studied transcription factor remain unknown. To provide unbiased and whole-genome level insight into KLF15 mechanism of action, including at the Serpina6 locus, we engineered a germline mouse strain harboring a 3x-FLAG epitope tag fused to the C terminus of endogenous KLF15 using Cas9-based genome editing of the endogenous locus . Anti-FLAG immunoblots from the liver tissue of these mice unambiguously confirmed robust detection of a specific band of the expected size , supporting that the Klf153xFLAG allele did not perturb the normal transcriptional function of KLF15.As KLF15 deficiency causes robust reduction in CBG abundance, we hypothesized that ted size . FLAG imted size . NotablyIvd  and Serpina6 loci, the former being a branched-chain amino acid metabolic gene known to be regulated by KLF15  in adult mouse liver tissue to determine genome-wide KLF15 enrichment. Concomitantly, we performed RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) in the liver tissue from adult KLF15-LKO versus control mice to assess the relationship between locus-specific KLF15 enrichment, gene expression, and chromatin accessibility . KL H3K4me3  (39). In H3K4me3 , top and H3K4me3 , bottom. H3K4me3 , a ubiquKlf15 strongly decreased its own transcript levels in our RNA-seq data. Among all detectable KLF family transcription factors, only Klf10 also exhibited a significant change in gene expression in the livers of KLF15-LKO mice (fig. S4A). We next investigated whether loss of KLF15 had a broader impact on the expression of its interactome, which was defined using experimentally validated and predicted interactions from the STRING database (fig. S4B and table S1), and found no KLF15 interactors as differentially expressed. These data suggest that hepatocyte Klf15 deficiency does not broadly affect the expression levels of KLF family genes or the predicted KLF15 interactome.To further dissect KLF15-dependent gene regulatory mechanisms, we analyzed changes in gene expression between KLF15-LKO and control mouse livers. We confirmed that hepatocyte-specific deletion of Nr3c1 expression itself was unchanged in KLF15-LKO versus control livers (fig. S5A). Next, we defined the directly bound targets of NR3C1 by curating a published dataset in which NR3C1 ChIP-seq was performed in the mouse liver (Klf15 deletion in the liver (fig. S5A). In addition, we compared the total promoter KLF15 signal to the total distal HNF4a signal among all expressed genes and found that many genes down-regulated in KLF15-LKO, including Serpina6, exhibit high levels of proximal KLF15 without any distal HNF4a (fig. S5B). This indicates that a specific subset of adult hepatocyte genes, including Serpina6, are more dependent on regulation by proximal promoter KLF15 binding than they are by tissue-specific distal enhancers or other transcription factor programs.We next defined the set of transcripts that are differentially expressed in KLF15-LKO versus control mice (fig. S4C) and analyzed the relationship between differential expression and local KLF15 binding (fig. S4D). Using gene set enrichment analysis (GSEA), we identified enrichment for metabolic pathways in the set of transcripts significantly down-regulated in LKO mice , consistent with the enrichment for metabolic genes bound by KLF15 in our ChIP-seq data (fig. S3B). Inflammation-related transcripts were up-regulated in KLF15-LKO mice ; however, the set of KLF15-bound genes was not enriched for these inflammatory targets (fig. S3B). We more closely examined the relationship between KLF15 promoter occupancy and the directional change in expression of the proximate assigned genes in KLF15-LKO mice . ConsistIvd and Serpina6 promoters, where KLF15 is normally bound   , indicatde (LPS) , KLF15-Lde (LPS) . In an iKlf15-deficient mice were caused by decreased CBG expression, we performed in vivo rescue experiments to restore CBG expression in the livers and plasma of Klf15-deficient mice. We generated adeno-associated virus serotype 8 (AAV8) harboring a complementary DNA (cDNA) encoding full-length murine CBG and first validated this reagent in Klf15−/− mice, whose highly penetrant defects in CBG expression and corticosteroid binding capacity allow for efficient physiological validation. AAV8-CBG administration via tail vein injection rescued CBG protein abundance in the livers and plasma of Klf15−/− mice  and completely rescued the defect in corticosteroid binding capacity in the plasma of Klf15−/− mice, with a correction of free and total plasma corticosterone  gene promoter has been shown to be activated by HNF4a and repressed by COUP-TF, implicating competition between these two factors for critical cis-regulatory elements in the locus as a mechanism of regulation  and subsequent elimination in Klf15-deficient cells or animals. Furthermore, no direct molecular mechanism is shown by which endobiotic metabolism genes are up-regulated in Klf15-deficient animals, and no rescue of endogenous corticosterone homeostasis is demonstrated by specific inhibition of the endobiotic metabolism machinery. By contrast, our current study shows that the low total plasma corticosterone in Klf15-deficient mice is accompanied by a three- to fourfold elevation in free plasma corticosterone , while most of the putative repressive effects of KLF15 occur indirectly. Therefore, our data support the conclusion that many of the genes in the endiobiotic metabolism pathway are not direct targets of KLF15, suggesting that much of this gene up-regulation that occurs in KLF15-deficient livers may arise through secondary or indirect effects.A recent study has suggested that KLF15 regulates total plasma corticosterone abundance via repressive effects on genes involved in metabolism, biotransformation, and elimination of endobiotics  at the University of California, San Francisco and conducted in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, eighth edition. Mice were housed in a temperature- and humidity-controlled pathogen-free facility with 12-hour light/dark cycle and libitum access to water and standard laboratory rodent chow.Klf15 and Serpina6 in primary hepatocytes after LPS exposure . For survival analysis of KLF15-LKO mice, statistical analysis was performed using a log-rank test. For cytokine measurement, two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test was applied to determine the effect of genotype on cytokine induction after LPS injection. For the down-regulation of exposure , a one-wKlf15−/− mice have been previously described 21Mgn/J; the Jackson Laboratory, catalog no. 003574] were crossed with Klf15flox/flox mice . The control mice for KLF15-LKO mice are of genotype Klf15flox/flox. All mice were in a pure C57Bl/6 background, and our experiments were performed in male mice aged 14 to 20 weeks. All experiments were performed using littermate controls.Klf153xFLAG allele in the germ line, we used CRISPR-Cas9–based genome editing of mouse blastocysts (which were in a pure C57Bl/6 background) using direct injection of purified Cas9, guide RNA, and single-stranded DNA repair template, as previously described (Klf15 locus. The DNA repair template (5′-ACACATCAAAGTGCATCGCTTCCCACGAAGCAGCCGCGCAGTACGCGCCATCAACGGAGGCGGTGGAGCCGACTACAAGGACCACGACGGCGACTACAAGGACCACGACATCGACTACAAGGACGACGACGACAAGGGGCCTGTTTGAGCGCAGTGGCCGCCCTTCCCTCCCCCAGCTCCACGTTTTGTTTTTAAATGCA-3′) encodes a cassette with a short linker sequence followed by a 3xFLAG tag and stop codon. Approximately 50–base pair (bp) flanking homology arms are present on both sides of the 3xFLAG cassette (0 (mosaic) offspring were screened for the presence of the 3xFLAG cassette using polymerase chain reaction (PCR) amplification of tail genomic DNA, TA cloning, and DNA sequencing. Three founder lines that had high levels of mosaicism were crossed with male C57BL/6 mice from the Jackson Laboratory  to generate F1 offspring, which were screened for germline transmission by PCR and sequencing of TA-cloned PCR amplicons from tail genomic DNA. Of these lines, we chose one line that was confirmed by PCR, sequencing of genomic DNA, and detection of the KLF15-3xFLAG fusion protein by FLAG Western blot. All Klf153xFLAG mice were in a pure C57Bl/6 background, and our experiments were performed in male mice aged 12 to 16 weeks. Genotyping primers used in fig. S2A are as follows: 5′-CGCTCAGATGAGTTGTCAAGG-3′ (forward) and 5′-GTGGCTCTGGGTCATACCT-3′ (reverse).To generate mice harboring a g for 15 min at 4°C to separate plasma from blood cells. This plasma sample was used for the measurement of total corticosterone concentration. To measure free plasma corticosterone concentration, we used an aliquot of plasma and separated the protein-bound fraction by ultracentrifugation using a Centrifree ultrafiltration device . Briefly, plasma was transferred into the ultrafiltration tubes and centrifuged at 2000g for 30 min. The ultrafiltrated and non-ultrafiltrated plasma were assayed for free or total corticosterone, respectively, by the Mouse Metabolic Phenotyping Center (MMPC) at the University of California, Davis using standardized radioimmunoassay. Non-ultrafiltrated plasma was also assayed for ACTH and aldosterone concentration by the Vanderbilt MMPC Hormone Assay and Analytical Services Core using standardized radioimmunoassays. For plasma corticosterone measurements, blood was taken from mice at 4:00 p.m. to coincide with the time of daily peak plasma corticosterone in mice.Mice were euthanized, after which blood was immediately collected through the abdominal aorta (approximately 0.7 ml) and transferred into EP tubes that contained 500 mM EDTA . The tubes were then centrifuged at 1500m/z) (collision energy of 24.63 V) and 351/121 m/z (collision energy of 25.56 V), respectively. Standard curves were prepared in acetate buffer using purified corticosterone standard and extracted as described above. Free and total corticosterone plasma concentrations were calculated using the ratio of corticosterone peak area/corticosterone-d4 peak area for each sample.A total of 20 μl of EDTA-plasma was used for total corticosterone measurement, and 150 to 200 μl of ultrafiltrated plasma were used for free corticosterone measurement in LC-MS experiments. Plasma corticosterone extraction was performed on the basis of previous reports with some modifications (Klf15−/− and Klf15+/+ mice and processed into 10-μm-thick cryosections on microscope slides. Slides were fixed for 5 min in 0.2% (w/v) glutaraldehyde . Slides were washed three times in wash buffer . Each section was covered with LacZ Staining Solution , dissolved in wash buffer, and incubated in the dark at 37°C overnight. Afterward, slides were washed three times for 5 min each in wash buffer followed by a 2-min wash in phosphate-buffered saline (PBS). Next, sides were dehydrated through sequential washes in increasing concentrations of ethanol and cleared with xylene . Slides were mounted with coverslips using VECTASHIELD hard-set antifade mounting medium . Images were then captured using a light microscope.The adrenal gland was dissected from adult l-lysine, and 0.04% azide (pH 7.4)] for 1 hour at room temperature. Slides were then washed twice for 7 min each in wash buffer. Primary antibody {anti–beta-galactosidase  in antibody buffer } was then added onto sections and incubated for 2 days at 4°C. Subsequently, slides were washed in wash buffer three times for 10 min each followed by a 1-hour incubation at room temperature with secondary antibody  and 10-min incubation with 4′,6-diamidino-2-phenylindole at room temperature and mounted with Fluor gel . Images were then captured with a Keyence fluorescence microscope. This analysis was replicated in at least three independent samples.Liver, hypothalamus, and pituitary gland were dissected from adult mice and processed into 10-μm cryosections on microscope slides. Slides were washed twice for 7 min each in wash buffer (PBS and Tween 0.2%) followed by blocking sections with blocking buffer  using a Dounce homogenizer and then rotated at 4°C for 10 min. The pellet was spun down and resuspended in lysis buffer 2  for 10 min at 4°C. The pellet was spun down and resuspended in 1 ml of sonication buffer . The sonication buffer with chromatin was then sheared on a BioRuptor , with samples continuously cooled at 4°C. The BioRuptor settings used were 30 s on and 30 s off with the output setting on “high” for a total of 12 cycles. The sonicated chromatin was immunoprecipitated overnight at 4°C using 5 μg of anti-FLAG  monoclonal antibody–bound Dynabeads , followed by extensive washing and elution. Input and immunoprecipitated chromatin were then reverse cross-linked followed by purification of genomic DNA, which was then subjected to library preparation and next-generation sequencing. Purified DNA was quantified using Qubit fluorometric analysis and assessed for quality using Agilent Bioanalyzer Nano DNA Chip. Library preparation was performed using the NEBNext Ultra II DNA Library Prep Kit from Illumina . Libraries were sequenced on a NextSeq 500/550 at the Genomics Core of Gladstone Institutes .ChIP was performed from mouse liver tissue as previously described  according to the manufacturer’s instructions. RNA-seq libraries were prepared with the Ovation Solo RNA-Seq kit (NuGEN). The final libraries were analyzed by Agilent Bioanalyzer Nano RNA Chip and quantified by KAPA qPCR. The library was then sequenced on an Illumina HiSeq 2500 at the Genomics Core of Gladstone Institutes.Total RNA was extracted from liver tissues of KLF15-LKO mice and Klf15flox/flox control mice as described above. For each sample, 50,000 cells were pelleted after washing once with 1 ml of PBS. Cell pellets were then resuspended in 50 μl of cold lysis buffer  and incubated on ice for 3 min. A total of 1 ml of cold wash buffer  was then added to the lysis. The sample was centrifuged at 500g for 10 min at 4°C. The pelleted nuclei were resuspended in 50 μl of transposition mixture  and incubated at 37°C for 2 hours in a ThermoMixer with 1000 rpm mixing. Transposed DNA was purified with the MinElute Reaction Cleanup Kit  and eluted in 21 μl of water. Purified DNA was amplified using 25 μl of the NEBNext High Fidelity 2x PCR Master Mix, 1.25 mM Nextera custom primers with unique barcodes, and nuclease-free water. Samples were amplified using the following PCR conditions: 72°C for 5 min; 98°C for 30 s; and cycled at 98°C for 10 s, 63°C for 30 s, and 72°C for 1 min. Half of each sample was amplified for 12 cycles, purified, and assessed by Bioanalyzer (Agilent) for library quality. Sample concentration was quantified by Qubit (Invitrogen) before pooling. Pooled samples were sequenced on a NextSeq 500/550 at the Genomics Core of Gladstone Institutes .Library preparation and ATAC-seq were performed as previously described  gene annotations.RNA-seq was aligned to the mm10 genome using STAR (version STAR_2.5.1b) (https://github.com/BradnerLab/pipeline/wiki/bamliquidator). Briefly, ChIP-seq reads aligning to the region were extended by 200 bp, and the density of reads per base pair was calculated. The density of reads in each region was normalized to the total number of million mapped reads producing read density in units of reads per million mapped reads per base pair (rpm/bp).We calculated the normalized read density of a ChIP-seq dataset in any genomic region using the Bamliquidator (version 1.0) read density calculator  for each sample was visualized as a heatmap.Microarray gene expression profiles from 2 ratio of classes as the metric for ranking genes. The hallmark gene sets were used for leading edge enrichment analysis, and enriched gene sets (FDR < 0.05) for each condition were visualized as a bar graph.GSEA (https://github.com/kundajelab/chipseq_pipeline). Briefly, fastq files were aligned to the mm10 genome using BWA  thresholded peaks were used as the final peak set. H3K27ac, H3K4me3, RNA Pol II, H3K4me1, and H3K27me3 ChIP-seq were obtained directly from the ENCODE database  , and GR/NR3C1  ChIP-seq data were processed using the AQUAS Transcription Factor and Histone ChIP-seq processing pipeline (https://github.com/kundajelab/atac_dnase_pipelines). Data were processed as described for ChIP-seq, with the addition of TN5 shifting of reads following read filtering and deduplication.ATAC-seq data were processed using the Kundaje laboratory ATAC-seq/DNase-seq pipeline , we measured the distance (within 1 Mb) to the nearest TSS (as previously defined), and the results were visualized as a histogram. Statistical comparison of distributions was done using the Epps-Singleton test.The closest genes (within 1 Mb) for each KLF15 peak were identified, and gene ontology enrichment analysis for gene ontology biological process terms was conducted using the PANTHER overrepresentation analysis –subtracted area under the curve (AUC) of all KLF15 peaks within the promoter (±2 kb around TSS) of a gene. The statistical significance of changes in distributions among up-regulated, down-regulated, and nondifferentially expressed genes was assessed using a Mann-Whitney Functional and physical protein associations were obtained from the STRING database with medium confidence level (0.4) and interactions from both experimental sources and computational predictions. Both first and second shell interaction partners were captured.2 fold change of gene expression level in each bin was plotted, along with a trend line of the median log2 fold changes in each bin.Expressed genes were identified as above and binned by KLF15 promoter signal or proximal GR/NR3C1 signal to generate a waterfall plot. The logProximal (promoter) KLF15 signal was computed as described above. Distal HNF4a signal is defined as the sum of the background-subtracted AUC for all HNF4a peaks within 1 Mb of a gene and not within the promoter region (±2 kb).2 fold change of control signal over KLF15-LKO signal was calculated for each gene and used to rank genes by change in ATAC-seq. Using GSEA  of a gene. Logn = 2) for a given condition. Peaks within the promoter region were selected, and control rank versus KLF15-LKO was visualized as a scatterplot. Points were colored by KLF15 AUC in the open chromatin region defined by the ATAC-seq peak.ATAC-seq peak sets under control and KLF15-LKO conditions were merged, and peaks were ranked in ascending order by their mean AUC (Motif co-occurrence with KLF15 peaks was generated by first filtering for actively expressed transcription factors with a known motif in the HOCOMOCO v11 core mouse transcription factor database (