CV777) and variant strain SD2014 (SSD2014) were mapped using biosynthetic peptides and rabbit anti-PEDV S serum. Seventy-nine and 68 linear BCEs were identified from SCV777 and SSD2014, respectively. While 66.2% of the BCEs of SSD2014 could be recognized by anti-SCV777 serum and 67.1% of SCV777 BCEs could be recognized by anti-SSD2014 serum, more than 40% of the BCEs identified using anti-SCV777 serum on SCV777 could not be recognized by anti-SSD2014 serum and vice versa. The completely shared BCEs took low percentages of 29.4% and 25.3% for SSD2014 and SCV777, respectively. These results indicate a low conservation of antigenicity of the S protein compared to a relatively high amino acid sequence similarity of 92.2% between the two strains. The study provided a BCE shift reference of PEDV antigenic escape and surveillance control.The spike protein (S) plays a crucial role in porcine epidemic diarrhea virus (PEDV) infection and induces neutralizing antibodies. Mutations of the S protein are supposed to provide the main antigenic shift leading to the antigenic escape of PEDVs. It is therefore a significant question how much accumulation of antigenic shift could lead to the antigenic escape of the variant PEDV. To provide an answer in the study, B cell epitopes (BCEs) on the S protein of the PEDV vaccine strain CV777 (S Alphacoronavirus in the family Coronaviridae, causes acute and highly contagious enteric disease in pigs and is designated as porcine epidemic diarrhea (PED). The pathogen was first detected in Europe in the early 1970s [Porcine epidemic diarrhea virus (PEDV), a member of ly 1970s . Since tly 1970s . In lately 1970s ,4,5. VarAmong the structural proteins of PEDV, the spike (S) protein plays pivotal roles in PEDV attachment, receptor binding, and virus–cell membrane fusion during PEDV invasion into host cells . The PEDImportant neutralizing regions had been screened out from the PEDV S protein, such as the N-terminal domain ,10, CO-2SD2014) has the typical insertion and deletion found in the S protein of current epidemic strains. In the present study, fragments of S proteins of the PEDV classical vaccine strains CV777 (SCV777) and SSD2014 were expressed and purified to immunize rabbits. Using the polyclonal sera acquired, the linear B cell epitopes (BCEs) were identified from serial glutathione S-transferase (GST)-fusion expressed 16-mers covering the entire sequences of SCV777 and SSD2014. Recognition efficiency of the anti-sera to the BCEs was analyzed on the two S proteins. The results confirm the low conservation of antigenicity of the S protein of the wild-type PEDV strain SD2014 compared with the classical strain CV777. The study provided a clear BCE comparison of the S proteins of the two PEDV strains, which laid a solid foundation for antigenicity variation and an antigenic escape analysis of wild-type PEDV.The highly virulent PEDV SD2014 strain was the etiology of a PED epidemic in a farm in Shandong Province of China in 2014. Whole-genome phylogenetic analysis showed that PEDV SD2014 belongs to the Genotype II subclass, clustered together with the re-emergent virulent variant PEDV strains, including US strains. Compared to the S protein of the classical strain (Genotype I), the S protein of SD2014 strain (SCV777 (ALS35469.1) and SSD2014 (AND76936) was performed with Mega 7 . Online server IEDB ) was used for BCE prediction. Seven residue-length epitopes were viewed as adequate to persuade defensive immune reaction. Only those epitopes predicted by no less than three algorithms were chosen. VaxiJen 2.0 server (http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html (accessed on 24 January 2022)) was utilized for antigenicity analysis of chosen epitopes.Sequence alignment of SCV777 or SSD2014) employed as the immunization antigen was prepared as described previously [S gene was synthesized, cloned into pET-28a or pET-32a in three segments , and expressed in Escherichia coli (E. coli) BL21. After separation of the expressed protein by SDS-PAGE and purification by electro-elution of the excised gel band, the protein segment was concentrated with PEG 8000 and stored at −80 °C prior to use.Recombinant S protein  were purchased from the Shanghai SLAC Laboratory Animal Co. Ltd. . The trial was approved by the Institutional Animal Care and Use Committee of Shanghai Academy of Agricultural Sciences  , and animals were treated in accordance with the regulations and guidelines of the committee. Eighteen rabbits were used for the immunization with the six S protein fragments (SFs) prepared as in 2.2 . Three rabbits were injected intramuscularly with 0.5 mg of each purified recombinant SF  emulsified in complete Freund’s adjuvant (CFA)  at multiple sites on the back of each animal. Three booster injections of 0.25 mg of the same antigen per injection in incomplete Freund’s adjuvant were administered at two-week intervals. Animals were bled 7 days after the third booster, and the serum was separated and stored at −80 °C until use. The mixed sera from 3 rabbits immunized with each SF were used in the following-up tests. Non-immune serum from the remaining one rabbit that received only CFA was used as the negative control.CV777 (or 172 16-mers and one 10-mer for SSD2014) were expressed as fusion protein as described earlier [BamH I and TAA-Sal I cohesive ends on their 5′- and 3′-terminals, respectively, were inserted into the BamH I and Sal I sites of plasmid pXXGST-1. The confirmed recombinant plasmids were transformed into E. coli BL21 and temperature-induced to express the fusion peptides. Positive clones were selected by SDS-PAGE. Cell pellets containing each fusion peptide were stored at −20 °C until BCE mapping by Western blot (WB).The truncated glutathione S-transferase  was used as carrier for the expression of 16-mer peptides. One hundred and seventy-one 16-mer peptides and one 14-mer peptide with an overlap of 8 aa residues covering the entire S earlier . Briefly®-PSQ, Merck Millipore Ltd., Darmstadt, Germany). Membranes were then blocked with 5% (w/v) skimmed milk powder in tris-buffered saline with 0.1% tween 20 (TBST), incubated sequentially with anti-S serum  and goat anti-rabbit IgG conjugated to horseradish peroxidase  , and visualized by enhanced chemiluminescence using ECL plus Western blot detection reagent  according to the manufacturer’s instructions.Cell pellets were resolved by SDS-PAGE using 15% gels. Gels were either stained with Coomassie Brilliant Blue G-250  for confirming the bands of fusion proteins or processed for WB by electro-transferring the proteins onto 0.2 μm polyvinylidene difluoride (PVDF) membrane  when the cells reached approximately 80% confluence. At 18 h post infection, the cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.1% Triton X-100 for 15 min at room temperature. After being washed three times in phosphate-buffered saline (PBS), the cells were blocked with 5% goat serum in PBS for 1 h at 37 °C and then incubated with rabbit anti-SFs serum  and S1A domain (23/80), while the 28 differential residues in the S2 subunit scatter over different functional domains except fusion peptide (FP) and transmembrane domain (TM)  .CV777 and SSD2014, respectively , while the strongest antigenicity on SSD2014 was predicted at position 133 to 146 aa with an antigenic value of 1.0705 , S1B (E14 and E17), ahead of FP (E21), and heptapeptide repeat 1 (HR1) (E27) domains, respectively, and elimination of six previous BCEs, which were distributed on the S10 (E5 and E8), S1A (E14 and E17), S1CD (E21), and HR1 (E29) domains. Similarly, amino acid variation on SSD2014 generated nine new dominant epitopes, distributed on the S10 , S1A (E11), S1B (E14 and E17), ahead of FP (E20 and E21), and HR1 (E27) domains and eliminated five previous dominant BCEs, which were distributed on the S1A (E17), S1CD (E22), and ahead of heptapeptide repeat 2 (HR2) (E30 to E32) domains.Compared to SBefore identifying linear BCEs on the S protein with the prepared rabbit anti-S sera, IFA and IHC were firstly performed to investigate whether the six anti-S segment sera could react with PEDV DR13. As shown in CV777 and SSD2014, the purified S protein segments were separated through SDS-PAGE and transferred onto PVDF film for WB with the sera, respectively. As shown in To test the binding ability of the prepared rabbit sera with SCV777, serial 16-mers and a 14-mer overlapping 8 aa were GST-fusion expressed in E. coli. Among the 171 16-mers and the single 14-mer segments covering the entire SCV777, four 16-mers  in TM domain and cytoplasmic tail (CT) could not be expressed. A little surprisingly, P168 (1337–1352 aa) at the junction of TM and CT was efficiently expressed. The GST-fusion 16-mers were expressed in size about 22.4 kD  by WB. Among 168 16/14-mers screened, 60 positive 16-mers and 1 positive 14-mer were identified by rabbit anti-SCV777 serum  and 18  were specific for the anti-SCV777 serum and the anti-SSD2014 serum, respectively. Therefore, 77.2% of the 79 positive 16/14-mers of SCV777could be bound by its anti-serum, and 67.1% could be bound by anti-SSD2014 serum, indicating an obvious overlapping of antigenicity between the two S proteins.In order to comprehensively study the BCE distribution on S77 serum B,E,H,K, 14 serum C,F,I,L. 0 domain, including three anti-SCV777 serum-specific (P8–P9 and P16) and one anti-SSD2014 serum-specific (P18)  and 5  were anti-SCV777 and anti-SSD2014 sera-specific, respectively. The other nine  were Co-RecoMers  were Co-Recomers (CV777 serum-specific (P102 and P107) and three anti-SSD2014-specific (P96–P98) 16-mers and one Co-RecoMer (P110)  were Co-RecoMers  were Co-RecoMers  were Co-RecoMers (SD2014 serum (P160), were identified in HR2 domain  out of the 79 positive 16/14-mers were located in the S1ic (P18) A. TwentyRecoMers A,B. Four777serum B. Four pRecomers B. Six por (P110) B. Four pRecoMers B. Six poRecoMers B,C. Six RecoMers C. Ten 16RecoMers C. Two 162 domain C. One 1677 serum C. No posCD2014, both anti-SCV777 and anti-SSD2014 sera were used to screen the positive peptides fusion-expressed with GST tags. Except for the 4 16-mers (P167–P170), which span part of the TM domain and its immediate downstream, the other 168 GST-fusion 16-mers or one 10-mer were successfully expressed  were anti-SSD2014 serum-specific. Therefore, 68 positive 16-mer were totally identified on SSD2014, of which 66.2% could be recognized by anti-SCV777 serum, and 82.4% could be bound by their own anti-serum.Using the same strategy as in 3.4 to identify linear BCEs on Sxpressed . From thectively . Among tectively . Twelve CV777, no positive 16-mer was located at the SP and TM domain of SSD2014. Ten positive 16-mers  were in the S10 domain, among which P19 is anti-SSD2014-specific, and the other nine were Co-RecoMers , 5  anti-SCV777 serum-specific, and 4 Co-RecoMers  . Two (P71–P72) and one (P76) positive 16-mers in the S1B domain were specific for anti-SSD2014 and anti-SCV777 sera, respectively  were Co-RecoMers  were Co-RecoMers  were Co-RecoMers  were Co-RecoMers (SD2014 serum-specific (P150)  was identified (Like the distribution of positive 16-mers on SRecoMers A (9/10).P60–P61) A,B. Thatectively B (1/3). RecoMers B (4/4). RecoMers B (4/8). RecoMers B (2/2). RecoMers B (4/5). RecoMers C (3/6). c (P150) C (7/8). c (P150) C (1/1). entified C (1/1).0 (5) and S1A (4).Among the 68 Co-Recomers found in the two S proteins, only 20  were Co-Recomers on both S proteins (coined as Shared Co-RecoMers here) . Nine ofCV777 were no longer Co-RecoMers on SSD2014. These 16-mers on SSD2014 could only be bound by anti-SSD2014 serum  or anti-SCV777 serum (P88). Likewise, eight Co-RecoMers on SSD2014 were not Co-RecoMers on SCV777. These 16-mers on SCV777 could only be recognized by anti-SCV777 serum  or anti-SSD2014 serum   .CV777 serum. On the opposite, eight 16-mers  on the two proteins could only be bound by anti-SSD2014 serum  on the two S proteins could only be bound by their own anti-sera. Six positive 16-mers  on the two proteins could only be bound by anti-S14 serum . TherefoCV777, which included 10 Co-RecoMers , 13 anti-SCV777 serum-specific , and 5 anti-SSD2014 serum-specific  , 7 anti-SSD2014 serum-specific , and 5 anti-SCV777-specific   . Meanwhind P119) .SD2014, only 13 (19.1%) Shared Co-Recomers in the S10 (5), S1A (2), S1CD (1), ahead of FP (1), FP (2), and ahead of HR2 (2) domains manifested the same antisera binding property as the corresponding Shared Co-Recomers on SCV777, which indicate that the incidence of exactly conserved BCE shared by the two S proteins was only 19.1%. The remaining 55 BCEs in the S10 (5), S1A (18), S1B (3), S1CD (3), ahead of FP (7), ahead of HR1 (5), HR1 (6), ahead of HR2 (6), HR2 (1), and CT (1) domains exhibited more or less difference in antisera binding ability compared with the corresponding 16-mers on SCV777, even with the other 7 Shared Co-Recomers. Furthermore, 28 BCEs on SCV777  were missing on SSD2014, while 17 new BCEs formed on SSD2014 . The above analysis demonstrates that the BCE conformity rate between the two strains was only 19.12% (13/68), albeit 66.2% of BCEs on SSD2014 could be recognized by anti-SCV777 serum. A total of 35.4% (28/79) of the BCEs distributed on nearly all functional domains (except FP and HR2) of SCV777 were obliterated from SSD2014, but at the same time, a quarter (17/68) of BCEs were newly formed on the latterCollectively, among the 68 BCEs identified on SCV777) and a virulent variant strain SD2014 (SSD2014) using a bioinformatics approach and experimental methods. We found that with a sequence similarity of 92.21% between SCV777 and SSD2014, only 66.2% polypeptides (16-mers) of SSD2014 could be recognized by the anti-SCV777 serum. The parallel existing Co-Recomers (shared Co-RecoMers) took percentages of 29.4 and 25.3 for SSD2014 and SCV777, respectively. The absolute BCE conformity rate between the two strains was only 19.12%. All these figures indicate a low conservation of antigenicity between the two S proteins and a considerate decrease in antigenicity if PEDV CV777 was the vaccine strain for wild-type PEDV prevention.Since late 2010, the highly virulent PEDV variants have led to heavy economic loss to the world swine industry due to the high mortality rate of infected newborn piglets . The vacCV777 and 52.9% (18/34) of the predicted BCEs on SSD2014, and more than half of positive 16-mers were identified from non-predicted BCE locations of both proteins (43/79 on SCV777 and 36/68 on SSD2014). Even the 16-mers containing sequences of some of the top five antigenic epitopes (antigenicity > 0.8) predicted on SCV777  or on SSD2014  were negative or weakly positive , and each genotype can be further divided into different subtypes, cross-protection between GI and GII was widely reported in previous studies ,28,29,30chniques . WB resu as well . All thehe study . In addihe study . A compa0 domain was shown to bind sialic acid to facilitate virus infection [A domain was speculated to interact with cell surface heparan sulfate as an attachment factor based on its structural similarity with the other alphacoronavirus S1A domain, the S1B domain was known as a receptor binding domain [CV777, 13 (16.5%) of the 79 BCEs were not bound by anti-SSD2014 serum, and 28 (35.4%) BCEs did not have counterparts on SSD2014. On the side of SSD2014, 16 (23.5%) of the 68 BCEs cannot be bound by anti-SCV777 serum, and 17 (25%) BCEs were newly formed compared with SCV777. The missed or non-bound BCEs scattered in different functional domains of the PEDV S protein except HR2. Especially in the receptor binding domain S1B where the core neutralizing epitope is located, there was no shared Co-RecoMer in this domain. Furthermore, three out of the four BCEs of SCV777 were missing on SSD2014. Likewise, two out of the three BCEs in the S1B domain of SSD2014 do not exist on SCV777. It is well known that change or deletion of antigenic epitopes in the neutralizing epitope region might lead to the immune escape of the variant strain against the classical strain-based vaccine [The PEDV S protein contains different domains, whose roles in virus infection have been elucidated to varying degrees. The S1nfection ,35. Whilg domain ,26,36,37g domain ,36. Antig domain ,11,12,13g domain ,33,38,39g domain . In the  vaccine ,28,29. TBCEs are generally divided into linear BCEs and conformational BCEs . Linear In this study, rabbits instead of pigs were selected to generate antibodies because clean rabbits are easier to obtain than clean pigs, and rabbits were suitable for immune experiments to prepare a certain amount of serum based on economic consideration. As the natural host of PEDV, the sera from pigs immunized with PEDV S protein or more ideally, convalescent pigs infected with PEDV are theoretically preferred for BCE identification on the S protein. Future experiments will focus on identifying BCEs of the S protein by using high-quality porcine serum and check the possible bias from the findings using rabbit-generated antibodies.CV777 and SSD2014, respectively, among which 20 BCEs were shared Co-Recomers. More than 40% of the BCEs recognized by anti-SCV777 serum on SCV777 cannot be recognized by anti-SSD2014 serum and vice versa. Therefore, compared with that of the classical vaccine strain CV777, antigenicity and immunity of S protein of variant PEDV strain SD2014 had changed significantly. Epitopes identified in this study provided a solid foundation for the rational design of multi-epitope-based vaccines and diagnostic techniques.Seventy-nine and sixty-eight linear BCEs (positive 16/14-mers) were identified on S


Gene therapy is a promising approach for treating tumors. Conventional approaches of DNA delivery depending on non-viral or viral vectors are unsatisfactory due to the concerns of biosafety and cell-targeting efficiency. The question how to deliver DNA into tumor cells efficiently and selectively is a major technological problem in tumor gene therapy. Here, we develop a vector-free gene transfer strategy to deliver genes effectively and selectively by taking advantage of targeting nucleolin. Nucleolin, a shuttle protein moving between cell membrane, cytoplasm and nuclei, is overexpressed in tumor cells. It has a natural ligand G-quadruplex (Gq). Gq-linked DNA (Gq-DNA) is likely to be internalized by ligand dependent uptake mechanisms independently of vectors after neutralizing negative charges of cell membrane by targeting nucleolin. This strategy is referred to as Gq-DNA transfection. Benefiting from its high affinity to nucleolin, Gq-DNA can be effectively delivered into nucleolin-positive tumor cells even nuclei. Gq-DNA transfection is characterized by low cytotoxicity, high efficiency, ease of synthesis, high stability in serum, direct access into nuclei, and specific nucleolin-positive tumor cell targeting. One of the hallmarks of cancer is genome mutation . To targNucleolin, a shuttle protein moving between cell membrane, cytoplasm and nuclei, is overexpressed on cell membrane in many kinds of cancer cells ,12. ThusHerein, we developed a low cytotoxic, effective and selective vector-free gene transfer strategy by directly linking Gq to DNA (Gq-DNA). The Gq-linked enhanced green fluorescent protein (EGFP) and luciferase (Luc) genes were specifically taken in by nucleolin-positive tumor cells, and exhibited higher transfection efficacy than commercial transfection reagents, such as polyethyleneimine (PEI) and Invitrogen Lipofectamine 2000 (Lipo2000). Importantly, Gq-DNA transfection showed higher transfection efficiency in hepatoma cell line HepG2 than normal hepatocytes L02, demonstrating its tumor targeting capability. Thus, Gq-DNA transfection provided a new transfection method for nucleolin-overexpressing cancer cells.® Max Super-Fidelity DNA Polymerase . The PCR products were analyzed on 1.5% agarose gel and recovered with a Gel Extraction Purification Kit .To prepare Gq-linked DNA, linear DNA fragments were amplified by conventional Polymerase Chain Reaction (PCR) from plasmid pEGFP-C1 or pCMV-tag2B-luciferase with the primer TTTTGCTCACATGTTCTTTC (forward) and ATTTACGCGTTAAGATA (reverse). The PCR procedure was carried out by PhantaTaking the linear DNA fragment as the template, the single strand DNA was amplified with the asymmetric PCR which only used forward or reverse primer separately. The asymmetric PCR reaction system was as follows: 25 μL of 2 × Phanta Max Buffer, 1 μL of dNTP Mix (10 mM each), forward primer (10 μM) or reverse primer (10 μM) 2 μL, template (100~200 ng), Phanta Max Super-Fidelity DNA Polymerase 1 μL and adding ddH2O up to 50 μL. The PCR procedure was as follows: 95 °C for 5 min; 35 cycles of 95 °C for 30 s, 50.6 °C for 30 s and 72 °C 105 s, 72 °C 5 min. The primer sequences for asymmetric PCR were GGTGGTGGTGGTTGTGGTGGTGGTGGTTTTTTTTTTTTTGCTCACATGTTCTTTC (forward) and GGTGGTGGTGGTTGTGGTGGTGGTGGTTTTTTTTTATTTACGCGTTAAGATA (reverse).2) [The two products of each single strand were mixed by the ratio of 1:1 per 100 μL mixture of the single strand products and 4 μL AS1411 (100 μM) strands were dissolved in 25 μL 5× annealing buffer  ,22. The 2.Human hepatoma cell line HepG2, human colon cancer cell line HCT116, human breast cancer cell line MCF7 and mouse melanoma cell line B16F10 were obtained from ATCC. Human normal liver cell line L02 was obtained from CCTCC. The normal immortalized colonic epithelial cell line NCM460 was obtained from INCELL. All these cells were incubated in DMEM  containing 10% fetal bovine serum, 100 IU/mL penicillin and 100 μg/mL streptomycin at 37 °C in incubator with 5% COGq-DNA or DNA were incubated in DMEM containing 5% fetal bovine serum. After incubation for 12, 24, 36 and 48 h at 37 °C, the samples were analyzed by DNA electrophoresis in a 1.5% agarose gel and stained with ethidium bromide (EB).4 cells were seeded on 24-well plates and grew to 70% confluence. The medium was changed with serum-free DMEM half an hour before transfection. Then, 0.8 μg DNA and 3 μL PEI were sufficiently mixed into 50 μL serum-free DMEM in tube, and incubated at room temperature for 20 min. Then, the mixture was dripped into cell culture well slowly. Six hours later, the medium was changed into DMEM containing 10% fetal bovine serum.The conventional transfection with commercial reagent Lipofectamine 2000  followed the provided instructions. For transfection with polyethyleneimine , 5 × 10For Gq-DNA transfection, cells were seeded on 24-well plates and grew to 70% confluence. The cells cultured in DMEM containing 10% fetal bovine serum or serum free DMEM were incubated with polybrene (5 μg/mL) and 0.8 μg Gq-DNA (or DNA as control) for 6 h. Then, the medium was changed into fresh DMEM containing 10% fetal bovine serum.HepG2 or B16F10 cells were transfected with DNA to express GFP or DNA stained with YOYO-1 . The samples were harvested, fixed with 4% paraformaldehyde and stained with DAPI  for 15 min. The fluorescent images were obtained by Nikon Ti-U microscope equipped with a CSU-X1 spinning-disk confocal unit  and an EM-CCD camera .The samples were harvested and washed with FACS buffer containing 5% FBS for 3 times and fixed with 2% paraformaldehyde diluted with FACS buffer containing 5% FBS. The results were analyzed by Flow cytometry using BD LSRFortessa X-20 flow cytometer.HepG2 or B16F10 cells were seeded on 24 well plates and transfected with DNA to express luciferase. Then, 24 h later, cells were lysed with 100 μL lysis buffer on the ice for 30 min. The mixture was centrifuged for 5 min at 12,000 rpm in 4 °C. Additionally, the supernatant was collected to detect the luciferase activities. Luciferase activities were normalized with protein concentrations.To test cellular uptake efficiency of the traditional transfection method and Gq-DNA transfection, DNA and Gq-DNA stained with YOYO-1  were transfected with the two methods for 6 h and 13 h, then the samples were analyzed by confocal microscopy, fluorescence microscope and flow cytometer.Quantitative Real-time PCR was used for the analysis of the nucleolin mRNA of HepG2 and L02. The RNA of HepG2 and L02 cells were extracted with RNAiso Plus . The cDNA was synthesized by M-MLV Reverse Transcriptase . Reverse transcription PCR procedure was two-step method. The first step was 65 °C for 5 min. The second step was 42 °C for 60 min and 70 °C for 10 min. Quantitative Real-time PCR was performed by AceQ qPCR SYBR Green Master Mix . The primer sequences for Nucleolin for qRT-PCR are CCACTTGTCCGCTTCACACT (forward) and AGGAGCCATTTTCTTGGGGT (reverse).HepG2 cells were seeded on 96 well plates with 5000 cells per well, incubated with different concentrations of polybrene and treated with 10 µL MTT -2,5-diphenyl-2-H-tetrazolium bromide) solution (5 mg/mL) for 4 h. After the supernatant was discarded, 100 μL DMSO (dimethyl sulfoxide) was added to dissolve the formazan. The absorbance was determined at 570 nm.HepG2 cells were seeded on 24 well plates and incubated with different concentrations of polybrene for 6 h. The supernatant was collected and centrifuged at 1000 rpm. The LDH was detected with fully automatic analyzer method .The sequences of siRNA are 5-aagacagugaugaagaggagg-3 (siNCL-1#), 5-agagaucgaugggcgaucuauu-3 (siNCL-2#) and 5-uucuccgaacgugucacgutt-3 . We detected expression of NCL at 48 h.The statistical analysis was made by the IBM SPSS Statistics 19. The unpaired student’s t-test was used to compare two group. All data was showed as means ± standard deviation.We introduced Gq structures, previously identified as AS1411 ,21,22, oThe DNA gel electrophoretic assay showed that Gq-DNA traveled more slowly than the control linear DNA A, probabTo determine whether Gq structure affected DNA expression, we transfected Gq-CMV-EGFP, Gq-CMV-Luciferase (Gq-CMV-Luc), and the control linear DNA into HepG2 cells using commercial transfection reagents. The activities of luciferase and EGFP fluorescence were detectable and even more effective than the control linear DNA C,D, demoGiven that Gq structure could bind to nucleolin, we speculated Gq-DNA could transfect tumor cells without assistance of transfection reagents. After incubation of HepG2 cells with Gq-CMV-EGFP, HepG2 cells expressed no EGFP fluorescence A. We susTo assess the cytotoxicity of Gq-DNA transfection, we used MTT assay and LDH leakage assay. The concentration of polybrene we used (5 μg/mL) showed little cytotoxicity D,E. We tTo verify the tumor targeting capability of Gq-DNA transfection, we compared the transfection efficiencies in human hepatocellular carcinoma cell line HepG2 and normal liver cell line L02. We firstly tested the expression of nucleolin and found that nucleolin level in HepG2 cells was 3- to 4-fold higher than that in L02 cells A. AccordThe efficiency of conventional approaches of DNA delivery usually depends on cell proliferation and mitosis . Thus, wTo confirm the function of Gq structures in Gq-DNA transfection, we added excessive free Gq as the antagonist to block the binding of nucleolin with Gq-DNA. The results showed that excessive free Gq greatly reduced the efficiency of Gq-DNA transfection of CMV-EGFP A. To furAs reported, Nucleolin is a shuttle protein moving between cell membrane, cytoplasm and nuclei . Thus, GThe major drawbacks of conventional virus-mediated gene delivery are immunogenicity and the risk of randomly insertional mutagenesis. As to non-viral methods, low transfection efficiency and poor selectivity are the main bottleneck . To overFirst, a special amplifying method of PCR was used to generate Gq-DNA that could transfect cells overexpressing nucleolin. In nucleolin-overexpressing cells, Gq-DNA bound to nucleolin on cell membrane after the negative charges of cell membrane were neutralized. Gq-DNA entered cells and reached nuclei along with nucleolin that shuttled between cell membrane and nuclei ,32. ThenNuclear entry is the last intracellular barrier of exogenetic gene expression because of the nuclear envelope. Nuclear envelopes break down during the process of cell divisions. Fast dividing cells were transfected more efficiently than slow dividing cells. Conventional transfection methods only deliver nucleic acids into cytoplasm, which means that their transfection efficiency depends on mitosis. Thus, direct delivery of nucleic acids into nuclei would be an improved strategy . Gq-DNA More importantly, the efficiency of Gq-DNA transfection is dependent on the expression of nucleolin, which means that Gq-DNA transfection can target tumor cells with high-level nucleolin. Nucleolin, besides high expression on the tumor cell surface, was also found to express on the surface of endothelial cells during angiogenesis . To suppUnlike the traditional chemical transfection methods, Gq-DNA entered cells and nucleus by ligand dependent uptake mechanisms after neutralizing negative charges of cell membrane. The advantages of Gq-DNA transfection are high efficiency (comparing with PEI and Lipo2000) and low cytotoxicity. Correspondingly, the disadvantages of Gq-DNA transfection are that Gq-DNA is in an unfolded loose state which will increase the steric hindrance and decrease the diffusion rate. The longer the strand of dsDNA, the greater the impact on steric hindrance and diffusion rate. Thus, there would be a maximum size for delivery. However, we cannot confirm exact value of the maximum size.Additionally, Gq-DNA is delivered directly into nuclei which depends on the shuttle of nucleolin between cell membrane, cytoplasm and nuclei. Thus, we speculated that the genes that might affect shuttle of nucleolin were inappropriate to Gq-DNA transfection. Similar to traditional chemical transfection methods, Gq-DNA transfection is transient transfection and not superior in durability.Indeed, the expression of nucleolin is significantly enhanced in several solid tumors such as pancreatic cancer, hepatocellular carcinoma, colorectal cancer, thyroid cancer, glioma, breast cancer, prostate cancer, gastric cancer, melanoma and leukaemia . Gq-DNA In this study, Gq-DNA transfection was designed as a vector-free gene transfer strategy for selective delivery of DNA to cancer cells. Our work demonstrates that: (i) Gq-DNA transfection is a low cytotoxic and efficient strategy for gene delivery; (ii) Gq-DNA transfection selectively deliver DNA into tumor cells with high-level nucleolin; (iii) Gq-DNA transfection work independently of cell division by directly delivering DNA into nuclei; (iv) Gq-DNA transfection is characterized by ease of synthesis and low cost (Cost Accounting is in the


Tetragenococcus halophilus No. 1 in miso was found to possess salient immunomodulatory functions. Recently, we also demonstrated its effect on boosting immunological robustness. Although the consumption of miso is suggested to affect health over generations, such a long-term experiment has not been conducted until now. Thus, we evaluated the effects of miso-derived T. halophilus No. 1 over generations on the immune system of mice. As the generations increase, the proportion of germinal center B cells tends to increase. Furthermore, we found that CD4+ T cells expressing CD69, an activation marker, were increased in the third generation of mice. In addition, the proportion of follicular helper T cells and regulatory T cells tended to increase. Among the subsets of CD4+ T cells in the fourth generation, effector T cells and effector memory T cells tended to increase. In contrast, central memory T cells and naive T cells decreased. Moreover, autoimmunity was suppressed by long-term administration of T. halophilus No. 1. Based on these findings, we believe that the long-term administration of T. halophilus No. 1 over generations promotes immune activation and tolerance and enhances immunological robustness.Japanese people have been consuming miso soup over generations; it is beneficial for health and longevity. In this study,  Aspergillus, lactic acid bacteria, and yeast, with salt and soybean or other grains, such as rice and wheat, as raw materials. The Japanese have been consuming miso, mainly in the form of miso soup, for a long time.The Japanese have a high life expectancy, possibly owing to their diet. A study investigating Japanese dietary habits and survival reported that people who consumed Japanese food frequently had a lower risk of death and a longer survival time. Japanese foods include miso soup in addition to rice, fish, and pickles . Miso isTetragenococcus halophilus No. 1 derived from miso as a potent immunomodulator [2+ signaling in intestinal epithelial cells in vivo, the response of lactic acid bacteria was found to be similar to miso [Based on the findings of some cohort studies, miso soup was found to reduce the risk of death in patients with gastric cancer , 3 and todulator . Further to miso , 12. BasAll mice were maintained under specific pathogen-free conditions following the animal care guidelines of Tokyo Medical and Dental University. The Tokyo Medical and Dental University  approved all animal experiments.T. halophilus No. 1 isolated from miso was prepared [T. halophilus No. 1 used in the experiment affected the immunity of mice [prepared . Because of mice .T. halophilus No. 1 for 2 weeks for short-term administration testing.C57BL/6 mice (8 weeks old) were fed either CE-2 (CLEA Japan) as a control diet or CE-2 containing 1% heat-killed T. halophilus No. 1. The progeny produced was designated as the first (F1) generation in each group. After continued administration of the abovementioned diet, the second (F2), third (F3), and fourth (F4) generations of mice were obtained. At 16 weeks of age, all mice were analyzed.For long-term administration over generation testing, male and female C57BL/6 mice of the exact parental origin were divided into two groups. One group was fed CE-2 (CLEA Japan) as a control, and the other group was fed CE-2 containing 1% heat-killed ™ 450-conjugated anti-B220 (Tonbo Biosciences); Alexa Fluor 647-conjugated anti-CD4 (BD Pharmingen); Brilliant Violet 510™-conjugated anti-CD4 (BioLegend); Brilliant Violet 510™-conjugated anti-CD8a (BioLegend); Alexa Fluor 647-conjugated anti-GL7 (BioLegend), phycoerythrin (PE)-conjugated anti-CD62L (BD Biosciences); allophycocyanin (APC)-conjugated anti-CD44 (eBioscience); APC-conjugated anti-CD86 (Tonbo Biosciences); PE-conjugated anti-CD69 (BioLegend); PE-conjugated anti-PD-1 (Tonbo Biosciences); Brilliant Violet 421™-conjugated anti-C-X-C chemokine receptor type 5 (BioLegend); Alexa Fluor 647-conjugated anti-inducible T cell co-stimulator (BioLegend); eFour450-conjugated anti-CD127 (eBioscience); PE-conjugated anti-CD25 (Tonbo Biosciences); and APC-conjugated anti-CTLA4 (Tonbo Biosciences). In addition, dead cells were excluded by propidium iodide staining. FlowJo  was used to conduct data analysis.A MACSQuant Flow Cytometer (Miltenyi Biotec) was used to analyze whether the cells utilized the following specific antibodies: Violet FluorAs previously described, the serum immunoglobulin levels were measured  using ant-test for unpaired data for two groups and the Tukey–Kramer test for three or more groups.The experimental data were represented as the mean ± standard deviations (SD). Statistical significance was analyzed using a two-tailed Student’s T. halophilus No. 1, as previously reported, eased the upregulation of activation markers, such as CD86 and CD69, on B cells and T cells in vitro [T. halophilus No. 1 for 2 weeks. To examine the changes caused by short-term administration, mice spleen cells were analyzed. No difference was observed in the ratio of activation markers on B cells and T cells  B cells in the spleen. The proportion of GC B cells was found to increase as the generations progressed . B cells+ T cells. First, follicular helper T (Tfh) cells involved in B cell activation were analyzed. We found that the proportion of Tfh cells did not differ in the F2 and F3 generations; however, their proportion increased in the F4 generation  and central memory T cells (TCMs). We examined the proportion of naive T cells, effector T cells, TEMs, and TCMs. In the F4 generation, wherein T cells are considered sufficiently activated by ecreased .T. halophilus No. 1 in mice over generations promotes immune activation and immune tolerance.These results suggested that long-term administration of T. halophilus No. 1. Anti-DNA IgM levels in the F2 generation showed no difference, but anti-DNA IgM levels in the T. halophilus No. 1 administration group tended to increase in the F4 generation  The proportion of B cells among the total lymphocytes. (B) The proportion of CD4+ T cells among the total lymphocytes. (C) The proportion of CD86+ among B cells. (D) The proportion of CD69+ among B cells. (E) The proportion of CD69+ among CD4+ T cells. Bars indicate the mean ± SD. P-value is relative to the control based on the two-tailed Student’s t-test.The proportion of spleen B cells and T cells in mice was examined. Mice (n = 12) were fed a diet with or without 1% (TIF)Click here for additional data file.S2 FigExamples of the measurement of flow cytometry in (TIF)Click here for additional data file.S3 Fig+. (B) PD-1 and ICOS in CD4+. (C) CD127 and CD25 in CD4+. (D) CD127 and CTLA4 in CD4+CD127−CD25+. (E) CD44 and CD62L in CD4+.Examples of the measurement of flow cytometry in (TIF)Click here for additional data file.


Enveloped virus entry requires fusion of the viral envelope with a host cell membrane. Herpes simplex virus 1 (HSV-1) entry is mediated by a set of glycoproteins that interact to trigger the viral fusion protein glycoprotein B (gB). In the current model, receptor-binding by gD signals a gH/gL heterodimer to trigger a refolding event in gB that fuses the membranes. To explore functional interactions between gB and gH/gL, we used a bacterial artificial chromosome (BAC) to generate two HSV-1 mutants that show a small plaque phenotype due to changes in gB. We passaged the viruses to select for restoration of plaque size and analyzed second-site mutations that arose in gH. HSV-1 gB was replaced either by gB from saimiriine herpesvirus 1 (SaHV-1) or by a mutant form of HSV-1 gB with three alanine substitutions in domain V (gB3A). To shift the selective pressure away from gB, the gB3A virus was passaged in cells expressing gB3A. Sequencing of passaged viruses identified two interesting mutations in gH, including gH-H789Y in domain IV and gH-S830N in the cytoplasmic tail (CT). Characterization of these gH mutations indicated they are responsible for the enhanced plaque size. Rather than being globally hyperfusogenic, both gH mutations partially rescued function of the specific gB version present during their selection. These sites may represent functional interaction sites on gH/gL for gB. gH-H789 may alter the positioning of a membrane-proximal flap in the gH ectodomain, whereas gH-S830 may contribute to an interaction between the gB and gH CTs. For most alphaherpesviruses, the core viral fusion machinery is comprised of the receptor-binding protein glycoprotein D (gD), the gH/gL heterodimer, and the fusion protein gB , 2. In tMultiple structures of gD, gH/gL, and gB have been determined –7, but t–10–in vitro natural selection to identify functional interactions between gB and other entry glycoproteins. Two versions of gB with impaired fusion function were introduced into herpes simplex virus 1 (HSV-1) and the viruses were passaged to select for second-site mutations that rescue entry function. The first approach used a previously studied HSV-1 gB mutant and the second approach used gB from another species of alphaherpesvirus.The goal of this study was to use Approach 1: Using structure-based mutagenesis, we previously identified three mutations in gB (I671A/H681A/F683A) that inhibit fusion . These mApproach 2: Herpesvirus entry glycoprotein interactions are often species-specific –21. We p–Using both approaches, serial passage of viruses carrying impaired gB resulted in the acquisition of second-site mutations. Characterization of these mutations demonstrated that two mutations in gH (gH-H789Y and gH-S830N) partially restored gB function in fusion and plaque formation. Interestingly, rather than having a global hyperfusogenic effect, both gH mutations partially rescued the function of the specific gB version that was present during their selection. The results suggest that more than one domain of gH functionally interacts with gB during entry.Based on previous cell-cell fusion data showing that SaHV-1 gB coexpressed with HSV-1 gD, gH, and gL did not mediate high levels of fusion , we hypoVero cells were infected with the BAC-derived HSV-SaHVgB or WT HSV-1 viruses at 0.01 PFU/cell. HSV-SaHVgB virus exhibited a growth defect, requiring 7 days to reach full cytopathic effect (CPE), compared to only 3 days for WT virus. As expected, HSV-SaHVgB virus formed small plaques . Three dWe hypothesized that the HSV-SaHVgB virus plaque size would be increased by mutations that enhance fusion, specifically by mutations that enhance interactions between SaHV-1 gB and the HSV-1 entry glycoproteins, such as gH/gL. The locations of these mutations could reveal sites of functional importance for glycoprotein interaction and/or regulation.pass.To select for mutations in SaHV-1 gB or HSV-1 entry glycoproteins that could restore plaque size, HSV-SaHVgB was passaged serially in Vero cells. Four independent virus samples were passaged in parallel . Cells wIn our previous work, HSV-1 encoding gB3A in place of WT gB (gB3A virus) exhibited delayed entry and a small plaque phenotype . The smaTo produce ΔgB/gB3A virus, a Vero cell line expressing gB3A was generated and designated Vero-gB3A . The gB3pass.To select for mutations in entry glycoproteins that could restore plaque size, ΔgB/gB3A virus was passaged serially in Vero-gB3A cells. Four independent virus samples were passaged in parallel . Cells wpass.Using a similar logic and approach, gB3A virus (which carries the gB3A gene in place of gB)  also waspass virus (lineage D) on Vero-gB3A cells had increased by 9-fold. Consistent with an enhanced growth phenotype, the time required to reach full CPE after infection decreased as the number of passages for gB3Apass virus increased. Unexpectedly, the plaque size for ΔgB/gB3Apass virus did not increase noticeably after passaging on Vero-gB3A cells  or the gD-receptor HVEM (M1A cells). Cells were infected with the original stocks of gB3A virus or HSV-SaHVgB virus and with the passaged virus stocks. Entry was assayed by measuring β-galactosidase activity  and HSV-SaHVgBpass virus  showed enhanced entry compared to their parental strains, demonstrating a two to 4-fold increase in entry.We hypothesized that second-site mutations in the passaged viruses may have increased plaque size by rescuing an entry defect imparted by gB3A or SaHV-1 gB. To investigate an entry phenotype, we performed an entry assay using CHO cells that carry the activity , 23. As activity . The gB3pass gB3Apass, and ΔgB/gB3Apass viruses were harvested and genomic DNA was sequenced for four independent lineages of each. For comparison, HSV-SaHVgB and gB3A virus genomes also were sequenced. For each passaged virus stock, mutations were identified in multiple glycoproteins  were infected with the passaged viruses. We predicted that if the mutant gH proteins were promoting growth of these mutant viruses, for example through an enhanced interaction with the mutant gB, cellular expression of WT gH would compete with the mutant gH and reduce plaque size. Vero cells were used because they readily form plaques. Vero cells and Vero-VgHC4 cells were infected with equal number of PFU per well, as judged by titers on Vero cells. The plaques on Vero-VgHC4 cells were smaller on average than the plaques on Vero cells  were transfected with the four entry glycoproteins required for fusion , swapping in gH or gB mutants. C10 cells were used because they can form large syncytia. When coexpressed with WT gB, both gH-S830N and gH-H789Y showed reduced syncytium formation, with gH-S830N reducing the number of syncytia to below 20% of that seen with WT gH  was transfected with plasmids encoding T7 polymerase, gD, gL, gB, and gH, swapping in mutant gB or gH constructs. A second set of CHO-K1 cells (target cells) was transfected with plasmids carrying the luciferase gene under the control of the T7 promoter and nectin-1 receptor. Effector and target cells were mixed and luciferase activity was measured after 6 h to quantify cell-cell fusion. CHO-K1 cells were used because they provide strong signals in this assay. Cell surface expression of the WT gH and the gH mutants was determined by CELISA, using the MAb 53S on a duplicate set of effector cells.gH-S830N and gH-H789Y were expressed at WT levels . As previn vitro natural selection to examine how mutations impact glycoprotein interactions. Building on previous studies, we introduced functionally impaired gB mutants into the HSV genome and use serial passage to select for second site mutations that rescue entry function. In the case of the gB3A virus, to shift the selective pressure away from gB and explicitly select for mutations outside gB, we passaged virus on cells expressing gB3A. A similar approach was used previously to isolate extragenic suppressors of an HSV-1 UL34 mutant  have been demonstrated using a split-luciferase assay  and bimoPurified forms of the gD and gH/gL ectodomains have been shown to interact directly using surface plasmon resonance (SPR) , 11. AntThe gB interaction site on gH/gL is less well defined than the gD interaction site on gH/gL. Purified forms of the HSV gH/gL and gB ectodomains were shown to associate at low pH using a co-flotation liposome binding assay . SimilarAlthough the gB and gH/gL ectodomains appear to interact and a purified form of the HSV gH/gL ectodomain was sufficient to trigger low levels of cell-cell fusion , the gH 30–Recently, a gH/gL-gB interaction was shown using a split-luciferase assay and chimeric forms of gH comprised of combinations of HSV and Epstein Barr virus (EBV) segments . The gH/pass viruses because (i) gH-H789Y enhanced cell-cell fusion when coexpressed with SaHV-1 gB que size ; and , replacing the final three residues with nine residues. Previous work has shown that the gH tail residues past V831 also contribute to fusion. gH truncation mutants show that gH fusion activity positively correlates with gH tail length  were grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) , penicillin, and streptomycin. Vero-Cre cells were derived from Vero cells to express Cre recombinase (kindly provided by Dr. Gregory Smith at Northwestern University) and Vero-VgHC4 cells  were derPlasmids and bacterial artificial chromosomes (BACs) generated for this study include: pQF439 (pSG5-gB), pQF440 (pSG5-gD), pQF441 (pSG5-gH), pQF442 (pSG5-gL), pQF444 (pSG5-gH-S830N), pQF445 (pSG5-gH-H789Y), pQF356 (pTuner-gB3A), pQF397 (HSV-SaHV1gB BAC), and pQF395 (an intermediate construct for BAC construction).Plasmids encoding HSV-1 KOS strain gB (pPEP98), gD (pPEP99), gH (pPEP100), and gL (pPEP101) were previously described . To clonGGTTCTCCGGACAAATGTCCCGTTTTTTTG3’ and 5’CAAAAAAACGGGACATTTGTCCGGAGAACC-3′ and to generate pSG5-gH-H789Y (pQF445) using primers 5′-CAAACGGAACCGTCATTTATTTGCTAGCCTTTGAC-3′ and 5′-GTCAAAGGCTAGCAAATAAATGACGGTTCCGTTTG-3′.Site-directed mutagenesis of pQF441 was used to generate pSG5-gH-S830N (pQF444) using primers 5′-gB3A was expressed using the previously described plasmid pSG5-HSVgB-I671A/H681A/F683A . SaHV-1 pT7EMCLuc plasmid encoding a firefly luciferase reporter gene under the control of the T7 promoter and pCAGT7 plasmid encoding T7 RNA polymerase , 54 wereTo generate an inducible expression construct to make a Vero-gB3A cell line, gB3A was inserted into pTuner-IRES2 using the plasmid pUL34-IRES2-EGFP . This plR), was used for the BAC construction and was kindly provided by Dr. Gregory Smith at Northwestern University.pGS1439, a plasmid containing the kanamycin resistance gene (kanR construct (pQF395), as described below.gB3A virus and ΔgB/gB3A viruses were generated using the previously described HSV-1 gB3A BAC (pQF297) and gB-null BAC (pQF282), respectively . HSV-SaHThe HSV-SaHVgB BAC (pQF397) was generated using the gB-null BAC pQF282 . pQF282 R gene was PCR-amplified from pGS1439 using the primers 5′-TGTACAACGACCGCGCCCCGGTTCCAT TCGAAGAGATCACGGACGTGATCAAC GCCAAAGGATGACGACGATAAGTAGGGAACGGGTC-3′ and 5′-TGTACACAACCAATTAACCAATTCTGATGAACCGGGGCGCGGTCGT-3′. These primers consist of 5′ SaHV-1 gB sequence, including a BsrGI restriction site in bold, and 3′ kanR homology (underlined). This PCR product was digested with BsrGI and ligated into BsrGI-digested pQF77 (pCAGGS-SaHV-1 gB) to generate pQF395. The SaHV-1 gB gene containing a kanR insert was PCR-amplified from pQF395 using primers 5′-GTCCTCCAGCACCTCGCCCCCAGGCTACCTGACGGGGGGCACGACGGGCCCCCGTAGTCCCGCCATGGCGCCTCCGGCCGCCAAGAGC-3′ and 5′-AACAAACCAAAAGATGCACATGCGGTTTAACACCCGTGGTTTTTATTTACAACAAACCCCCCGCTACACAGCAGCGTCGTCTTCGTCC-3′. Using two-step red-mediated recombination, this PCR product was recombined into the gB-null BAC pQF282. Then the kanR cassette was recombined out to generate the BAC HSV-SaHVgB (pQF397) that carries the SaHV-1 gB gene in place of HSV-1 gB. The intermediate BAC with kanR insertion and the final BAC constructs were confirmed by at least four restriction enzyme digestions.The HSV-SaHVgB BAC was generated using a two-step red-mediated recombination strategy . First, gB3A virus and WT HSV-1 (GS3217) were generated from BACs previously . To geneVero cells in six-well plates were transfected with 1.5 μg/well pQF356 using Lipofectamine 2000. Then the cells were seeded in 100-mm plates in medium containing 800 μg/mL G418 to allow colonies to develop for two to 3 weeks. Approximately 100 individual colonies were seeded into 96-well plates. When the cells reached near confluence, they were transferred to 24-well plates. As these cells reached confluence, duplicate cultures were seeded in 24-well plates. One set of cells was saved and one was assessed for GFP expression after an overnight infection with WT HSV-1 at an MOI of 5. Cells that screened positive for GFP expression were subjected to two additional rounds of screening. Fluorescence microscopy was performed using an EVOS Cell Imaging Systems .Plaques were visualized 3 days postinfection using Giemsa staining and imaged with transmitted light microscopy using EVOS Cell Imaging Systems. The average radius of randomly selected plaques was calculated from two independent measurements. At least 50 plaques were measured for each virus sample. Using the average radius, the plaque area and the ratio of plaque size between WT and mutant viruses was determined, as described previously , 17.As previously described , M1A or GU734771.1) with the long- and short-terminal repeat regions deleted (KT899744.1) with the gB3A substitutions added. Using Geneious 8.1.9, sequencing reads were aligned to the reference genome and variants were identified. Variants present in both the revertant viruses and the original BACs were ignored, as were variants in the gJ gene due to the RFP insertion. Mutations identified in other glycoproteins are reported in pass viruses was confirmed using standard single pass DNA sequencing after PCR amplification.DNA was purified for sequencing from cells infected in six-well plates using a blood and cell culture DNA minikit (Qiagen). Samples were sequenced at the Northwestern University Genomics Core Facility using NextGen Illumina HiSeq SR500 sequencing. Unipro UGene  was used deleted . The gB pass, or HSV-SaHVgBpass virus. Alternatively, Vero cells seeded in six-well plates were transfected with 1.5 μg/well of plasmids encoding gH-S830N, gH-H789Y, or vector using 5 μL Lipofectamine 2000. After an overnight incubation, cells were infected at 150 PFU/well with gB3A or HSV-SaHVgB virus. Viral titers had been determined previously on Vero cells. Cells were stained with Giemsa 3 days postinfection to visualize plaques.Vero and VgHC4 (cells expressing WT gH) seeded in six-well plates were infected at 150 PFU/well with WT, gB3AThe syncytium formation assay was performed as described previously . C10 celThe fusion assay was performed as previously described . BrieflyTo evaluate the cell surface expression of the gH mutants, cell-based ELISA (CELISA) staining was performed as previously described . During Statistical comparison of the plaque areas was performed with a Mann-Whitney U test using SPSS (version 25). Analyses were performed using IBM SPSS statistics version 25 for Windows  and SAS 9.4 .


This investigation was proposed to evaluate the effect of intratracheally administered rat bone marrow-derived c-kit-); and PBS containing C-kit+ cells (S+C-kit+). After two weeks, circulatory CD4+/CD8+ T-cell counts and pulmonary ERK/NF-ƙB signaling pathway as well as the probability of cellular differentiation were assessed. Forty rats were randomly divided into 4 groups; healthy rats received phosphate-buffered saline (PBS) (C); sensitized rats received PBS (S); PBS containing C-kitˉ cells ; however, they diminished the level of CD8+ cells (P<0.01). Moreover, data demonstrated increased p-ERK/ERK ratio (P<0.001) and NF-ƙB level (P<0.05) in sensitized rats compared with the C group. The administration of C-kit+, but not C-Kit-, decreased p-ERK/ERK ratio and NF-ƙB level compared with those of the S group (P<0.05). The results showed that transplanted C-Kit+ cells engrafted into pulmonary tissue reduced the NF-ƙB protein level and diminished p-ERK/ERK ratio, leading to suppression of inflammatory response in asthmatic lungs. The study revealed that C-Kit Asthma is a complex pathological condition with an allergic response in the broncho-pulmonary segment that threatens human health , 2. It w+ cells are one of the stem cell subgroups with several regenerative capacities. It has been indicated that activation of the C-kit, a stem cell factor (SCF) receptor, can contribute to indispensable functions of effectors and signaling proteins inside human cells  and were confirmed by the Animal Care Committee of Tabriz University of Medical Sciences (No: IR.TBZMED.VCR.REC.1397.404).Experimental groups5 C-kitˉ cells (S+C-kit- group), and sensitized rats receiving 50 µl of PBS intratracheally containing 3×105 C-kit+ cells (S+C-kit+ group). The remaining rats were used for extraction of C-kit+ and C-kit- cells.In this study, fifty male rats  were enrolled in the study. These animals were purchased from the animal house of Tabriz University of Medical Sciences and kept in standard condition and permitted to access chewing food and water ad libitum. Fourteen days after accommodation, 40 rats were randomly selected and divided into the following groups; healthy rats receiving 50 µl of phosphate-buffered saline (PBS) intratracheally (C group); sensitized rats receiving 50 µl of PBS intratracheally (S group); sensitized rats receiving 50 µl of PBS intratracheally containing 3×10Induction of asthma th days. From the 14th day, animals were kept in a suitable whole-body inhalation exposure chamber with dimensions of 30×20×20 cm3 and exposed to 4% OVA aerosol produced by a nebulizer  for 5 min daily for 18±1 days . The control group was challenged with normal saline instead of OVA. On the 33rd day, PBS, PBS containing C-kitˉ and C-kit+ cells were injected into the trachea through a cervical incision in S, S+C-kit-, and S+C-kit+ groups, respectively. All animals were humanely euthanized two weeks later by cervical dislocation after a high-dose injection of xylazine and ketamine. The asthma model of the rat was established according to the previously used method , 30. RatMagnetic-activated cell sorting (MACS)+ cells, rats were humanely euthanized by cervical dislocation after high-dose injection of xylazine and ketamine. Femurs were completely removed and medullary cells flushed out by PBS solution containing 2% fetal bovine serum . The mononuclear cells were isolated by Ficoll (Sigma-Aldrich) gradient centrifugation at 400×g for 20 min. For the MACS procedure, harvested marrow mononuclear cells were incubated with 1% FBS for 30 min at 4 °C. After that, cells were exposed to anti-human C-kit microbead  and cells passed through the LS column (Miltenyi Biotec) to isolate the c-kit+ and c-kit- cells  and mononuclear cells were isolated using Ficoll solution as above-mentioned. Then, cells were incubated with FITC-conjugated mouse anti-rat CD4 (eBioscience) and CD8 (eBioscience) for 30 min at 4 °C. Finally, cells were analyzed using the BD FACSCalibur flow cytometry system and FlowJo software (ver. 7.6.1).The percentage of systemic CD4Western blottingThe right lungs of each group were homogenized using lysis buffer and then the samples were centrifuged at 4 °C for 10 min at 10,000 rpm. The supernatant containing protein was determined by Bradford’s method. Equal amounts of protein were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Polyvinylidene fluoride (PVDF) membranes . The membranes were washed with TBST buffer and blocked with 5% skimmed milk for 1 hr. After that, membranes were incubated with primary antibodies anti-β-actin , ERK 1/2 , p-ERK 1/2 , NF-ƙB p65  at 4 °C for 16–18 hr. Finally, the membrane was washed with TBST buffer and detected with an HRP-conjugated secondary antibody .Immunofluorescence imaging (IF)Fourteen days after injection of Dil-labeled cells, 5-6 μm thick cryo-sections were prepared from lung tissues. The samples were incubated with anti-cytokeratin 19 antibody for 1 hr. After three-time PBS washes, the samples were exposed to an appropriate FITC-labeled secondary antibody for 1 hr at room temperature. The nuclei were stained using DAPI . Here, we monitored the existence of Dil+/cytokeratin-19+ cells , indicating the differentiation of transplanted cells into the pneumocyte-like cells . Data analysisResults were displayed as mean±SEM and analyzed by one-way ANOVA with Tukey–Kramer post hoc test. Statistical P-values less than 0.05 were considered significant.Transplanted C-Kit+ cells were engrafted into pulmonary tissue and differentiated into epithelial cells- and C-Kit+ cells can lead to successful homing into the pulmonary tissues, we performed an IF analysis. Data showed red-colored C-kit- and C-Kit+ cells inside the lung parenchymal 14 days after intratracheal administration. In S + C-kit- and S + C-Kit+ groups, these cells were randomly distributed. It seems that the number of labeled cells was higher in the S+C-Kit+ group compared with the S+C-kit- group. These data showed that intratracheal administration of C-kit- and C-Kit+ cells led to successful recruitment into the pulmonary niche after 14 days in a rat model of asthma. We also performed an IF analysis based on Cytokeratin-19 staining. This factor is a typical pneumocytes protein marker. We found that C-kit cells had the potential to express Cytokeratin-19 fourteen days after transplantation into the rat model of asthma. Based on our data, the number of green-colored cells (Double green/red-stained cells) was high in the S+C-Kit+ group compared with S+C-kit- rats, showing the prominent trans-differentiation capacity of C-kit+ cells compared with the C-kit- subsets. As expected, labeled cells were not detectable in either sensitized (S) or control (C) groups. These data showed that C-kit+ progenitor cells can acquire a pneumocyte-like phenotype inside the asthmatic niche which can help the host lung to restore the function of resident pneumocytes  as compared with group C. According to our data, transplantation of C-kit- and C-Kit+ cells via intra-tracheal route increased the systemic number of CD4+ cells in comparison with the asthmatic profile . These changes were more evident in sensitized rats that received C-Kit+ cells (P<0.001). In contrast to dynamic changes of CD4+ cells, the blood levels of CD8+ cells were diminished after the onset of asthmatic changes compared with the control rats . The transplantation of both C-kit- and C-Kit+ cells reduced the abnormal elevation of CD8+ cells almost to near-to-control levels . These data showed that transplantation of C-Kit+ cells in sensitized rats can modulate the abnormal lymphocyte counts. Although C-kit- can alter the number of lymphocyte subsets under asthmatic conditions, these changes were less in sensitized rats that received C-Kit+ cells . We noted that administration of C-kit+ but not C-Kit- decreased abnormal phosphorylation of ERK effector compared with the sensitized group (P<0.05). Despite reduction of ERK phosphorylation in S+C-Kit- and S+C-Kit+ groups, the p-ERK/ERK ratio was higher compared with the normal condition  compared with the control (C) group (ondition . P<0.05). We showed that administration of C-Kit+ cells can reduce synthesis of NF-ƙB compared with the S group (P<0.05). Unlike the therapeutic effects of C-Kit+ cells, C-Kit- cells did not change NF-ƙB compared with the S group . The global increase of asthma and the absence of completely effective modalities increase the need to invent new therapeutic approaches. Previously, the existence of special progenitor types such as C-Kit lacking , 35. Somtitution -40. In t+ lymphocytes was declined during the asthmatic changes and intratracheal administration of C-Kit+ cells can, if not completely, restore the CD4+ lymphocyte numbers to near-to-normal levels. These effects were less in the sensitized group that received C-Kit- cells. In contrast to the changes of CD4+ lymphocyte number, the percent of CD8+ lymphocytes was increased after the onset of asthmatic changes. The transplantation of C-Kit+ cells, but not C-Kit- cells, in sensitized rats decreased the abnormal increase of CD8+ lymphocytes to the control levels. It was suggested that increased CD8+ cytotoxic T lymphocytes and reduction of CD4+ lymphocytes are closely associated with airway hyperreactivity and asthmatic pathologies . Murine pulmonary C-Kit+ cells are eligible to restore lung function by reducing the release of Th2 pro-inflammatory cytokines IL-4, -5, and -13, coincided with an elevation of type 2 macrophage IL-10 . The authors declare that no conflict of interest exists.


We retrospectively analyzed 82 patients who were discussed by the uro-oncology board of the University Hospital of Ferrara for primary staging newly diagnosed PCa (median age 72 (56–86) years; median baseline prostate specific antigen (PSA) equal to 8.73 ng/mL). Patients were divided into three groups based on the imaging performed: group A = only CI; group B = CI + [18F]F-choline PET/CT; group C = only [18F]F-choline PET/CT. All data on imaging findings, therapy decisions and patient outcomes were retrieved from hospital information systems. Moreover, we performed a sub-analysis of semiquantitative parameters extracted from [18F]F-choline PET/CT to search any correlation with patient outcomes. The number of patients included in each group was 35, 35 and 12, respectively. Patients with higher values of initial PSA were subjected to CI + PET/CT (p = 0.005). Moreover, the use of [18F]F-choline PET/CT was more frequent in patients with higher Gleason score (GS) or ISUP grade (p = 0.013). The type of treatment performed  did not show any relationship with the modality adopted to stage the disease. [18F]F-choline PET/CT induced a change of planned therapy in 5/35 patients in group B (14.3%). Moreover, patients investigated with [18F]F-choline PET/CT alone demonstrated longer biochemical recurrence (BCR)-free survival (30.8 months) in comparison to patients of groups A and B , probably due to a more accurate selection of primary treatment. Finally, total lesion choline kinase activity (TLCKA) of the primary lesion, calculated by multiplying metabolic tumor volume and mean standardized uptake value (SUVmean), was able to more effectively discriminate patients who had recurrence after therapy compared to those without (p = 0.03). In our real-world experience [18F]F-choline PET/CT as a tool for the initial management of PCa had a relevant impact in terms of therapy selection and was associated with longer BCR-free survival. Moreover, TLCKA of the primary lesion looks a promising parameter for predicting recurrence after curative therapy.Initial staging of prostate cancer (PCa) is usually performed with conventional imaging (CI), involving computed tomography (CT) and bone scanning (BS). The aim of this study was to analyze the role of [ Prostate cancer (PCa) is the most common malignancy in males, with an incidence of approximately 1.4 million cases per year worldwide . Indeed,11C or 18F [68Ga]Ga-PSMA-11, the most utilized, allows the detection of lesions expressing PSMA, which is directly related to the GS and to the aggressive behavior of PCa F-choline PET/CT in the initial management and outcome prediction of PCa patients by analyzing data from a multidisciplinary approach. As a secondary aim, we evaluated whether semi-quantitative parameters, extracted from [18F]F-choline PET/CT, could discriminate between patients who had recurrence after primary treatment.The aim of this study was to analyze the role of F-choline PET/CT); and group C (patients who only underwent [18F]F-choline PET/CT staging). For patients in groups A and B, the different diagnostic investigations were performed within a month. In group B patients, [18F]F-choline PET/CT was usually performed as a second-line exam after CI to investigate inconclusive findings. Conversely, [18F]F-Choline PET/CT alone was proposed by the multidisciplinary team in the case of high risk of metastasis at diagnosis . The number of patients included in groups A, B and C was 35, 35 and 12, respectively. We analyzed the patients’ distribution into the 3 groups in relation to baseline clinical pathological characteristics. Moreover, we investigated whether the different staging strategies were correlated with therapy decision and outcome. Finally, for patients included in group B, we analyzed whether [18F]F-choline PET/CT induced a change into multidisciplinary therapeutic decision in comparison to CI findings.Patients were divided into three subgroups based on the staging strategy decision: group A ; group B . Imaging analysis was performed by an expert radiologist with more than 10 years of experience in uro-oncologic disease.BS was performed on a Symbia T2 dual-head hybrid SPECT/CT system , 3 h after intravenous injection of 740–925 MBq of [99 mTc]Tc-hydroxydiphosphonate. The patient was required to hydrate with at least 1 L of water between the radiotracer injection and the diagnostic scan. BS images were evaluated by an expert nuclear medicine physician, with more than 20 years of experience.18F]F-choline injection (3 MBq/Kg) using a standard technique on a dedicated PET/CT system . After non-contrast-enhanced low-dose CT , PET was acquired in 3-dimensional mode  using a continuous scan with variable speed. The emission data were corrected for randoms and scatter, and decay and attenuation correction was performed using the non-enhanced low-dose CT data. Reconstruction was performed on a Syngo.via workstation . All images were processed and analyzed by two experienced nuclear medicine physicians. Circular regions of interest (ROIs) were drawn around the tumor lesions with focally increased uptake in transaxial slices and automatically adapted to a 3-dimensional the volume of interest (VOI) (Images were acquired from the mid-thigh to the skull vertex about 60 min after F-choline PET/CT was performed in 47 patients (57.3%), and every scan detected at least one pathological uptake indicative of PCa localization. In particular, in 28 cases (59.6%) [18F]F-choline PET/CT detected only primary tumor, while in 17 patients (36.2%) a pathological uptake was detected for prostate and lymph node  and/or bone metastases . In total, PET detected 56 metastatic lesions (n = 15 bone lesions and n= 41 metastatic lymph nodes). In two patients (4.3%) [18F]F-choline PET/CT detected only bone metastases, without significant uptake in the primary tumor. Finally, no patients showed visceral metastases. In [18F]F-choline PET/CT quantitative analysis, median MTV_WB and TLCKA_WB were 10.58 and 54.14, respectively.F-choline PET/CT was more frequent in patients with higher GS or ISUP grade (p = 0.013). The type of treatment performed did not show any relationship with the modality staging strategy. However, a slightly different treatment approach was used for the three groups. Patients from group A were more often treated by local therapies (surgery and RT) or active surveillance than groups B and C. Furthermore, group C patients were often subjected to systemic therapy. [18F]F-choline PET/CT induced a change of planned therapy in 5/35 patients in group B (14.3%). In particular, [18F]F-choline PET/CT upstaged four patients that advanced from radical treatment to systemic therapy due to metastatic disease. In the remaining patient, [18F]F-choline PET/CT did not show any uptake in skeletal lesions detected by CT. As a consequence, the patient underwent radical radiation therapy on the prostate bed that induced a fall in PSA values (from 3.64 ng/mL to 0.18 ng/mL). Notably, the patient had still not been incurred in BCR after the 26-month follow-up, and the skeletal lesions had not been modified on follow-up CT images. Moreover, patients who underwent only [18F]F-choline PET/CT demonstrated longer BCR-free survival (30.8 months) in comparison to patients of groups A and B .Results of a sub analysis, where patients were divided into three groups based on the staging strategy, are displayed in 18F]F-choline PET/CT (p = 0.03).The analysis of semiquantitative parameters extracted from [e PET/CT  demonstr18F]F-choline PET/CT over CI in primary staging of intermediate to high-risk PCa [18F]F-choline PET/CT was useful for excluding extra-prostatic disease spread in a prospective cohort of 132 patients . Their results are consistent with those published by Poulsen et al. [18F]F-choline PET/CT outperformed CI for N-staging . However, guidelines [18F]F-choline PET/CT in a limited number of cases, mainly on the basis of other studies whose results are in contrast with those listed above [18F]F-choline PET/CT, even though a direct comparison of the two radiotracers in the primary staging setting is yet to be published [18F]F-choline PET/CT staging was most frequently used in the case of GS > 7 or inconclusive CI results (p = 0.013), in line with current guidelines. Moreover, patients with higher values of baseline PSA underwent a higher number of imaging examinations (p = 0.005). Furthermore, we found that patients who underwent only [18F]F-choline PET/CT (group C) showed significantly longer BCR-free survival (30.8 months) in comparison to groups A and B (p = 0.006), even though their initial risk was higher. Moreover, contrary to our expectations, patients in group A had shorter BCR-free survival in comparison to those for whom [18F]F-choline PET/CT was associated with CI (15.5 vs. 23.5 months), despite starting with a lower PCa class of risk . To the best of our knowledge, this is the first paper reporting longer BCR-free survival in patients staged with [18F]F-choline PET/CT in comparison to those staged with CI. We believe that this result may reflect the better accuracy offered by [18F]F-choline PET/CT staging in our cohort of patients, which induced a better treatment selection and, consequently, a benefit in terms of outcome. Moreover, the evidence that group C patients showed longer BCR-free survival if compared to patients in group B suggests that CI does not provide an added value in PCa patients undergoing primary staging with [18F]F-choline PET/CT. If confirmed in larger studies, we could even speculate that CI represents a confounding factor in some subset of patients undergoing primary staging with 18F-choline PET/CT, inducing a subsequent inappropriate therapeutic decision. [18F]F-choline PET/CT induced a change in treatment selection in 14.3% of patients of group B, in line with previous results in the literature [18F]F-choline PET/CT ruled out suspected bone metastasis in another patient, allowing successful prostate radiation therapy.Correct primary staging is of great importance for offering the best therapeutic management to every newly diagnosed PCa patient. Much of the evidence in the literature agrees on the better accuracy of F-choline PET/CT staging is that it provides “one stop shop” imaging, as it allows a metabolic evaluation of the prostate, beside N- and M-staging. However, it is described in the literature that several pathological conditions can induce an increased prostate tracer uptake, both benign and malignant [18F]F-choline PET/CT showed an increased prostate uptake in 45/47 patients (95.7%). Nevertheless, the prostate pattern of uptake was very inhomogeneous, as it was focal and intense in some patients and mild and diffuse in some others. Laudicella et al. [68Ga]Ga-PSMA-PET/MRI compared to the expansive growth pattern. A similar correlation should also be investigated for [18F]F-choline PET/CT or possibly [18F]F-choline PET/MRI. Moreover, radiomics and artificial intelligence have been rarely applied to [18F]F-choline PET/CT [Another advantage of [alignant . In our a et al.  recentlye PET/CT . Future 18F]F-choline PET/CT. Tseng et al. [18F]F-choline PET/CT staging was a predictor of BCR after radiation therapy. TLCKA of the primary tumor reflects both the local extension of PCa as well as its choline metabolism. Interestingly, TLCKA_WB could not predict BCR. This means that in our cohort, the PET-characteristics of the primary tumor were more important than the PET whole body load of disease for discriminating patients who would experience BR.Volumetric parameters (VPs) extracted by PET images are promising and widespread diagnostic and prognostic tools in several types of cancers and for many PET radiotracers ,35. Howeg et al.  reportedg et al.  found a g et al.  who repoSome limitations should be considered when analyzing the results of this paper. Indeed, this study is retrospective, with a medium-sized sample. Prospective trials with larger cohort of patients to confirm our data would be desirable. Moreover, it was not possible to calculate the change of therapeutic management in patients in group C, due to the absence of comparative CI data available.18F]F-choline PET/CT was associated with longer BCR-free survival in comparison to CI. Moreover, we identified a prognostic factor in TLCKA of the primary lesion that was able to discriminate patients who would have recurrence after primary treatment. We are strongly convinced that multidisciplinary discussion is an added value for selecting the best primary staging scheme for every newly diagnosed PCa patient. Despite the rise of PSMA-ligand PET imaging, our real-world experience suggests that [18F]F-choline PET/CT should still be considered a valid and reliable diagnostic tool for primary staging of high-risk PCa patients or, in the case of inconclusive CI, providing an outcome improvement.This real world-experience showed that primary staging of PCa patients with [


Growth trait is one of the most important economic traits for meat animals. Somatostatin DNA vaccine has been proven to enhance the growth rate of animals. However, the growth-promoting effect is not ideal. This study aimed to evaluate the immune effects of a novel eukaryotic dual expression vaccine known as pIRES-S/CST14-S/2SS in mice. Firstly, we demonstrated pIRES-S/CST14-S/2SS could functionally express in GH3 pituitary cells by regulating the growth hormone (GH) and prolactin (PRL) productions. Secondly, we observed that all concentrations of pIRES-S/CST14-S/2SS vaccine could evoke anti-somatostatin (SS) antibodies, leading to a higher level of GH concentration. Notably, pIRES-S/CST14-S/2SS (10 μg/100 μL) immunized mice obtained maximum body weight gain in a booster vaccination period. Our results are helpful for better understanding of the relationship between SS and CST, and pIRES-S/CST14-S/2SS vaccine may be used as a promising alternative for developing growth-promoting vaccine.p < 0.05). Furthermore, the antibody level, hormone secretion, and weight gain in the mice injected with novel recombinant plasmids were also evaluated. The anti-SS antibodies were detectable in all vaccine treated groups, resulting in significantly higher levels of GH secretion (p < 0.05). It is worth mentioning that pIRES-S/CST14-S/2SS (10 μg/100 μL) vaccinated mice exhibited a higher body weight gain in the second immunization period. This study increases the understanding of the relationship between somatostatin and cortistatin, and may help to develop an effective growth-promoting DNA vaccine in animals.Animal growth traits are directly linked with the economics of livestock species. A somatostatin DNA vaccine has been developed to improve the growth of animals. However, the growth-promoting effect is still unsatisfying. The current study aimed to evaluate the effect of a novel eukaryotic dual expression vaccine known as pIRES-S/CST14-S/2SS, which encodes the genes obtained by fusing somatostatin (SS) and cortistatin (CST) into hepatitis B surface antigen (HBsAg). After transfection into GH3 cells with pIRES-S/CST14-S/2SS, green fluorescence signals were observed by fluorescence microscopy, suggesting the effective expression of CST and SS in GH3 cells using the IRES elements. Subsequently, both GH and PRL levels were found to be significantly lower in pIRES-S/CST14-S/2SS-treated cells as compared to the control group ( Somatostatin (SS) consists of 14 peptides and is a pleiotropic neuropeptide widely distributed throughout the organism, which is generally viewed as a crucial regulator of several hormones, including growth hormone (GH) , prolact−/− mice) provokes a higher GH plasma level compared to normal mice [Cortistatin (CST) is a neuropeptide that displays a high structural and functional homology with somatostatin . Cortistmal mice . Importamal mice . This remal mice . The in-mal mice . Those sBased on the above-mentioned information, we hypothesize that cortistatin may play a compensatory role in the process of in-vivo SS active immunization. We therefore constructed somatostatin and cortistatin eukaryotic dual expression plasmids using pIRES elements, which contain two multiple cloning sites (MCS) and an internal ribosome entry site (IRES), allowing for the high-level expression of two genes. Moreover, the biological functions of both SS and CST were detected in GH3 pituitary cells, and the growth-promoting effects were evaluated in mice.Nhe I and EcoRI, were fused into the 5′ and 3′ ends of S/CST14, respectively. The eukaryotic expression vector pIRES with double MCS was kindly provided by Dr. Qiuyang Li from Jinan University.The recombinant plasmid pCS/2SS encoding S/2SS gene obtained by fusing two copies of somatostatin with hepatitis B surface antigen (HBsAg) was constructed and described in the previous paper . NotablySal I site (underline) is 5′-GCGTCGACCTTCTGAGATGAGTTTTTGTTCThe schematic diagram for the construction of the recombinant co-expression plasmid pIRES-S/CST14-S/2SS is presented in NotI site (underline) is 5′-ATAAGAATGCGGCCGCAATTCCCTGCGCTGAACATG-3′. The amplification was carried out by Taq DNA polymerase  according to the following procedures: pre-denaturation at 94 °C for 4 min and then 35 cycles of denaturation at 94 °C with each cycle lasting 30 s; annealing at 68 °C for 30 s; extension at 72 °C for 1 min; and final extension at 72 °C for 10 min. After PCR amplification, the fragment was excised at the SalI/NotI sites, purified by gel electrophoresis, and then ligated into the pIRES vector to yield the pIRES-S/2SS plasmid. Similarly, the S/CST14 fragment was excised at the Nhe I/EcoR I sites, purified by gel electrophoresis, and then ligated into the pIRES vector and the pIRES-S/2SS plasmid to generate the pIRES-S/CST14 and pIRES-S/CST14 -S/2SS plasmids, respectively. The plasmids were then transformed into DH5α and preserved in the China Center for Type Culture Collection (CCTCC).TAC-3′, and a downstream primer with a in vitro on hormone secretion. GH3 cells were cultured in Ham’s F10 Nutrient Mixture  supplemented with 2.5% fetal bovine serum  and 15% horse serum . All cells were cultured at 37 °C in an incubator under a humidified atmosphere containing 5% CO2. For transfection experiments, the cells were grown until they were a 70–80% confluent monolayer.GH3 pituitary cells were purchased from the National Platform of Experimental Cell Resources (China) and used to detect the effect of the overexpression of SS and CST The plasmids pIRES-S/CST14-S/2SS, pIRES-S/CST14, pIRES-S/2SS, and pIRES in super-coiled form were obtained using the Ultra-pure Mini-plasmid Rapid Isolation Kit , and their concentrations were detected by Nanodrop spectrophotometer . The transfection was performed using Lipofectamine™ LTX with Plus™ Reagent  following the manufacturer’s instructions. After transfection for 6 h, the medium was replaced with fresh growth medium. After transfection for 48 h, cells were collected for cyclic adenosine monophosphate (cAMP) assay and the culture medium was collected for hormone detection.The expressions of SS and CST were identified by indirect immunofluorescence after transfection with the pIRES-S/CST14-S/2SS plasmid for 48 h as described in the previous study . After wGH3 pituitary cells were washed twice with 200 µL cold PBS after 48 h of transfection with plasmids, and then cAMP levels were measured by a cAMP measuring kit  according to the manufacturer’s protocol. The data derived from the cAMP measurement was subjected to a nonlinear regression analysis.n = 18 in each group) according to the initial weight and a primary intramuscular immunization was given with 100 μL of either pIRES-S/CST14-S/2SS in 50 μg, 20 μg, 10 μg, pIRES-S/CST14 in 20 μg, pIRES-S/2SS in 20 μg or saline solution (negative control), respectively. A booster immunization was conducted two weeks later. Body weights were measured each week after primary immunization. Blood samples were harvested from the tail vein of each mouse at 2, 4, and 8 weeks after the booster immunization. Following centrifugation at 4000 rpm for 5 min, the plasma was separated from the whole blood and stored at −20 °C for further use.A total of 108 three-week-old female Kunming mice were purchased from the Medical Laboratory Animal Center of Hubei Province  and housed in cages (five mice per cage) in an air-conditioned room at a constant temperature of 25 ± 2 °C with 12 h light/dark cycles and were provided with water and food ad libitum. The guidelines of the Committee on the Care and Use of Laboratory Animals of Huazhong Agricultural University were followed. The animals were divided into six groups  according to the manufacturer’s protocol. Somatostatin antibodies were measured as previously described by our lab [Growth hormone (GH) and the prolactin (PRL) concentrations in GH3 cells were measured by enzyme linked immunosorbent assay (ELISA) methods using commercial kits purchased from Wuhan Colorful Gene Biological Technology Co. LTD . The assay sensitivity of GH and PRL were 1.0 and 0.5 pg/mL, respectively. In addition, the GH and cortistatin concentrations in the mice plasma were measured by  our lab .P values of less than 0.05 and 0.01 were considered significant and extremely significant differences, respectively.There were at least three replicates for each treatment. The data are presented as mean ± SEM. Data analysis was performed using GraphPad Prism software. A comparison of the mean values among groups was carried out using one-way ANOVA followed by either Tukey’s post hoc test or LSD test. Nhe I and EcoR I digestion, 789 bp and 6808 bp segments by Sal I and Not I digestion, and 2200 bp and 5397 bp fragments by Nhe I and Not I digestion were obtained, respectively . However, no significant difference in cAMP levels was observed between the dual expression pIRES-S/CST14-S/2SS transfected group and other groups (The levels of GH and PRL hormone in GH3 cells were examined after transfection with pIRES-S/CST14-S/2SS for 48 h. The results showed that both GH and PRL hormone levels significantly decreased when compared to the control group (r groups III.p < 0.05, p < 0.01, p < 0.05, After immunization with different types of vaccines, anti-SS antibody in the plasma was measured. The results showed that the anti-SS antibodies were detected in all immunized groups, and the SS antibody levels in pIRES-S/CST14-S/2SS at a concentration of 10 μg was significantly higher than that in 50 μg of the pIRES-S/CST14-S/2SS group at week 4 after a booster immunization (p < 0.05, p < 0.05, In order to determine whether the changes in antibodies and GH production coupled to the increment of body weight, we further recorded the body weight and observed that mice immunized with 20 μg of pIRES-S/2SS showed a higher body weight at two weeks post-first immunization when compared to the pIRES-S/CST14 group ((Salmonella, the growth-promoting effect of DNA immunization is still unsatisfied [Although the previous studies have reported various somatostatin genetic vaccines including naked pCS/2SS plasmid and pVGS/2SS plasmid delivered by attenuated atisfied ,11. Cortatisfied . Based oatisfied , and theatisfied  were insThe biological functions of this novel plasmid were subsequently determined by detecting the secretion of GH and PRL in GH3 cells, which can express all five SSTRs . After tAfter the injection of mice with the CST and SS dual expression plasmid, we found that all does of pIRES-S/CST14-S/2SS immunized groups exhibited higher levels of GH hormone compared to the control group, which was consistent with the previous studies in SS and CST DKO mice . We alsoIt can be concluded that the novel dual expression pIRES-S/CST14-S/2SS plasmid was successfully constructed and could induce higher GH levels and enhance weight gain in mice in a dose-independent manner. This study helps us to better understand the relationship between somatostatin and cortistatin, and lays a foundation for the development of a growth-promoting DNA vaccine.


Introduction: immune-mediated necrotising myopathy (IMNM) is associated with pathogenic anti-signal recognition particle (SRP) or 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) antibodies, at least partly through activation of the classical pathway of the complement. We evaluated zilucoplan, an investigational drug, and a macrocyclic peptide inhibitor of complement component 5 (C5), in humanized mouse models of IMNM. Methods: purified immunoglobulin G (IgG) from an anti-HMGCR+ IMNM patient was co-injected intraperitoneally with human complement in C57BL/6, C5-deficient B10 (C5def) and Rag2 deficient (Rag2−/−) mice. Zilucoplan was administered subcutaneously in a preventive or interventional paradigm, either injected daily throughout the duration of the experiment in C57BL/6 and C5def mice or 8 days after disease induction in Rag2−/− mice. Results: prophylactic administration of zilucoplan prevented muscle strength loss in C5def mice  and wild-type C57BL/6  with corresponding reduction in C5b-9 deposits on myofibres and number of regenerated myofibres. Interventional treatment of zilucoplan after disease induction reduced the complement deposits and number of regenerated myofibres in muscles of Rag2−/− mice, although to a lesser extent. In this latter setting, C5 inhibition did not significantly ameliorate muscle strength. Conclusion: Early administration of zilucoplan prevents the onset of myopathy at the clinical and histological level in a humanized mouse model of IMNM. This pathogenic effect was markedly reduced in C3−/− mice [Immune-mediated necrotising myopathies (IMNM) belongs to idiopathic inflammatory myopathies (IIMs) which encompass a group of acquired muscle disorders, including dermatomyositis, inclusion body myositis, polymyositis and overlap myositis. IMNM are rare and severe diseases with symmetrical and proximal muscle weakness, elevated levels of creatine kinase reflecting the degree of muscle cytolysis and myogenic patterns in electromyography . IMNM ar−/− mice . These dTherapeutic approaches for IMNM include corticosteroids and immunosuppressants, such as azathioprine and methotrexate, that may be combined with rituximab and/or intravenous immunoglobulins . AlthougAbnormal complement activity is associated with autoimmune diseases and specific therapeutic drugs targeting complement are used. For instance, in systemic lupus erythematosus (SLE), a disease characterized by a wide range of clinical manifestations such as cutaneous lesions, arthritis, renal involvement and hematologic disorders, and the presence of anti-DNA aAbs , anti-C5The presumed role of the antibody-mediated activation of the complement provides a rationale to investigate therapeutics targeting the complement system in IMNM. Zilucoplan, an investigational drug, is a 15-amino acid macrocyclic peptide with high affinity and specificity to the human complement component 5 (C5). The binding of zilucoplan blocks the downstream assembly of the membrane attack complex  by (i) inhibiting the cleavage of C5 by the C5 convertase into C5a and C5b and (ii) binding to preformed C5b to sterically block interaction with C6, thereby inhibiting the formation of membrane pores and subsequent cell death.The goal of the present study was to pre-clinically evaluate the C5 inhibitor zilucoplan in a humanized mouse model of IMNM and to compare its efficacy when used in a preventive manner or therapeutic setting.Plasma was obtained after a first plasma exchange from a patient who fulfilled the consensus criteria for IMNM with the presence of anti-HMGCR aAb . The pla−/−) C57BL/6 and C57BL/10 B10.D2-Hc0 H2d H2-T18c/oSnJ, also called C5def mice, were purchased from Jackson Laboratories. The protocol for animal experimentation was approved by the institutional ethics committee n°054 under the reference 1415-201508071625487.Eight-week-old female C57BL/6 mice were purchased from JANVIER Labs, France. Rag2 deficient , controlled, and quantified, as previously described [Total IgG were purified from anti-HMGCRescribed . All sam−/− or C5-deficient mice were divided into groups of 8 animals and received daily intraperitoneal injections of 2 mg of IgG purified from anti-HMGCR+ or healthy donor for 8 to 14 days. To reduce anti-human xenogeneic immune responses, C57BL/6 wild type and C5-deficient mice received a single dose of 300 mg/kg of cyclophosphamide  at day-1.Eight- to 10-week-old female C57BL/6 wild type, Rag2The human complement was fresh serum depleted in IgG as described above; the experiments were performed at 4 °C to avoid complement degradation and stored at −80 °C until use. After IgG depletion, the haemolytic complement activity (CH50) was measured with the MicroVue CH50 Eq EIA assay . The mice were supplemented by daily intraperitoneal injections of 200 µL of IgG-depleted complement human serum.Complement-mediated haemolysis and human C5 and split products C5a and sC5b9 levels were monitored from mouse serum before and after the injection of pooled complement human serum.7 antibody-sensitized sheep red blood cells  were incubated with 20% C5-deficient B10.D2/0Sn mouse serum in the presence of 1.5% of C5-depleted serum  with GVB++ buffer . The plates were then incubated at 37 °C for 1 h and centrifuged at 1000× g for 3 min, and the supernatants were transferred to a new plate. To evaluate red blood cell lysis, the optical density was measured at 412 nm (OD412).In a 384-well flat bottom plate, 1.25 × 10Human C5 levels were assessed using a human C5 ELISA kit , in which mouse sera samples were diluted 400-fold. The absorbance at 450 nm was measured with a spectrophotometer  and the concentration of samples was calculated from the absorption and standard curve. Human C5 split products of C5a and sC5b9 levels were assessed using the MicroVue Complement Multiplex kit , where mouse sera samples were diluted 5-fold. The developed ELISA plates were read using a Q-view Imager Pro .−/− or C5-deficient mice receiving purified IgG (anti-HMGCR+ or control) and supplemented by IgG-depleted complement human serum. The dose of S.C. administration of zilucoplan was 10 mg/kg. The zilucoplan injection started at day 0 or 8, 30 min prior to the administration of the complement, and until the end of the experiment at day 7 or 14.Zilucoplan was administered subcutaneously daily to C57BL/6 wild type, Rag2Muscle strength was evaluated by measuring the in situ muscle contraction upon electrostimulation, and the analysis was performed on LabChart7 software , as previously described .gastrocnemius of mice receiving control IgG or anti-HMGCR+ IgG. The number of regenerating fibres was related to the surface of the cryosection.Mouse muscles were snap frozen into isopentane cooled in liquid nitrogen and cut at 8 μm. Cryosections were stained using hematoxylin and eosin (H & E). Regenerating myofibres defined as centronuclear cells were counted manually from a section of For immunofluorescence C5b-9 analysis, cryosections were incubated with rabbit anti-C5b-9 Ab , followed by AlexaFluor647-labeled anti-rabbit IgG Ab . For immunofluorescence foetal myosin heavy chain analysis, cryosections were incubated with mouse anti-myh3 Ab , followed by AlexaFluor647-labeled anti-mouse IgG Ab . For laminin staining, muscle sections were incubated with a rabbit anti-laminin Ab  followed by AlexaFluor488-labeled anti-rabbit Ab , and counterstained with DAPI. The controls for staining specificity (omission of the primary antibody) were always negative.Image acquisition was obtained by microscopy and the images were processed using ImageJ.p-value < 0.05 was considered statistically significant and represented as * p < 0.05, ** p < 0.01 or *** p < 0.001.Data were compared by the Mann–Whitney test using Prism 7 software . Data are presented as mean ± SD; a def mice followed by the administration of purified anti-HMGCR+ or control IgG every two days. The recipients of the anti-HMGCR+ IgG were treated with a daily administration of zilucoplan or vehicle control . The histological analysis of muscles from mice treated with zilucoplan highlighted fewer regenerated fibres (anti-HMGCR+ vs. anti-HMGCR+ + zilucoplan: p= 0.0401)  D,I, necr 0.0401) H and C5blucoplan J. Moreovlucoplan E. Consislucoplan F,J.def mice when injected daily from day 0, with less complement deposition on myofibres and consequently less necrosis/regeneration. These data support that C5 activation in this model is driven by transferred human C5 and that the C5 blockade is effective in preventing muscle strength loss in C5+-driven myopathy, we repeated the same experiment as above using C57BL/6 recipient mice (+: p = 0.0037) . Histological analysis of gastrocnemius muscles from mice treated with zilucoplan revealed a statistically significant reduction of regenerated myofibres as quantified by the proportion of centronucleated fibres/mm2 (anti-HMGCR+ vs. anti-HMGCR+ + zilucoplan: p = 0.0476)  C. In con 0.0476) D. We als 0.0476) E,F. More 0.0476) G.Together, these data confirm that C5 is required to induce myopathy and that the inhibition of human C5 activation is effective in preventing IMNM in this humanized mouse model.+ IgG . On day 15, zilucoplan-treated animals exhibited a modest recovery of muscle strength compared with controls, but this was not statistically significant (control vs. anti-HMGCR+ + zilucoplan: p = 0.1893 and anti-HMGCR+ vs. anti-HMGCR+ + zilucoplan: p = 0.0650). Compared with mice receiving anti-HMGCR+ IgG, gastrocmemius muscles from mice injected with anti-HMGCR+ IgG and zilucoplan have no more necrotic  anti-HMGCR+ patients [The anti-SRP and anti-HMGCR aAb titres correlate negatively with muscle strength and positively with serum creatine kinase levels in IMNM patients ,8. Thesepatients ,17.+ or anti-HMGCR+ patients induces myopathy in mice, since the C-terminal domains of human and mouse HMGCR and SRP54 share 98% and 100% homology, respectively, allowing human aAbs to react against their cognate murine target [−/− compared to wild-type mice [In a previous report, we demonstrated that the passive transfer of IgG purified from anti-SRPe target . Moreoveype mice . Togethehttps://clinicaltrials.gov/ct2/show/NCT04115293; https://www.ucb.com/stories-media/Press-Releases/article/UCB-announces-positive-data-in-myasthenia-gravis-with-zilucoplan-phase-3-study-results; accessed date 16 August 2022).In a randomized double-blind placebo-controlled phase II trial, zilucoplan (0.3 mg/kg), administered S.C. daily, yielded meaningful improvements in the treatment of acetylcholine-receptor autoantibody-positive myasthenia gravis . Acetylcdef mice supplemented with the human complement. Despite this preventive efficacy, zilucoplan injected 8 days after induction of myopathy did not statistically significantly ameliorate muscle strength loss in Rag2−/− mice. This is in line with the recent findings of a phase 2, randomized, double-blind, placebo-controlled, multi-centre trial in patients with IMNM, where zilucoplan failed to meet the primary and secondary endpoints .Here, we harnessed the pathogenicity of human aAbs in mice to evaluate the C5 inhibitor zilucoplan in a mouse model of IMNM. In this model of IMNM, we demonstrated herein that zilucoplan injected concomitantly with aAbs was effective in preventing muscle weakness and myopathy in wild-type C57BL/6 mice as well as in C5+ patients induced hypotrophic myotubes and myofibre atrophy [This discrepancy between the preventive and therapeutic effects of C5 inhibition with zilucoplan may be due to additional non-complement-dependent mechanisms that are likely involved in aAb pathogenicity. Indeed, incubation with plasma from anti-SRP+ and anti-HMGCR atrophy . This wa atrophy . This ph+ or anti-HMGCR+ IgG purified from different patients was able to induce myopathy in mice. Here, anti-HMGCR+ IgG from only one IMNM patient was used. Another limitation of this study is that the short 7-day or 15-day period of IgG injection does not fully recapitulate the chronic aAb exposure of IMNM patients and our model is therefore biased toward early pathological events.As a limitation of this study, it should be reminded that each single mechanism for Ab pathogenicity may be more or less prominent at different time points of the disease and likely from one patient to another. In fact, in our previous study , we obseThis work opens the perspectives that an early application of complement inhibition in the human disease course of IMNM may be beneficial. Indeed, we evidenced that the timing of complement inhibition in the disease course is of importance, i.e., Zilucoplan is effective in preventing myopathy if concomitantly applied with IgG inducing disease but less effective in already induced disease. Therefore, there would be a rationale for also pre-clinically evaluating the administration of the complement inhibitor in combination with other therapeutics.