Influenza A virus (subtype H3N2) causes seasonal human influenza and is included as a component of influenza vaccines. The majority of vaccine viruses are isolated and propagated in eggs, which commonly results in amino acid substitutions in the haemagglutinin (HA) glycoprotein. These substitutions can affect virus receptor-binding and alter virus antigenicity, thereby, obfuscating the choice of egg-propagated viruses for development into candidate vaccine viruses. To evaluate the effects of egg-adaptive substitutions seen in H3N2 vaccine viruses on sialic acid receptor-binding, we carried out quantitative measurement of virus receptor-binding using surface biolayer interferometry with haemagglutination inhibition (HI) assays to correlate changes in receptor avidity with antigenic properties. Included in these studies was a panel of H3N2 viruses generated by reverse genetics containing substitutions seen in recent egg-propagated vaccine viruses and corresponding cell culture-propagated wild-type viruses. These assays provide a quantitative approach to investigating the importance of individual amino acid substitutions in influenza receptor-binding. Results show that viruses with egg-adaptive HA substitutions R156Q, S219Y, and I226N, have increased binding avidity to α2,3-linked receptor-analogues and decreased binding avidity to α2,6-linked receptor-analogues. No measurable binding was detected for the viruses with amino acid substitution combination 156Q+219Y and receptor-binding increased in viruses where egg-adaptation mutations were introduced into cell culture-propagated virus. Substitutions at positions 156 and 190 appeared to be primarily responsible for low reactivity in HI assays with post-infection ferret antisera raised against 2012–2013 season H3N2 viruses. Egg-adaptive substitutions at position 186 caused substantial differences in binding avidity with an insignificant effect on antigenicity. Influenza A virus (IAV), subtype H3N2, causes seasonal human influenza and is included in trivalent and tetravalent vaccines containing H1N1, H3N2 and influenza B virus components. Influenza viruses undergo antigenic drift by mutation of the haemagglutinin (HA) gene, encoding the major protein target for immune responses, to evade pre-existing immunity. Accumulation of these mutations can result in the emergence of antigenically-distinct groups if certain amino acid substitutions are introduced in the HA glycoprotein. To ensure vaccines are as effective as possible, global surveillance and monitoring of circulating wild-type viruses has been carried out since 1948 under the auspices of WHO to monitor influenza virus evolution, and vaccine composition is reviewed by WHO twice annually.Most influenza viruses used for vaccines have been propagated in hens’ eggs. However, cultivation of clinical samples containing human influenza viruses in eggs can select for virus variants . HoweverVirus adaptation to propagation in eggs usually results in the introduction of amino acid (AA) substitutions in the HA glycoprotein. Due to the close proximity of the receptor binding site (RBS) and areas targeted by virus neutralising antibodies , these sEgg-adaptive AA substitutions can obfuscate the choice of vaccine viruses. In several 2012–2013 season H3N2 CVVs  derived from A/Victoria/361/2011 (Vic361), egg-propagation resulted in AA substitutions at the RBS and known antigenic sites , which wHere we report on the effects of egg-adaptive substitutions in HA of recent H3N2 vaccine viruses on SA receptor-binding using a recently developed surface biolayer interferom etry (BLI) assay . Three CVVs were produced from V361e: IVR-165, X-217 and NIB-79 . In the RG viruses were produced with either one or both of the R156Q and S219Y AA substitutions to define which was responsible for the lack of measurable binding to the receptor-analogues. Introduction of substitutions R156Q and S219Y independently into Vic361e HA resulted in some increased binding to 3-SLN , but decCVV NIB-79 encoded a D190E substitution in HA1. RG/Vic361e D190E, constructed to correspond to NIB-79, showed a similar binding profile to 3-SLN  with a sVic361e differs from the cell culture-propagated virus (Vic361c) at two AAs in the vicinity of the RBS, 156 and 186. Two RG viruses RG/Vic361e R156H and RG/Vic361e V186G were constructed to represent viruses with single substitutions. Both RG viruses failed to bind 3-SLN  and had In 2013, the H3N2 vaccine component recommendation changed from Vic361e to Tex50e. Tex50e showed no measurable binding to 3-SLN or 6-SLN (data not shown). CVV X-223, derived from Tex50e and carrying a substitution at residue 226 of HA1 (I226N), also failed to bind to 6-SLN (data not shown) but bound with high avidity to 3-SLN . The lacIn September 2014, a change in the recommendation for the H3N2 component of the influenza vaccine was made due to the emergence of antigenically-distinct viruses in early 2014. A/Switzerland/9715293/2013 (Switz13e) was recommended as the prototype virus. In the set of 2015 and 2015–2016 CVVs derived from Switz13e, X-247 and NIB-88, both carried AA substitutions at residue 219, S219F and S219Y respectively, compared to the egg-propagated prototype Switz13e. Both bound to 6-SLN with low avidity compared to the Switz13e virus , and botBoth the exclusively cell culture-propagated cultivar of Switz13 (Switz13c) and the RG/Vic361c H156R virus bound with high avidity to 6-SLN  but no oFailure of the majority of exclusively cell culture-propagated viruses to bind to receptor-analogues may be related to the AA at position 225. Previous BLI studies reported that representative H3N2 IAV collected between 2005 and 2010, propagated in cell culture, lost the ability to bind to 6-SLN by 2010. AA substitution D225N, occurring between 2004 and 2005, was demonstrated to be the major cause of a structural conformation change . HoweverRBC from different mammalian and avian species express cell-surface SAs of different linkages and modifications making them useful tools in indicating receptor-binding preferences of IAV  for antiserum raised against Vic361e, indicating antigenic similarity. However, 7 of the 19 test viruses were recognised at 4-fold reductions in titre (HI titres of 640) compared with the homologous titre of the antiserum, which is classed as being antigenically distinguishable from the homologous virus, but not enough to be defined as antigenically distinct. These viruses included Vic361c and RG viruses with AA substitutions at HA positions 156 and 219, RG/Vic361e R156Q, R156Q+S219Y, RG/Vic361c H156Q, S219Y, S219F, H156Q+S219Y. Thus reduced recognition by the antiserum raised against Vic361e can be associated withAA substitution at positions 156 and 219.Seven out of 19 viruses tested with antiserum raised against cell culture-propagated Vic361c gave HI titres that were significantly reduced (160) compared to the homologous titre (640), and CVV NIB-79 was found to be poorly recognised by this antiserum at an 8-fold reduction in HI titre (80) indicating it was antigenically distinct from Vic361c. NIB-79 contains a D190E substitution, also shared with RG/Vic361e D190E, but not present in any of the other viruses tested. RG/Vic361e D190E showed reduced recognition by the antiserum (4-fold reduced over the homologous titre) indicating that it may be responsible for the change in antigenicity.Antiserum raised against NIB-79 had a high homologous titre (1280) and CVV IVR-165 and cell culture-propagated Vic361c were poorly recognised (160). Moreover, egg-propagated RG virus RG/Vic361e R156Q+S219Y and cell culture-propagated RG viruses RG/Vic361c H156Q, S219F, S219Y, and H156Q+S219Y have significantly lower HI titres of 320 when tested against NIB-79 antiserum. Antisera raised against either IVR-165 or X-217 recognised NIB-79 and RG/Vic361e D190E at titres of 320, 4-fold reduced compared to the homologous titre.RG/Vic361e I226N reacted well (HI titre 1280) with antiserum raised against Vic361e (homologous titre 2560), indicating that the CVV X-223 is antigenically similar to the Tex50 virus (Table S1) from which it was derived. Similarly, the I226N substitution has previously been reported to improve virus replication in eggs of a cold-adapted H3N2 IAV A/Brisbane/10/07 without affecting virus antigenicity  or receptor-binding properties (V186G and G186V) or both, as seen in viruses with HA1 position 156 substitutions.The HA of H3N2 IAV has evolved continuously over the last 47 years resulting in changes in receptor-binding preference and avidity, and antigenic properties. Despite this, H3N2 viruses show no decrease in their ability to circulate within the human population  contribute to HI titres, as discussed in . As it iThe 2014–2015 influenza season was more severe than the previous season, with H3N2 viruses circulating as the dominant subtype . Clearly-1 penicillin-streptomycin  and 1  mg mL−1 G418 Sulphate  at 37 °C, 5 % CO2.MDCK-SIAT1 cells engineered to over-express α2,6-sialyltransferase (Details of viruses used (A/Victoria/361/2011 (Vic361), A/Texas/50/2012 (Tex50), A/Switzerland/9715293/2013 (Switz13), and CVVs IVR-165, NIB-79, X-217, X-223, X-247, and NIB-88) are listed in Table S2.An eight-plasmid reverse genetics rescue system . HA and Cell culture-propagated viruses were cultivated in MDCK-SIAT1 cells (Haemagglutination assays were performed with suspensions of 1 % (v/v) guinea pig , 1 % horse, 0.5 % chicken , or 0.5 % turkey  red blood cells (RBC). HI assays were performed using WHO recommended methods  in the pVirus binding to polyacrylamide-linked, 20 % (w/w) carbohydrate, polyvalent receptor-analogues, α2,3-sialyl lactosamine (3-SLN) or α2,6-sialyl lactosamine (6-SLN) , was measured using BLI assays on an Octet® RED platform  as previously described (


We found that the non-phosphorylated (NP)-Dvl3 was more stable than the phosphorylated form, more active in activating β-catenin transcriptional activity, and more potent in enhancing self-renewal ability in HCC cells. Mechanistically, we confirmed that the homeodomain-interacting protein kinase-2 (HIPK2) and E3 ubiquitin ligase ITCH were able to physically bind to Dvl3 protein. Knockdown of HIPK2 and the protein phosphatase regulatory unit C-alpha (PP1Cα) resulted in sustained Dvl3 phosphorylation and hence decrease in the NP form of Dvl3. On the other hand, knockdown of E3 ubiquitin ligase ITCH reduced the phosphorylation-induced degradation and stabilized the phosphorylated Dvl3 protein. Furthermore, the NP-Dvl3 enhanced the LGR5 promoter activity to upregulate LGR5 expression, which was associated with increased cancer stemness in HCC. Our findings established that HIPK2/PP1Cα/ITCH axis sustains the de-phosphorylation of Dvl3. This post-translational modification of Dvl3 in turn maintains LGR5 expression and enhances the cancer stemness properties in HCC.Dishevelled-3 (Dvl3) is regarded as a binding hub with many different interacting partners. However, its regulation and mechanism on cancer stemness remain to be explored. In this study, we showed that Dvl3 was significantly overexpressed in human hepatocellular carcinomas (HCCs) and promoted cancer stemness both  Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide . One of Dishevelled (Dvl) protein is an important mediator of the Wnt/β-catenin signaling pathway. It is a family of three members, namely Dvl1, 2, and 3, and sharing three conserved domains, DIX, PDZ and DEP domains . Dvl3 hain vivo [LGR5 has been identified as a marker of crypt-locating intestinal stem cells , 10 and in vivo . A previin vivo  and pro-in vivo . These sin vitro sphere formation assays and in vivo tumorigenicity assays in immunodeficient NOD-SCID mice.In this study, we found that, in human HCCs, Dvl3 was overexpressed at protein level as compared with the corresponding non-tumor livers. The NP form of Dvl3 had enhanced stability and activity in HCC cells in promoting sphere forming ability in HCC cells. Our data also suggest that HIPK2/PP1Cα/ITCH participates to mediate Dvl3 phosphorylation and degradation to upregulate LGR5 expression. This in turn promotes cancer stemness and HCC formation, as we demonstrated in P < .001)    Figure . Success) Figure . The upp) Figure  and the ) Figure . Further) Figure . This le) Figure . This ph) Figure . In addi) Figure . Importa) Figure . These fWe explored the possible regulatory mechanism by HIPK2-PP1Cα-ITCH axis on Dvl3 in HCC cells. First, using co-immunoprecipitation (Co-IP) assay, we found that HIPK2 protein was able to physically bind to Dvl3 protein in Huh-7 cells Figure . We thenP = .01)  Figure . The datP = .001)  Figure . In addi) Figure . On the 2 = .262, P = .001)    Figure . Besides = .001) . Similar) Figure . In orde) Figure . These dP = .001)   Figure . Corresp) Figure  also sig) Figure . This inP = .037 and .008, respectively) as compared to the non-target control (NTC)   Figure . Further) Figure  and enhactively) . For thep Figure . A similor onset . It was or onset  as we moP = .034 and P = .001, respectively)  Figure . Those c) Figure . Further) Figure . The knoormation .In this study, we found that Dvl3 expression was upregulated in human HCCs. We have shown that Dvl3 promoted cancer stemness in terms of enhanced tumorigenicity, sphere formation ability, chemoresistance, and expression of stemness genes. However, how its stability is controlled has not been previously clarified.When Dvl3 is phosphorylated upon Wnt stimulus, it appears as a more slowly migrating band (P-Dvl3) as compared to the faster migrating band (NP-Dvl3) in Western blots , 26–29, Such dynamics may represent a built-in feedback regulatory mechanism of the signaling. While Wnt ligands activate the signaling, they may also trigger Dvl phosphorylation to prime part of the Dvl protein pool to become less active. In this way, less amount of active Dvl3 will be available to convey further signals downstream to prevent prolonged activation of the pathway. This may allow time for degradation of the accumulated β-catenin to return the pathway activity to the initial basal level in order to prepare the cells for subsequent round of activation. This appears to be critical to the concise temporal and spatial Wnt-mediated biological processes.The presence of HIPK2/PP1Cα and ITCH may offer additional layers of regulation on Dvl3 quantity and activity to help modulate the Wnt signaling pathway activity: (1) when the P-Dvl3 resulted from Wnt stimulation is de-phosphorylated by HIPK2, such Dvl3 protein can return back to the initial pool of active NP-Dvl3 to maintain sustainable Wnt signaling activity; (2) when the P-Dvl3 resulted from Wnt stimulation cannot be efficiently de-phosphorylated by the HIPK2/PP1C, this may indicate an already excessive Wnt stimulation and ITCH will remove the excessive P-Dvl3 , 8 in orLGR5 is a β-catenin target gene as well as a marker of crypt-locating intestinal stem cells , 10 and All HCC specimens and their corresponding non-tumorous liver tissues were resected from Chinese patients between year 1992 to 2000 at Queen Mary Hospital, Hong Kong. Thirteen patients were male and seven were female. Their ages ranged from 38 to 71 years (mean = 56.1 years). Sixteen (80%) of the 20 patients had chronic hepatitis B viral infection with positive serum hepatitis B surface antigen (HBsAg) status. None of these patients received any other therapies including chemoembolization or chemotherapy prior to hepatic tumor resection. Following surgical resection, all specimens were either snap-frozen immediately in liquid nitrogen and stored at −80°C, or fixed in buffered 10% formalin for paraffin embedding. The use of the clinical specimens was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority. Human HCC cell lines , human embryonic kidney cells HEK-293, human hepatoblastoma cell line HepG2, mouse L-cells and Wnt3a-producing L-cells were obtained from American Type Culture Collection. MHCC-97L was a gift from Dr. Z.Y. Tang of Fudan University, Shanghai. BEL7402, SMMC-7721, LO2 and MIHA were obtained from Shanghai Institute of Cell Biology, Chinese Academy of Sciences. All cell lines were maintained as described .The Dvl3-expressing construct (Dvl3/pcDNA) was a gift from Dr M. Semenov . The N-terminally Flag-tagged and myc-tagged Dvl3 was cloned from Dvl3/pcDNA to FLAG/pcDNA3.1 and 2x-myc/pcDNA3.1 plasmids respectively. Phospho-defective mutant of Dvl3 (P2A) was created using mutational primers 5′-AGCGAAGGCAGTCGGAGCAGTGGCGCCAACCGTGCCGGCAGCGATCGGAGG-AAGGAGAAG and 5′-CTTCTCCTTCCTCCGATCGCTGCCGGCACGGTTGGCGCCA- CTGCTCCGACTGCCTTCGCT. The N-terminally GFP-tagged HIPK2 was made by cloning HIPK2 to pEGFP-C1 plasmid. The N-terminally FLAG-tagged HIPK2 constructs were gifts from Dr. L. Schmitz . The N-terminally myc-tagged ITCH was created by cloning ITCH to 2x-myc/pcDNA3.1. Super8X TOP Flash and Super8X FOP Flash constructs were gifts from Dr. R. Moon  for TOP/FOP reporter assays. pRL-CMV construct with a CMV promoter-driven renilla luciferase reporter was transfected as reference for normalization. LGR5 promoter was cloned to pGL3-Basic plasmid. Small interfering RNA (siRNA) targeting Dvl3 was purchased from Dharmacon, Inc while those targeting HIPK2, PP1Cα and ITCH were from Integrated DNA Technologies, Inc. based on sequence available from literature., 8 TransTRIzol reagent  and Reverse-Transcription kit  were used to extract total RNA and generate complementary DNA respectively according to the manufacturers' protocols. RT-qPCR was carried out using Taqman probes for Dvl3 and HPRT  in the 7900HT Fast Real-Time PCR system (Applied Biosystems). SYBR green RT-qPCR was used to analyze gene expressions in cell lines. The list of primers is shown in TM software . Co-IP was performed in Huh-7 cells at 48 hours after transfection, as previously described [Lysate preparation and SDS–polyacrylamide gel electrophoresis for Western blotting were performed as described , 32. Antescribed .Briefly, cells were suspended in 0.25% methyl cellulose in serum-free DMEM/F12 in wells pre-coated with 1% polyHEMA to form spheres in presence of the necessary supplement as described . At end The LGR5 promoter-driven cyan fluorescent protein (CFP)-containing construct was transfected to BEL-7402 cells and cell sorting was performed as described  48 hrs aCommercial kit  was used according to the manufacturer's instruction. By using a FACSCalibur flow cytometer and CellQuest software (BD Biosciences), 10,000 events were acquired per sample under proper gating for PI and annexin V signals for analysis.To examine the self-renewal ability of the injected cells to generate tumors in immunodeficient mice , indicatTOP/FOP reporter assays were performed  using tht or Mann–Whitney test was used for continuous data. Tests were considered significant when the P-value was < 0.05.Chi-squared test or Fisher's exact test was used to analyze categorical data, while Student's


Lamin A is a component of the nuclear matrix that also controls proliferation by largely unknown mechanisms. NF-Y is a ubiquitous protein involved in cell proliferation composed of three subunits (-YA -YB -YC) all required for the DNA binding and transactivation activity. To get clues on new NF-Y partner(s) we performed a mass spectrometry screening of proteins that co-precipitate with the regulatory subunit of the complex, NF-YA. By this screening we identified lamin A as a novel putative NF-Y interactor. Co-immunoprecipitation experiments and confocal analysis confirmed the interaction between the two endogenous proteins. Interestingly, this association occurs on euchromatin regions, too. ChIP experiments demonstrate lamin A enrichment in several promoter regions of cell cycle related genes in a NF-Y dependent manner. Gain and loss of function experiments reveal that lamin A counteracts NF-Y transcriptional activity. Taking advantage of a recently generated transgenic reporter mouse, called MITO-Luc, in which an NF-Y–dependent promoter controls luciferase expression, we demonstrate that lamin A counteracts NF-Y transcriptional activity not only in culture cells but also in living animals. Altogether, our data demonstrate the occurrence of lamin A/NF-Y interaction and suggest a possible role of this protein complex in regulation of NF-Y function in cell proliferation. NF-Y is an heterotrimeric transcriptional factor composed of 3 proteins, the regulatory subunit NF-YA, NF-YB and NF-YC. The three proteins are transcribed from independent genes highly conserved from yeast to human. All 3 proteins are necessary for NF-Y binding to the target DNA sequence, the CCAAT-box. The NF-YA subunit contain a DNA binding domain, an activation domain and the binding site for the dimer NF-YB/-YC. The NF-YB and -YC subunits contain histone-like domains . A bioinin vivo NF-Y/mutant p53 complex able to increases DNA synthesis, in a NF-YA dependent manner [It has been reported the presence of two major NF-YA isoforms, “long” and “short”, the short isoform lacking a 28-amino acid within the NF-YA amino-terminal domain . Expresst manner . Clinicat manner . Analysit manner . In agret manner . AlthougLMNA gene, are expressed only in differentiated cells, while Lamin B1 and lamin B2, encoded by LMNAB1 and LMNAB2 genes, are expressed throughout development. Prelamin A (the precursor of lamin A protein) and lamin C are produced by an alternative splicing within exon 10. The two proteins differ in the carboxyterminal domain where the human lamin A (646aa) contains 80 unique amino acids and lamin C (572aa) contains 6 unique amino acids. It has been shown that lamin A/C stabilizes the nuclear lamina and chromatin, preventing DNA breaks and favouring epigenetic stabilization.The major components of the nuclear lamina are lamins. These type V intermediate filament (IF) proteins play important roles in nuclear architecture, mechanosignaling  and chroThe nuclear lamina interacts with large genomic regions, called lamina-associated domains (LADs). LADs are often located in repressive chromatin structures that appear principally at the nuclear periphery , 26. BesLamins A and C are implicated in epigenetics, heterochromatin organization and are shown to complex with histones and key regulator of transcription such as pRB (retinoblastoma-associated protein), MOK2 (zinc finger transcription repressor), several components of the Pol II (RNA polymerase II) complex . Lund etUsing a combination of biochemical, cell biology and molecular imaging techniques, we demonstrate here that NF-Y, a master regulator of cell proliferation, forms a complex with a component of the nuclear lamina, lamin A. This interaction impacts on the expression of NF-Y target cell cycle regulatory genes and consequently cell proliferation.To get clues on NF-Y function(s) in cancer cells, we performed a mass spectrometry screening of a pool of proteins that co-precipitate with the long NF-YA isoform overexpressed in human breast cancer, SKBR3 cells. By this screening we identified lamin A, but not lamin C, as a novel putative NF-YA interactor Figure .The occurrence of laminA/NF-Y interaction was also validated by coimmunoprecipitation experiments between endogenous proteins. As already described it has been reported the presence of two NF-YA isoforms, “long” and “short”. As shown in Figure Reciprocal immunoprecipitation experiments performed with anti-lamin A/C antibody further validated the occurrence of endogenous lamin A/NF-YA in all tested cell lines Figure . ApparenTo demonstrate the specificity of lamin A/NF-YA interaction we evaluated the interaction of lamin A with p53, a well known NF-Y interactor. The results presented in To study the function of a complex often is critical the identification of cellular compartment (s) where the interaction between the two proteins occurs. To get clues on lamin A and NF-YA physical interaction, we isolated different lamin enriched nuclear fractions . We isolRecent data support a role for lamin A in gene regulation through its interaction with chromatin . Thus weTo start to investigate whether lamin A localizes on euchromatin, where NF-Y plays a key role as transcription factor, we first isolated euchromatin fraction from several cell lines . To thisBased on this result, we digested for 5 minutes chromatin from several cell lines and analyzed it by agarose gel . In all in vivo recruitment . Specific primers were used to amplify DNA regions encompassing NF-Y consensus sites on several NF-Y target promoters. Our experiments performed with two different antibodies against lamin A, demonstrate that it associates, although at different extent, with several promoter regions carrying the CCAAT-boxes, such as CCNB2, DHFR, CCNA2, CDK1, CCNB1, CDC25C, TOPO2A and PCNA promoters Figure . As expet Figure . MoreoveTo directly assess the role of NF-Y in the recruitment of lamin A to the NF-Y target promoter regions carrying CCAAT boxes, we performed ChIP experiments upon overexpression of dnNF-YA mutant protein in SW480 cells. ChIP-qPCR analysis shows that the overexpression of dnNF-YA induces a decreased recruitment of NF-Y Figure , and thiVery recently, it has been demonstrated that lamin A/C is able to bind to specific DNA sequences . We inveTaken together, our data demonstrate that the binding of lamin A to CCAAT containing promoter regions of several cell cycle genes is largely dependent on the binding of NF-Y complex, and indicate that lamin A binds these promoters through its ability to bind NF-Y.To characterize the functional role of the lamin A binding to NF-Y target promoters, we investigated the chromatin structure of the promoter regions bound by lamin A/C and NF-Y. We performed ChIP-qPCR experiments using antibodies against H4K20me3 and H3K14ac, hallmarks of closed and open chromatin structure, respectively. Previous analysis demonstrated an excellent correlation between NF-Y binding and regions enriched for histone modifications associated with active transcription . As expeThus, we asked whether the depletion of lamin A affects histone modifications and we compared the recruitment of H3K14ac on several NF-Y target promoter regions in cells silenced for lamin A. On this purpose, we produced stable siLMNA SW-480 and SAOS-2 cells (LMNA-KD cells) showing a reduction in the protein level of lamin A of about 60% and 70%, respectively, compared to control cells transfected with the same vector carrying a non-targeting artificial siEmGFP-miRNA Figure . ResultsCCNB2 promoter as prototype of NF-Y target gene  are recruited to all the analyzed regions, thus further indicating gene activation Figure . The samget gene . AltogetWe confirmed the involvement of lamin A on NF-Y target gene transcription using CCNB2 promoter driven luciferase reporter construct as a sensor of NF-Y activity. We transiently overexpressed this construct in SW-480 cells together with a vector expressing NF-YA and/or a vector that allows the simultaneous expression of an artificial miRNA able to target the mRNA of lamin A, and the EmGFP reporter gene as control (pcDNA6.2-GW/EmGFP-miR-LMNA) . Our datNext, we stably transduced in SW-480 cells a human lamin A (lamin A-res) expressing vector resistant to siRNAs [in vivo the impact of lamin A on NF-Y transcriptional activity we took advantage of the MITO-Luc mouse model, that we recently developed, carrying a luciferase reporter driven by a promoter strictly dependent on NF-Y allowing us to monitor the NF-Y activity within the entire living organism in a spatiotemporal manner [To demonstrate l manner . We demol manner . We injel manner . Of note2O2). The growth of SW-480 cells was partially reduced upon growth factor deprivation , in which an NF-Y–dependent promoter drives luciferase expression. Data obtained strongly support the physiological impact of lamin A expression in cell proliferation.In our study, an inverse correlation between lamin A and several NF-Y target genes expression level was observed, thus supporting the role of lamin A as regulator of NF-Y transcriptional function. We validated these evidences by NF-Y is one of the transcription factors responsible for aberrant oncogenic transcription occurring in several cancers . Loss ofFurther exploration to uncover the molecular mechanism(s) by which NF-Y/ lamin A complex acts as crucial regulator in diverse cellular processes and, in particular, in cancer could be important to improve and potentially provide new clues into new therapeutic approaches for cancer treatment.Lysates (2 mg/sample) were precleared with Protein-G Agarose (Pierce) and incubated 4h at 4°C with an anti-NF-YA polyclonal antibody-protein-G complexes (50ul proteinG + 5ug antibody/sample) previously crosslinked by DMP (Sigma). We used two negative controls : 50ul of anti-NF-YA polyclonal antibody-protein-G complexes immunoprecipitated without extract (ctr ab), 2 mg of cell extract immunoprecipitated with 50ul of protein-G without the antibody (ctr cell extract).Immunoprecipitated proteins were resolved on a 12%T- 3.3%C SDS–PAGE separating gel (1+18+18 mm), revealed by Sypro Ruby staining and visualized using a Typhoon 9200 laser scanner . Proteins were excised from the gel using Investigator ProPic spot picker (Genomic Solutions) and transferred to small tubes (0.2 ml). Protein-containing gel pieces were destained with 50 ml of 0.1M ammonium bicarbonate (5 min at RT). After two washes with 50% acetonitrile/0.05M ammonium bicarbonate, gel plugs were shrunk by addition of 100% acetonitrile. The dried gel pieces were re-swollen with 4.5 ng/ml trypsin in 50mM ammonium bicarbonate and digested overnight at 378C. Peptides were concentrated with ZipTipmC18 pipette tips (Millipore). Coelution was performed directly onto a MALDI target with 1 ml of a-cyano-4-hydroxycinnamic acid matrix . Proteins were identified by Matrix Assisted Laser-Desorption Ionization (MALDI)-MS and MALDI-MS/MS . Data were acquired in positive MS reflector mode. Five peptides  were used as calibration standards. Mass spectra were obtained from each sample by 30 sub-spectra accumulation (50 laser shots each) in a 750 to 4000. mass range. Five signal-to-noise best peaks of each spectrum were selected for MS/MS analysis. For MS/MS spectra, the collision energy was 1 keV and the collision gas was air. The interpretation of both the MS and MS/MS data was carried out by using the GPS Explorer software , which acts as an interface between the Oracle database containing raw spectra and a local copy of the MASCOT search engine (Version 1.8). Peptide mass fingerprints obtained from MS analysis were used for protein identification in the NCBI non redundant database. All peptides mass values were considered monoisotopic and mass tolerance was set at 50 p.p.m. One missed cleavage site was allowed, cysteines were considered carboamidomethylated, methionine was 0assumed to be partially oxidized and serine, threonine and tyrosine partially phosphorylated. Mascot (Matrix Science) scores >61 were considered significant (P<0.005). For MS/MS analysis, all peaks with a signal-to-noise ratio >5 were searched against the NCBI database using the same modifications as the MS database, with a fragment tolerance <0.3 Da.2 atmosphere.Human breast cancer cell lines MCF-7 and SKB-R3, human colorectal carcinoma cell lines HCT-116 and SW-480, human osteosarcoma with osteoblastic properties cells Saos-2, human primary fibroblasts (HF), and human cervical adenocarcinoma HeLa cells were all cultured in Dulbecco's modified Eagle's medium  supplemented with 10% fetal calf serum and antibiotics in a humidified 5% COCells, harvested in cold phosphate-buffered saline, were extracted for 30 min at 4°C in lysis buffer  containing 1 mM DTT, protease inhibitor cocktail (Roche) and phosphatase inhibitors . After centrifugation, supernatants were collected as the total protein extract and stored at -80°C. Protein concentrations were measured using the Bradford-type protein assay (Bio-Rad).Cells were resuspended in hypotonic buffer  and nuclei separated by centrifugation. Nuclei were then incubated 30 min in moderate-salt buffer buffer  and centrifuged  to obtain a lamin-enriched pellet. This pellet was incubated on ice 30 min in high pH/high detergent buffer  followed by centrifugation  to yield a supernatant of solubilized lamins (soluble lamins) and an insoluble pellet.Cells were lysed by mechanical homogenization in isotonic- sucrose based buffer  followed by centrifugation . The pellet, containing the nuclei, was resuspended and lysed in lysis buffer  and centrifuged . The supernatant corresponds to the nucleoplasmic fraction and the pellet to the chromatin fraction.Chromatin fraction, obtained as described above, was digested im MNase buffer  with Micrococcal nuclease to a final concentration of 1.2 units/mL for 5 minutes, and the reaction was quenched by 1 mM EGTA on ice for 10 minutes. The sample was centrifuged at 1,000× g for 5 minutes at 4°C to generate the supernatant, corresponding to the euchromatin fraction. The insoluble chromatin pellet was resuspended in 15 mM Tris, pH = 7.5, 0.5% SDS. Extraction of DNADNA was isolated from chromatin samples by extraction with an equal volume of phenol/chloroform (1:1), extraction with an equal volume of chloroform, ethanol-precipitated and then resuspended in water and loaded onto a 2% agarose gel.For immunoprecipitation experiments, lysates were clarified and immunoprecipitated at 4°C overnight in lysis buffer by adding protein G-agarose after 2 h of incubation with 2 μg of antibody. Proteins were resolved by SDS-PAGE and electro transferred to nitrocellulose. Each membrane was blocked with 5% non-fat dry milk in Tris buffered saline-Tween-20 (TBST) for 1 h at room temperature and subsequently incubated with primary antibody for 16 h at 4°C. The following antibodies were used: anti-NF-YA polyclonal (Santa Cruz), anti NF-YA monoclonal (Santa Cruz) and anti-lamin A (Santa Cruz) and anti-lamin A/C (Santa Cruz), anti-β actin . Immunoreactivity was detected by sequential incubation with HRP-conjugated secondary antibody.For tissue total exstract preparation, after tissue dissection, the organs were immediately frozen on dry ice. Bone marrow was flushed out from the femur and tibia, as previously described . Tissue Cells were fixed with 2% formaldehyde, permeated with 0,05% Triton X-100, blocked 1h with 5% BSA, and subjected to staining using anti-lamin A (Santa Cruz), anti-NF-YA monoclonal (Santa Cruz), Cy3-conjugated donkey anti-mouse and Cy2-conjugated donkey anti-rabbit (Jackson Immuno Research Laboratories). Slides were analyzed within 24 h. As control, single immunofluorescence labeling for each antibody, and immunofluorescence labeling without primary antibody was performed (data not shown). All experiments were performed several times with similar results. Images were recorded by using a Zeiss LSM 510 Meta confocal laser scanning microscope equipped with a 20X and 60X/1.23 NA oil immersion objective. As laser (488 and 514 nm), and HeNe laser (543 nm) were used to excite the fluorophores. The LSM 510 R. 3.2 META (Zeiss) image analysis software was used.Plasmids used in transfections were as follows: NF-YA and empty vector , wt cyclCells were transfected with Lipofectamine LTX and Plus reagent (Invitrogen) following the manufacturer's instructions.Stably transfected cells were selected for by culturing the cells in the presence of blasticidin at10 μg/mL (Invitrogen). The transfected cells were screened using western blotting and real-time RT-PCR assays to determine the levels of lamin A expression and the miRNA expression was monitored by checking the simultaneous coexpression of the EmGFP reporter gene by fluorescence microscopy. For reporter assay luciferase activity was measured using the dual luciferase assay system (Promega) according to the instructions of the manufacturers. All transfections were done as cotransfections with a CMV-driven plasmid expressing Renilla luciferase as internal control to standardize transfection efficiencies.1% formaldehyde was added directly to the cells and incubated at 22 °C for 10 min. The reaction was stopped adding 0.125 m glycine. Then, the cells were rinsed with cold 1× PBS, incubated with 0.2× trypsin-EDTA in 1× PBS, and scraped. cells were centrifuged, washed in cold 1× PBS plus 0.5 mm PMSF and resuspended in lysis buffer  mm KCl, 0.5% Nonidet P-40). Next, nuclei were solicited in the sonication buffer , 0.5% deoxycholic acid) for 10 min by using a microultrasonic cell disruptor. The chromatin was sheared to an average size of 500 base pairs, and immunoprecipitation was performed with protein G-agarose (KPL). The chromatin solution was precleared by adding protein G for 1 h at 4 °C and incubated at 4 °C overnight with 4 μg of antibody or non-specific immunoglobulins  as negative control. Input was collected from a control sample supernatant (not immunoprecipitated antibody). Immunoprecipitates were recovered by incubation for 2 h at 4 °C with protein G-agarose precleared previously in immunoprecipitation buffer . Reversal of formaldehyde cross-linking, RNase A, and proteinase K treatments were performed. DNA was phenol-extracted, ethanol-precipitated, and analyzed by PCR. DNA representing 0.005–0.01% of the total chromatin sample (input) or 1–10% of the immunoprecipitates was amplified using specific primers indicated in Table Total RNA was extracted using the Trizol reagent (Gibco BRL) following the manufacturer's instructions. The first-strand cDNA was synthesized according to the instructions for the M-MLV RT kit (Invitrogen). Quantitative PCR (qPCR) was performed using SYBR Green (Applied Biosystems) on an ABI Prism 7500 apparatus (Applied Biosystems). mRNA expression was normalized for β-actin levels. Primers used are listed in Table Phoenix-ampho cells (American Type Culture Collection) were transfected with pMXIH-V5 or pMXIH-V5 Lamin A. The supernatant medium containing the emerging retrovirus was collected 48 and 72 h after transfection. The supernatants were pooled and the retroviral particles were concentrated by ultracentrifugation at 22.000 rpm for 2 h, resuspended in cold PBS and stored at -80°C. The viral titer was determined infecting NIH3T3 murine fibroblasts at different serial dilutions. The retroviral vectors were injected into the tail vein in adult mice with 5.25x10^2/mouse.MITO_Luc C57Bl/6j  were intAll animal studies were approved by the Institutional Animal Care of the Regina Elena National Cancer Institute and by the Government Committee of National Minister of Health and were conducted according with EU Directive 2010/63/EU for any-animal experiments http://ec.europa.eu/environment/chemicals/labanimals/legislation en.htm.


This study aimed to report the frequency of the DI*A and DI*B alleles in a Central Thai population and to compare them with those of other populations previously published. Altogether, 1,011 blood samples from unrelated healthy blood donors at the National Blood Centre, Thai Red Cross Society, Bangkok were included. Only 391 samples were tested with anti-Dia by conventional tube technique. All samples were genotyped for DI*A and DI*B alleles using an in-house polymerase chain reaction with sequence-specific primer (PCR-SSP) technique. The DI phenotyping and genotyping results were in 100% concordance. The DI*A and DI*B allele frequencies among 1,011 Central Thais were 0.0183  and 0.9817 , respectively. Allele frequencies were compared between Central Thai and other populations. Our data shows that DI*A and DI*B allele frequencies are similar to Southeast Asian, Brazilian, Southern Brazilian and American Native populations; whereas, these frequencies significantly differ from those reported in East Asian, Italian, Alaska Native/Aleut, Hawaiian/Pacific Islander and Filipino populations (P<0.05), corresponding to the results of a matrix of geometric genetic distances. This study confirms that the prevalence of DI*A and DI*B alleles among Central Thais is similar to Southeast Asians and different to others populations of the world. A PCR-based identification of DI genotyping should overcome some of the serological limitations in transfusion medicine and provides a complementary tool for further population-genetic studies.Alloantibodies to the Diego (DI) blood group system, anti-Di In gea and Dib) are the products of DI*A and DI*B alleles caused by a single nucleotide polymorphism, SNP (c.2561C>T) in the human erythrocyte membrane anion-transport protein gene (SLC4A1). This gene encodes a substance called band 3 protein, which is expressed on the surface of red blood cells (RBCs) and plays a central role in mediating the transport of carbon dioxide in the blood. A single amino acid substitution in position 854 results in a leucine corresponding to Dia antigen and proline to the Dib antigen [Antithecal antigens (Di antigen –13.a is more frequently found than anti-Dib among pregnant women and multitransfused patients [a using the indirect antiglobulin test (IAT). However, some limitations of serological techniques occur in patients who have previously received blood transfusions or have RBCs that give a positive direct antiglobulin test (DAT) result. Various polymerase chain reaction (PCR)-based techniques have been implemented for Diego blood group genotyping such as PCR-restriction fragment length polymorphism (PCR-RFLP), PCR with sequence-specific primer (PCR-SSP) as single or multiplex assays, real time quantitative PCR, high-resolution melting analysis and DNA microarray hybridization [DI*A and DI*B alleles are beneficial not only in transfusion medicine but also in human genetic research because the DI*A allele has been recognized as a genetic marker in biological anthropology among populations [DI*A and DI*B alleles in the Central Thai population and to compare them with those of other populations previously published.Because Di(b+) antigen is highly prevalent in different populations, anti-Dipatients , 14. Roudization –25. Thesulations , 27. ThePeripheral venous blood was collected in EDTA-anticoagulated blood from 1,011 unrelated healthy Thai blood donors from the National Blood Centre, Thai Red Cross Society, Bangkok, Thailand from May to December 2015, and the study was conducted until April 2016. Written informed consent was obtained from each subject. Samples demonstrating the presence of infectious markers such as syphilis, hepatitis B and C as well as HIV were not included in this study. This study was approved by the Committee on Human Rights Related to Research Involving Human Subjects, Thammasat University, Pathumtani, Thailand. Genomic DNA was extracted from peripheral blood samples using the Genomic DNA extraction kit  and then stored at -20°C until use for genotyping.Eleven known DNA samples of 1 Di(a+b-), 5 Di(a+b+) and 5 Di(a-b+) phenotypes were provided by Mr. Morakot Emthip, Histocompatibility and Immunogenetics Laboratory, National Blood Centre, Thai Red Cross Society and were used as controls. All samples were confirmed by DNA sequencing.a  was added to a test tube and then 1 drop of 2–3% RBC suspension in 0.9% normal saline (NSS) was added. Thereafter, one drop of 22% bovine albumin  was added. The test tube was mixed and incubated at 37°C for 30 min. The cells and serum mixture were washed 3 times with NSS and 2 drops of the antiglobulin serum  were added. After centrifugation, the reactions were read macroscopically and the agglutination reactions were graded as 4+, 3+, 2+, 1+ and w+. After reading the negative reaction under microscope, the IgG-coated RBCs were added to check the validity of the antiglobulin test. Negative, Di(a-b+) and positive, Di(a+b+) control cells were also tested in parallel. In addition, DAT was performed for all donor samples that gave positive results to rule out false positive results, if any. A total of 391 blood samples from Thai blood donors were tested for Dia antigen phenotyping.Briefly, 1 drop of human anti-DiDI*A and DI*B alleles was performed by PCR-SSP technique. Primers and amplification conditions were designed. Briefly, 1 μL of genomic DNA (50 ng/μL) was amplified in a total volume of 20 μL using 5 μM of DI-AB-F forward primer 5’-GGTGTGATAGGCACTGACCC-3’, 1 μL and 5 μM DI-A-R reverse primer 5’-GGGCCAGGGAGGCCA-3’, 1 μL for DI*A allele detection and 5 μM of DI-AB-F forward primer, 1 μL and DI-B-R reverse primers 5’-GGGCCAGGGAGGCCG-3’, 1 μL for DI*B allele detection. Co-amplification of the human growth hormone (HGH) gene using 6 μM HGH forward primer 5’-TGCCTTCCCAACCATTCCCTTA-3’, 1 μL, and 6 μM HGH reverse primer 5’-CCACTCACGGATTTCTGTTGTGTTTC-3’; 1 μL was run as the internal control and 5 μL of PCR grade water. The PCR was performed with 10 μL of 2X PCR reaction mixture  in a G-STORM GS1 thermal cycler .Genotyping of DI*A and DI*B alleles was 130 bp, whereas that of the internal control, the HGH gene was 434 bp.PCR was performed under the conditions described below, i.e., 95°C for 30 sec . The cycle parameters of the PCR program began with the first step of 10 cycles of 30 sec at 95°C and 60 sec at 69°C, then 20 cycles of 10 sec at 95°C, 50 sec at 62°C and 30 sec at 72°C. The last step was final extension 5 min at 72°C and the sample was kept at 4°C. PCR products were electrophoresed at 100 volts with 1.5% agarose gel using 1X TBE buffer and were visualized under blue-light transilluminator. The PCR product size of both DI*A and DI*B allele detection using PCR-SSP.Known DNA controls of Di(a+b-), Di(a+b+) and Di(a-b+) phenotypes were used as the controls. Altogether, 1,011 DNA samples of Thai blood donors were tested for DI*A/DI*A, 20 DI*A/DI*B and 15 DI*B/DI*B) was sequenced to confirm the results of PCR-SSP. A fragment of 598 bp containing both SNPs (c.2561C/T) was obtained from PCR amplification of genomic DNA using the forward primer 5’-TTAGGGGTCCAGCTCACTCA-3’ and reverse primer 5’-TGACCGCATCTTGCTTCTGT-3’ and using similar PCR conditions to genotype DI*A and DI*B alleles.Genomic DNA of 36 genotyped blood donors (one 2) test was used to evaluate whether the observed genotype frequencies agreed with the expected ones under the Hardy-Weinberg equilibrium. The χ2 test of homogeneity was used to determine the difference between Central Thai and those previously reported in other populations including 9 Asians: Han Chinese (Shanghai) [DI*A and DI*B allele frequencies were used to compute a matrix of Cavalli-Sforza chord distances for 9 Asian and 6 non-Asian populations [Gene frequencies were calculated by gene-counting method. The Chi-square (χhanghai) , Chinesehanghai) , Chinesehanghai) , Brazilihanghai) , Japaneshanghai) , Korean hanghai) , Korean hanghai) , Southeahanghai)  and Filihanghai)  and 6 nohanghai) , Brazilihanghai) , Italianhanghai) , Americahanghai) , Alaska hanghai)  and Hawahanghai) , respectulations , 23–25 bulations . A metria antigen among 391 Thai blood donors was studied. It was found that 13 of 391 (3.32%) were Di(a+) and 378 of 391 (96.68%) were Di(a-). Moreover, 1,011 DNA samples included 391 known Dia antigen phenotype samples and an additional 620 samples were genotyped for DI*A and DI*B alleles by in-house PCR-SSP. The DNA controls were tested with two sets of primer combinations and the results agreed. According to the interpretation of PCR-SSP, homozygous DI*A/DI*A and DI*B/DI*B samples were positive with only the set of DI-AB-F and DI-A-R primers and the set of DI-AB-F and DI-B-R primers, respectively. In addition, heterozygous DI*A/DI*B samples were positive with both sets of primers, as shown in The distribution of DiDI*A and DI*B genotyping among 391 known Dia antigen phenotype samples were computed to predicted phenotypes and were in agreement when compared with the phenotyping results by IAT. Additionally, 620 DNA samples were then tested for DI*A and DI*B genotyping using PCR-SSP. It was found that DI*B/DI*B was the most common , followed by DI*A/DI*B ; whereas, DI*A/DI*A was not found in this study. Moreover, validity testing of the in-house PCR-SSP was performed using thirty-six randomly chosen DNA samples and the genotyping results showed 100% concordance between PCR-SSP and DNA sequencing . Allele frequencies of DI*A and DI*B among Central Thais were 0.0183  and 0.9817 , respectively. DI*A and DI*B allele frequencies between Central Thai and other populations were compared. Allele frequencies were similar to Southeast Asian, Brazilian, southern Brazilian and American native populations [The determined ulations , 20, 25.ulations , 23–25, DI*A and DI*B allele frequencies of Central Thai blood donors were compared with 15 other blood donor populations examined in previous studies [DI*A and DI*B as shown in Regarding an analysis of a matrix of geometric genetic distances,  studies , 23–25. a antigen is notably high among Asians of Mongoloid origin like Japanese, Chinese and Korean populations and the DI*A allele has never been found in unmixed European and African descendants [DI*A and DI*B alleles among populations can provide information for those working with anthropology and transfusion medicine [a antigen phenotype was studied in 391 Central Thai blood donors and the prevalence of Di(a+) was 3.32%, similar to a recent study in the Thai population [b is not a marketed product. Moreover, in a previous study in Thailand, alloantibodies specific to the Diego blood group system, found in multitransfused patients were only anti-Dia (1.2%) consisting of a single antibody (1.08%) and combined with other antibodies (0.12%) [The prevalence of Dicendants . The stumedicine , 13, 14.pulation . Accordi (0.12%) . A posit (0.12%) , 16.a antigen phenotype samples, the predicted phenotypes were in 100% concordance with the phenotyping results by IAT. Validity of in-house PCR-SSP for DI genotyping was performed and confirmed by DNA sequencing. The results of DNA sequencing were consistent with determined genotypes by PCR-SSP. Because the homozygous DI*A/DI*A alleles were not found among 1,011 Central Thais, finding extended-matched donors for a patient with anti-Dib would be difficult. In this case, autologous or family-related donations are recommended. Moreover, screening for DI*A/DI*A individuals by PCR-SSP in a larger sample size may be helpful.After DI genotyping using in-house PCR-SSP was implemented in 391 known DiDI*B allele was the most common and its frequency was similar to those observed among other populations [DI*A and DI*B allele frequencies are significantly different and geometric genetic distance showed that Filipino was moderately related to Central Thais. This may be due to the combined effects of group isolations and cultural practices such as within-group marriages that may have influenced those group differentiations [DI*A and DI*B allele frequencies and geometric genetic distances were highly related to the Central Thai population. This may be because the analysis of a single gene is subjected large stochastic errors. Therefore, further studies of other relevant blood group alleles with larger sample sizes are required to confirm the relationships among populations.This is the first report of DI blood group allele frequencies in the Thai population. The ulations , 23–25. ulations , 20, 25.ulations . Althougtiations . Howevertiations . For theDI*A and DI*B allele frequencies, geometric genetic distances and MDS analysis of the Italian (Naples) populations were unrelated to the Central Thai and other Asian populations, similar to another study using the bootstrap tests with multiple genetic loci [Owing to the MDS analysis, East Asian populations including Japanese, Brazilian Japanese Descendant, Han Chinese (Shanghai), Korean (Seoul), Korean and Chinese (Chengdu) were moderately related to Central Thais and seemed to be all clustered together, which are consistent with population ethnicity and regions of residence. Notably, tic loci .DI*A and DI*B alleles between Central Thai and Southeast Asian populations was demonstrated. A PCR-based identification of DI genotyping should overcome some of the serological limitations in transfusion medicine and provides a complementary tool for further population-genetic studies.In conclusion, the similar prevalence of


The brain stem contains important nuclei that control cardiovascular function via the sympathetic nervous system (SNS), which is strongly influenced by nitric oxide. Its biological activity is also largely determined by oxygen free radicals. Despite many experimental studies, the role of AT1R-NAD(P)H oxidase-superoxide pathway in NO-deficiency is not yet sufficiently clarified. We determined changes in free radical signaling and antioxidant and detoxification response in the brain stem of young and adult Wistar rats during chronic administration of exogenous NO inhibitors.G-nitro-L-arginine-methyl ester , a nonspecific NOS inhibitor, and with drinking water (Control group) during 6 weeks. Systolic blood pressure was measured by non-invasive plethysmography. Expression of genes  was identified by real-time PCR. NOS activity was detected by conversion of [3H]-L-arginine to [3H]-L-citrulline and SOD activity was measured using UV VIS spectroscopy.Young (4 weeks) and adult (10 weeks) Wistar rats were treated with 7-nitroindazole , a specific nNOS inhibitor, with NWe observed a blood pressure elevation and decrease in NOS activity only after L-NAME application in both age groups. Gene expression of nNOS (youngs) and eNOS (adults) in the brain stem decreased after both inhibitors. The radical signaling pathway triggered by AT1R and p22phox was elevated in L-NAME adults, but not in young rats. Moreover, L-NAME-induced NOS inhibition increased antioxidant response, as indicated by the observed elevation of mRNA SOD3, HO-1, AT2R and MDR1a in adult rats. 7-NI did not have a significant effect on AT1R-NADPH oxidase-superoxide pathway, yet it affected antioxidant response of mRNA expression of SOD1 and stimulated total activity of SOD in young rats and mRNA expression of AT2R in adult rats.Our results show that chronic NOS inhibition by two different NOS inhibitors has age-dependent effect on radical signaling and antioxidant/detoxificant response in Wistar rats. While 7-NI had neuroprotective effect in the brain stem of young Wistar rats, L-NAME- induced NOS inhibition evoked activation of AT1R-NAD(P)H oxidase pathway in adult Wistar rats. Triggering of the radical pathway was followed by activation of protective compensation mechanism at the gene expression level.The online version of this article (doi:10.1186/s12929-017-0366-4) contains supplementary material, which is available to authorized users. The sympathetic nervous system (SNS) is one of the autonomic nervous pathways with a dominant role in the regulation of short- and long-term blood pressure. SNS regulates heart rate, contractility (systolic volume) and vasoconstriction on the periphery through adrenergic receptors . ExperimThe SNS activity is strongly influenced by nitric oxide (NO) produced in the nuclei of the brain stem: nucleus tractus solitarii (NTS), and rostral ventrolateral medulla (RVLM) .2+- dependent neuronal NOS  primarily expressed in neurons and glial cells, endothelial NOS  present in endothelium, platelets and cardiomyocytes (both in low nanomolar concentrations) -L-Arginine  to 3H–L-Citrulline  with sma4 cells per well and incubated for 3 days. After seeding, the cells were treated with NO inhibitors L- NAME (100uM) and 7-NI (50uM) during 24 h. After treatment, the cells were stained with 10uM DAF-FM diacetate (30 min in 37 °C in CO2 incubator), rewashed by DMEM buffer (without FBS and phenol red) and measured by fluorescence miscroscope exc/em 488/510 nm. Image J program was used for image analysis.Indicator for nitric oxide determination: 4,5-diaminofluorescein diacetate . The non-fluorescent dye is pass across cell membrane. The cell’s estherase activity turns the non fluorescent dye into a weakly fluorescent form and nitric oxide binds to this intracellular dye and increases the fluorescence activity in cells. The effect of NO inhibitors (7-NI and L-NAME) on NO production in the SH-SY5Y cell model was detected using an inverted fluorescence microscope (NIKON Eclipse Ti-E) and the NIS Elements AR program. The neuroblastoma cells of the SH-SY5Y line containing PgP and MRP proteins were seeded into 24-well plates, with 6 × 10Total SOD activity was analyzed by the SOD Assay Kit (Fluka) in 0.5% homogenates in Tris-HCl with the addition of protease inhibitors according to the manufacturer’s protocol. Activity of SOD was measured as inhibition of production of formazan (WST-1-formazan) from tetrazolium salt (WST-1), which reacts with superoxide anions. Samples were incubated 20 min at 37 °C. Absorbance was measured on a spectrophotometer (Thermo Scientific Multiskan FC) at 450 nm. The resulting values were calculated using a standard curve and expressed as U / mg (Unit / milligram) of protein.The total RNA from the brain stem samples (50–100 was isolated by TRIsure reagent (Bioline) according to the manufacturer’s protocol. Isolated total RNA was quantified spectrophotometrically at 260/280 nm on Nanodrop 2000c (ThermoSci). Purity of the RNA was evaluated by the ratio between the absorbance values at 260 nm and 280 nm (A260 / A280). We worked with the pure RNA with the ratio of the optical densities in the range 1.9 to 2.1. TetrocDNA Synthesis kit (Bioline) was used for the reverse transcription and SensiFAST SYBRNoROX kit (Bioline) was used for the real-time polymerase chain reaction (PCR) according to the manufacturer’s protocol. Amplification of the cDNA was performed on a BioRad CFX96 Realtime system. All primers (100 pmol/ul) used for amplification of the studied genes  were used according to Dovinova et al. . Primer extremevalues package [glht method from the R’s multcomp package [p-values were calculated using the Westfall-Young maxT free step-down permutation algorithm to account for the large number of comparisons, correlations among comparisons and slightly non-gaussian distribution of model residuals [Data of adult and young animals were processed separately. The gene expression data for a whole set of genes were processed by fitting a 3-factor linear model (3- way ANOVA) using a script written in R . The mod package , with de package  to calcuesiduals .Only chronic administration of L-NAME changed systolic blood pressure in young and adult Wistar rats. Blood pressure was increased in L-NAME experimental groups at the end of experiments. Inhibition of neuronal NOS by 7-NI did not invoke this increase in any group Table . In younGene expression of mRNA(messenger RNA) nNOS was decreased in young Wistar rats after administration of 7-NI and L-NAME. On the contrary, significant increase was observed in adults after L-NAME treatment. We observed no changes after 7-NI treatment. On the other hand, we observed changes in gene expression of mRNA eNOS only in adults, where expression significantly declines after both types of inhibitors Fig. .Fig. 1EfWe observed that mRNA AT1R was overexpressed in adult rats treated with L-NAME Fig. , while tThe compensative effect of AT2R was also observed in adult rats. We found a significant increase of this gene in L-NAME (63.5%) and 7-NI (57%) group.There was no age-dependent effect on mRNA of AT1R/NADPH oxidase Table .Table 2TP < 0.05, L-NAME vs. control, #P < 0.05, 7-NI vs. L-NAME. 7-NI (7-nitroindazole), L-NAME (NG- nitro–L–arginine methyl esther), GAPDH , AT1R (angiotensin receptor 1).Expression of mRNA of AT1R and, p22phox subunits of NAD(P)H oxidase was normalized on expression of GAPDH in brain stem. Data represent mean ± std. dev., *In the case of gene expression of individual SOD isoforms, we noticed major changes in SOD1 expression only in young rats. We observed that 7-NI and L-NAME significantly decreased expression of SOD1 and treatment with 7-NI markedly attenuates this expression compared with L-NAME Fig. . DiffereActivation of Nrf2 was detected through HO-1 expression. mRNA expression of HO-1 was normalized to “housekeeper” GAPDH. In adult rats we found stimulation of HO-1 gene expression significantly increased only in the L-NAME group . 7-NI did not influences HO-1 expression, however a difference in 7-NI and L-NAME group  was observed.An age-dependent effect on mRNA SOD isoforms has been observed. We found changes among young and adult rats in mRNA SOD2 and SOD3, while SOD1 was not affected .p < 0.05)  and in L-NAME, a significant decrease of 34,3% ,while adult rats achieved the level of high blood pressure characteristic for hypertensive animals  (Tab. 1). These changes in blood pressure were associated with differences in other measured parameters  in young and adult rats.7-nitroindazole is used as a specific blocker of neuronal NOS at certain concentrations . In our Several studies have shown that NOS inhibition stimulates the presence of oxidative stress. The administration of L-NAME is often associated with increased production of secondary products of oxidative stress –80. A shOur results show that stimulation of the radical signaling pathway through AT1R-NAD(P)H-oxidase is different for various age groups and NOS inhibitors. This radical pathway in the brain stem is not significantly influenced in young rats Fig. , while iG-nitro-L-arginine (L-NNA) stimulated SOD activity, but not catalase activity in brain [2∸ levels, protecting endogenously produced NO at a sufficient level to maintain cerebral vascular tone and reactivity. SOD3 was found to increase the vasodilatory effect of endogenously produced NO in the brain [NOS inhibition affects oxidative stress in the brain and also induces changes in the antioxidant response. Previous studies showed changes in the total plasma activity of superoxide dismutase after L-NAME treatment , 95. Chrin brain . These din brain –102, whein brain , 104. Itin brain .We obserin brain . In normhe brain . Stimulahe brain , 38, 39.Cytoprotective responses in brain were observed through the Nrf2 activation pathway (detoxification phase II) and/or MDR1 pathway (detoxification phase III). The Nrf2/ Keap1/ARE signaling pathway is an important regulator of cellular oxidants and electrophile stress through induction of antioxidant and detoxification genes such as SOD3 and HO-1 . ActivatMDR1a and MDR1b genes in rodent brain, but only MDR1a is localized in brain capillaries. This efflux transporter mediates the export of drugs from the blood-brain barrier (BBB), where it prevents many drugs from entering into the CNS [MDR1a genes in the brain stem of young animals  used in our study is a commonly used method for studying NO-deficient hypertension. Direct action of the pharmacological agents on brain were expected based on chemical ADMET  properties of 7-nitroindazole and N(G)-Nitro-L-arginine methyl esther. The ADMET structure-activity relationship server (admetSAR) is a comprehensive knowledge base and a tool for predicting ADMET properties of drug candidates . ADMET PThe effect of orally administered NO inhibitors in brain stem critically depends on their ability to cross the blood brain barrier (BBB). The BBB protects the brain from potentially harmful substances  and is fhttp://www.simulations-plus.com/software/admet-property-prediction-qsar/) provides predictions of BBB crossing probabilities for drugs and other substances. For L-NAME and 7-NI, it predicts BBB crossing probabilities of 72.1% and 97.45%, respectively.The ADMET database  has age-dependent effect on radical signaling (AT1R-NAD(P)H oxidase pathway) and antioxidant (Nrf2 activation) and detoxification (MDR1a transporters) response in Wistar rats. In young rats, major effect wasobserved in antioxidant response (SOD activity) and expression of AT2R induced by 7-NI. This stimulation of antioxidant system suggests aneuroprotective effect of 7-NI in brain stem of young Wistar rats. In adult rats, treatment with L-NAME led to activation of AT1R-NAD(P)H oxidase pathway following stimulation SOD3 expression as a protection of NO in brain stem, AT2R expression as a protection against brain damage;other lines of the defense system were activated as well Nrf2-ARE pathway and efflux transporter. Our data suggest that changes of radical signaling, antioxidant and detoxification response can be important in the development of hypertension.


DNA orientation-specific adhesion and patterning of living mammalian cells on self-assembled DNA monolayers. e.g. poly-l lysine, PLL) substrates. Furthermore, we could manipulate cell adhesion by tuning the length of poly adenine (polyA) in the DNA sequence. We also prepared a DNA aptamer-based SAM to regulate cell adhesion by exploiting stimuli-responsive conformational change of the aptamer. By using the well-established DNA spotting technology, we patterned cells on DNA-SAMs to form a spot matrix and four English letters “CELL”. Our findings suggest that DNA-SAMs on gold substrates are potentially useful for making smart surfaces for cell studies, thus introducing a new platform for cell/tissue engineering research.To better understand cell behaviors on substrates, the precise control of density and orientation of cell-specific ligands remains a great challenge. In this study, we established an easy-to-use approach to manipulate the adhesion and patterning of mammalian cells on gold substrates. We prepared DNA self-assembled monolayers (DNA-SAMs) on gold substrates and found that the sequence-specific orientation of DNA-SAMs played an important role in modulating cell adhesion. We also found that the DNA-SAMs on gold substrates could be used as a potentially universal cell culture substrate, which showed properties similar to cationic polymers ( Arg-Gly-Asp, RGD peptide) or growth factor (e.g. fibroblast growth factor 2) have proven to be successful for cell adhesion with improved orderliness.e.g. cell-specific aptamers) to capture and manipulate cells.Self-assembled monolayers (SAMs) hold great promise for controlling molecular structures on surfaces. Prior efforts in fabricating SAMs incorporating ECM-derived biomolecules  were self-assembled on gold and were then co-assembled with a hydrophilic synthetic polymer, (2-[2-(1-mercaptoundec-11-yloxy)-ethoxy]-ethanol) (SH-OEG). SH-OEG modified substrates are well known to be non-fouling, surface-resistant to the adhesion of proteins or cells. Indeed, when the SH-OEG modified substrate was incubated overnight with a human breast adenocarcinoma cell line, MCF-7, in culture medium containing 10% v/v fetal bovine serum (FBS), we did not observe any cell adhesion on this surface . In conte.g. poly-l lysine, PLL) are often employed to coat substrates for cell culture in biological studies. However, DNA is an anionic polymer in nature. To explore the properties of cells grown on these reversely-charged substrates, we studied MCF-7 cells grown on T20-based SAMs and the commonly used PLL-coated substrates. Cells adhered on substrates were stained with phalloidin–TRITC (for actin) and Hoechst 33258 (for cell nuclei), and then imaged using a fluorescence microscope. As shown in Fig. S1,Cationic polymers  and β-actin proteins at constant levels in normal cells. Western blot analysis demonstrated that the expressed protein levels of GAPDH and β-actin expressed in adhered cells on DNA-SAM and PLL substrates were nearly the same Fig. S2, which pHaving established that DNA-SAMs provide a new type of substrate for cell adhesion, we attempted to pattern cells on substrates by exploiting the convenience in spotting DNA arrays of arbitrary shapes. We spotted T20 on a gold substrate to form a spot matrix. When MCF-7 cells were seeded on this substrate, we observed that cells specifically adhered to the spotting areas with T20, forming an array of cells exhibiting green fluorescence of phalloidin–TRITC that is stained on actin . As a fuFurthermore, we started to investigate DNA SAMs assembled with thiolated A20. Previous studies have established that consecutive adenines possess greater affinity on gold as compared to other base combinations.To further substantiate the observation that the orientation of DNA SAMs exerts a great influence on cell adhesion, we varied the length of non-adsorbed polyT to co-assemble with SH-OEG. Our rationale is that short polyT would be embedded in the OEG layer, resembling surface-adsorbed polyA, whereas long polyT would protrude out of the OEG layer, allowing efficient cell adhesion. A series of polyT of different lengths  of the same concentration was co-assembled with SH-OEG on gold. As shown in Furthermore, we studied the effect of DNA density on cell adhesion. T20 with different concentrations  was co-assembled with SH-OEG on gold. When the assembly concentration of T20 was higher than 100 nM, we observed significant adhesion of MCF-7 cells with comparable amount to each other Fig. S6. RemarkaOur extensive studies on the effects of DNA density, length and sequence on the adhesion properties of cells inspired us to manipulate the cell adhesion by programmably tuning the length of poly adenine (polyA) in the DNA sequence, through which we expected to tune the cell adhesion on DNA SAMs. We inserted different lengths of polyA (from 0 to 80 nt) between thiol group and random sequence, which were used to prepare DNA SAMs  interactions.Because we can finely modulate cell adhesion by changing the density, length and sequence of DNA, we further explore whether we can use these DNA-SAMs to control cell adhesion molecules (CAMs) that are In this study, we have demonstrated that DNA-SAMs can be used as a type of facile and highly tunable substrate for cell culture. Furthermore, we can manipulate cell adhesion by tuning the length of poly adenine (polyA) in the DNA sequence and, in turn, the cell adhesion on the DNA SAM can be programmably regulated. We also prepared a DNA aptamer-based SAM to regulate cell adhesion by exploiting stimuli-responsive conformational change of the aptamer. These DNA-SAM-based substrates showed comparable properties for cell adhesion and growth to commercially available PLL substrates. Our extensive studies on the effects of DNA density, length and sequence on the adhesion properties of cells reveal that DNA orientation on gold plays an important role in modulating cell adhesion. This provides a versatile approach to manipulate the adhesion of cells on substrates, which is potentially useful for making smart surfaces for cell studies. We also demonstrated the ability to precisely control the density of RGD, which should be readily adaptable in cell/tissue engineering. By taking advantage of the well-established DNA spotting technology, we can readily form microarrays of living cells with predefined patterns, which is useful for manipulating the growth and differentiation of cells and realizing artificial extracellular matrix and even tissues/organs on chips.11-EG2) was purchased from Prochimia. SH-PEG (MW 5000) was purchased from Laysan Bio. Inc. Mercaptohexanol (MCH), Hoechst 33258, phalloidin–tetramethylrhodamine B isothiocyanate (TRITC) and SMCC were purchased from Sigma. Cyclic peptide RGDfK-NH2 was purchased from Peptides International, Inc.DNA oligonucleotides were purchased from Takara (purified by HPLC). OEG 2-[2-(1-mercaptoundec-11-yloxy)-ethoxy]-ethanol (HS-CDNA sequences are as follows:SH-A20: SH-AAAAAAAAAAAAAAAAAAAASH-T20: SH-TTTTTTTTTTTTTTTTTTTTSH-G20: SH-GGGGGGGGGGGGGGGGGGGGSH-C20: SH-CCCCCCCCCCCCCCCCCCCCSH-random sequence: SH-GTGTCGTGCCTCCGTGCTGTGSH-DNA: SH-CACAGCACGGAGGCACGACACSH-A10-random: SH-AAAAAAAAAAGTGTCGTGCCTCCGTGCTGTGSH-A20-random: SH-AAAAAAAAAAAAAAAAAAAAGTGTCGTGCCTCCGTGCTGTGSH-A30-random: SH-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGTGTCGTGCCTCCGTGCTGTGSH-A40-random: SH-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGTGTCGTGCCTCCGTGCTGTGSH-A80-random: SH-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGTGTCGTGCCTCCGTGCTGTGSH-ATP aptamer: SH-ACCTGGGGGAGTATTGCGGAGGAAGGT2 (pH = 8.0). All solutions were prepared with deionized water.TM buffer was prepared from 10 mM of Tris and 5 mM of MgCll-glutamine (2 mM) at 37 °C in humidified environment containing 5% CO2. For different cells, different media were used. MCF-7 was cultured with RPIM 1640; HeLa, PC12 and HacaT were cultured with DMEM; CHO was cultured with F12-K.Cells were cultured in medium supplemented with 10% fetal bovine serum, penicillin/streptomycin (100 units per mL) and Gold films were cleaned by sonicating in ethanol and water for 15 min, respectively, followed by irradiation under UV for 15 min to make them asepsis. 3 μM of thiol-modified DNA in TM buffer was added on the gold surface for 4 h for immobilization. After removing surplus liquid, the gold surface was immersed in 4 mM of OEG for 4 h, so that the gold surface that was not covered by DNA was grafted with a monolayer of OEG. For cell experiments, the gold films were washed extensively with PBS.For cell pattern experiments, a DNA microarrayer was used to spot SH-T20 on gold substrate with a distance of 500 μm for four English letters: “CELL”. After assembly for 4 h, gold substrates were passivated with OEG.2 and SH-DNA were first coupled through SMCC . After annealing with 5′-thiol-labeled complementary DNA strands (SH-random sequence) in TM buffer, the product was added on the gold surface to assemble.For RGD-DNA-SAMs, RGDfK-NHIn the experiments of controlled cell adhesion by ATP, 1 μM thiol-labeled DNA was incubated at 37 °C with or without the presence of 1 mM ATP for half an hour. Then DNA was grafted on the gold surface for an hour followed by passivation with SH-PEG5000 for 4 hours to perform cell experiments.DNA was assembled on the gold surface as described above. Briefly, 3 μM of Cy3-labeled thiol-modified DNA in TM buffer was added on the surface for 4 h for immobilization. After passivation in 4 mM OEG for 4 h, the surface was imaged with fluorescence microscopy (Leica epi-fluorescence with EMCCD).4 cells per well for HeLa, MCF-7 and 10 × 104 cells per well for PC12, HacaT and CHO. For the ATP aptamer experiment, MCF-7 cells were seeded either with or without 1 mM ATP. At specific time points, cells were washed three times with pre-warmed PBS, and fixed with paraformaldehyde/sucrose (4% w/v) in PBS at room temperature for 15 min. Then cells were washed with PBS and permeated with TritonX-100 (0.1%) in PBS for 15 min. After blocking with BSA (1%) for 30 min, the actin cytoskeleton was labeled with 1 μg mL–1 of phalloidin–TRITC for 20 min and cell nuclei were labeled with Hoechst 33258 for 10 min at room temperature. Finally, the cells were washed extensively with PBS before observation by microscopy.Cells were seeded on PLL-coated coverslips or DNA self-assembled monolayers in 24-well culture plates at a density of 6 × 10To study the process of cell adhesion on the DNA surface, cells were fixed at various time points  after seeding.MCF-7 cells were seeded on DNA-SAMs and PLL-coated substrates and cultured overnight. Cells were then harvested and cell numbers counted. For protein extraction, equal numbers of cells were used. Whole lysate was resolved by SDS-PAGE, transferred onto PVDF membranes. GADPH and actin were immunoblotted by specific primary antibodies and secondary antibodies (Santa Cruz).Cell numbers were determined by counting cell nuclei in the 200× magnified field of view and projected cell areas were determined based on an algorithm using ImageJ. Data are presented as mean ± SD.Supplementary informationClick here for additional data file.


Several of the figures in the published article had been generated using bands or after carrying adjustments to the original blots. The authors are issuing this Correction to provide the following corrected figures:Figure 1: Duplicate bands from the original blot were cut to generate the figure. We report the entire blot for IL-8 at 6 h and at 24 h, indicating the different concentrations of CSE in duplicate. Since the bands increased, two panels have been added instead of one (Panel A: 6 h and panel B: 24 h). The revised figure legend is as follows:'Figure 1. Effects of Cigarette smoke extract (CSE), alone and in combination with lycopene, on IL-8 production in human THP-1 cells. Panels A-E: intracellular IL-8 production; panel F: IL-8 production in culture medium. Panel A: effects of different CSE concentrations for 6 h, panel B: effects of different CSE concentrations for 24 h; panels C, D, E: effects of a pre- treatment for 6 h with lycopene (2 mM) followed by a 24-h CSE (0.5%) exposure; panel D: effects of a pre-treatment with different concentrations of lycopene followed by a 24-h CSE (0.5%) exposure; Panels A, B, C, D: representative Western Blot analyses; the values indicated represented the ratio of IL-8 and actin. Panel E: mRNA levels by reverse transcription polymerase chain reaction.Panel F: Chemiluminescence Immunometric Assay. In the different panels, the values were the means ± SEM of three independent experiments. Values not sharing the same letter were significantly different .'Figure 2D. In the published figure, the bands for the different raw blots  had been cut and pasted to align the bands in the figure, we attach the original blot.Figures 2C, 3C, 5B, 5D and 6D. The published figures were processed to eliminate background, we report the original blots.Figure 5C. In the published figure the actin bands were rotated, we present the corrected figure. Updated Versions of Figures:


Insulin and insulin-like growth factors I and II are closely related protein hormones. Their distinct evolution has resulted in different yet overlapping biological functions with insulin becoming a key regulator of metabolism, whereas insulin-like growth factors (IGF)-I/II are major growth factors. Insulin and IGFs cross-bind with different affinities to closely related insulin receptor isoforms A and B (IR-A and IR-B) and insulin-like growth factor type I receptor (IGF-1R). Identification of structural determinants in IGFs and insulin that trigger their specific signaling pathways is of increasing importance in designing receptor-specific analogs with potential therapeutic applications. Here, we developed a straightforward protocol for production of recombinant IGF-II and prepared six IGF-II analogs with IGF-I-like mutations. All modified molecules exhibit significantly reduced affinity toward IR-A, particularly the analogs with a Pro-Gln insertion in the C-domain. Moreover, one of the analogs has enhanced binding affinity for IGF-1R due to a synergistic effect of the Pro-Gln insertion and S29N point mutation. Consequently, this analog has almost a 10-fold higher IGF-1R/IR-A binding specificity in comparison with native IGF-II. The established IGF-II purification protocol allowed for cost-effective isotope labeling required for a detailed NMR structural characterization of IGF-II analogs that revealed a link between the altered binding behavior of selected analogs and conformational rearrangement of their C-domains. The insulin-insulin-like growth factor (IGF)3–Both IGFs are essential for embryonic development and are present in serum at nanomolar concentrations in adults  with IGF17–IGF-I and IGF-II are single chain peptides composed of 70 and 67 amino acids, respectively. Mature IGFs consist of four domains: B, C, A, and D in order from the N terminus. IGF-I and -II share over 60% sequence identity, mostly in the B- and A-domains that correspond to the B and A chains in insulin . The 3D 24–IR-A, IR-B, and IGF-1R are homodimeric, and each monomer consists of an extracellular subunit (α) and transmembrane subunit (β) that are linked via four disulfide bonds into a functional β-α-α-β homodimer –40. The –41–44–51–Both insulin and IGF-I have been extensively studied through the preparation and functional analysis of numerous analogs  (−1 of culture) of IGF-II analogs with only a single additional glycine residue cloning artifact at the N terminus. The fusion protein was successfully expressed in E. coli and purified using immobilized metal ion affinity chromatography . Two major peaks were observed; the first eluted at lower concentrations of imidazole  and consisted of folded and misfolded monomeric IGF-II with slightly different migration of two bands observable in non-reducing SDS-PAGE . The second peak eluted at higher concentrations of imidazole and consisted of multimeric forms . Both monomeric and multimeric fusion proteins were subsequently cleaved using TEV protease under redox conditions of oxidized and reduced glutathione. Interestingly, the moderate reducing environment triggered disulfide bond reshuffling that resulted in liberation of monomeric IGF-II from multimeric aggregates. Following cleavage, IGF-II was separated from the His6-tagged GB1 and TEV by immobilized metal ion affinity chromatography. RP-HPLC of this crude IGF-II product consisted of one major peak and two to four minor peaks . The retention time of the major protein peak was nearly identical to that observed for native human IGF-II, and the correct molecular weight of the recombinantly produced purified IGF-II protein with formed disulfide bonds was confirmed by high resolution mass spectrometry. Both forms, monomeric and multimeric, yielded the desired product of correct mass and were combined after the correct protein fold was confirmed by 1D 1H NMR  and 1H-15N HSQC that was highly similar to the previously published data  , 64. Thihed data .29 for Asn ([N29]IGF-II), (ii) an insertion of Gly-Ser after Arg34 ([R34_GS]IGF-II), (iii) an insertion of Pro-Gln after Ser39 ([S39_PQ]IGF-II), (iv) a combination of both insertions , (v) a combination of S29N mutation with Pro-Gln insertion , and (vi) a combination of S29N mutation with both insertions . All analogs gave comparable RP-HPLC elution profiles (data not shown) with that of IGF-II  with one major product and several minor peaks. The characterization of minor by-products was prevented by their relatively low yields.In total, six IGF-II analogs were designed to determine the effects of IGF-I motif incorporation into IGF-II. The modifications were as follows: (i) a point mutation at position Ser1H NMR and far-UV circular dichroism as illustrated in supplemental Figs. S2 and S3. The CD spectra obtained for prepared analogs are similar to the broadly α-helical secondary structure profile obtained for non-modified IGF-II. The presence of the expected tertiary structure was further confirmed by 1D 1H  NMR spectra, and each analog compared well with the native IGF-II profile.The structural integrity of the six analogs was confirmed using supplemental Figs. S4–S6.The binding affinities of the IGF-II analogs toward human IR-A and IGF-1R together with binding affinities of selected analogs to IR-B are summarized in All modifications led to a significantly impaired IR-A binding, ranging from 4.2 to 1.1% of the insulin affinity when compared with IGF-II (7.9%). The [N29]IGF-II B-domain mutant gave a 2-fold reduction in IR-A affinity, whereas the analogs with C-domain insertions exhibited stronger negative effects. [R34_GS]IGF-II showed an almost 3-fold reduction in binding (2.8%), whereas [S39_PQ]IGF-II showed an 8-fold reduction. All of the analogs bearing the Pro-Gln motif were significantly less active (1.1–1.8%), and further combinations did not appear to have any additive effect.An insertion of IGF-I-like features, S29N, Gly-Ser, Pro-Gln alone, or a combination of Gly-Ser and Pro-Gln, within the IGF-II molecule led rather unexpectedly to a moderate decrease of binding potency toward IGF-1R . Howeverm; m).Both reference molecules, commercial IGF-II and our recombinant IGF-II, show similar binding potency for IR-B compared with IGF-I  and IGF-II (with decreased IR-A and enhanced IGF-1R binding)  for NMR 15N- or 13C/15N-labeled IGF-II analogs to unlabeled D11 led to a significant line narrowing of the NMR signals as illustrated in supplemental Fig. S7 despite the more than a 2-fold increase in the total molecular mass of the system. First, we determined the structure of the D11-bound unmodified IGF-II that was utilized in the structural analysis of IGF-II analogs. As expected, it is highly similar to the previously published structure . Although significant chemical shift perturbations were observed over the A-domain and the first 75% of the B-domain, the regions containing the mutations showed very small or negligible chemical shift perturbations.Undesirable dynamic and aggregation behavior of IGF-II severely affects the quality of NMR spectra of this protein and would prevent the accurate structural determination required for a detailed comparison between these analogs. Previously, it has been shown that upon binding to an engineered high affinity Domain 11 (D11) of the IGF-2R the spectral properties of IGF-II improve dramatically . The factructure  with som39 led to increased conformational freedom within the C-loops of both analogs that generated a rearrangement stabilized by several new packing interactions in the remote part of the C-domain. In the native IGF-II sequence, Tyr27 points away from the C-loop and forms hydrophobic contacts with Ala61, whereas the C-loop is unrestrained by additional contacts to the other parts of IGF-II (A). By contrast, the aromatic ring of Tyr27 forms contacts to the methyl group of Ala32 in [S39_PQ]IGF-II (B) and Arg30 and Pro31 in IGF-II (C). Arg30 is no longer unrestrained in these analogs and interacts with the aromatic ring of Tyr61 (Tyr59 in unmodified IGF-II) via a cation-π interaction. These new hydrophobic contacts lead to the formation of a better defined C-loop that bends around the bulky side chains of Tyr27 and Tyr61 of both C-domain-modified analogs (B and C). In comparison with unmodified IGF-II, the extended C-domain in both analogs is spatially constrained and bent toward the triad of aromatic residues at the C terminus of the B-domain . Ser29 in IGF-II (A) is located at the hinge of the semiflexible loop with no significant contacts to neighboring residues. The Pro-Gln extension in [S39_PQ]IGF-II led to the repositioning of Ser29 in close proximity to Tyr27, although there are no observed NOE contacts between Ser29 protons and Tyr27 or surrounding residues. However, the hydroxyl proton from its side chain may be involved in hydrogen bonds, e.g. with the backbone carboxyl groups either from Pro31 , which is closer in the extended loop, or from Arg42 (<2.8 Å in a quarter of the structures) at the opposite side of the loop  that were not observed for the [S39_PQ]IGF-II analog.Both analogs, [S39_PQ]IGF-II and IGF-II, preserved their overall structural organization with the three highly conserved α-helices further stabilized by three disulfide bonds. As expected, the D11 binding interface on the IGF-II analogs was not perturbed by the modifications, and structural changes were restricted to the modification sites . In bothf IGF-II A. By conQ]IGF-II B and ArgQ]IGF-II C. Arg30  analogs , B and Cn IGF-II A is locathe loop B and 5. 30/Pro31 C that wedK ∼ 3 nm; m; in vivo , which may affect the structure of the C-domain main chain and therefore binding to IR-A.Our initial goal to reduce IR-A affinity of IGF-II was successful as all six IGF-II analogs showed reduced IR-A binding  with fouAlthough we only tested a single analog for binding to IR-B, the 2-fold reduction in binding observed for [N29]IGF-II suggests a similar sensitivity to changes in the C-domain . Hence, e.g. IGF-I Tyr31 . Non27 and Ala32 and a hydrogen bond between Ser29 and Pro31 or Arg42 in [S39_PQ]IGF-II (B and 31 packing against Tyr27 (C). A comparable decrease in IR-A binding affinities of [S39_PQ]IGF-II and IGF-II in comparison with IGF-II indicates it is caused mainly by their similarly altered C-loop structures rather than S29N mutation, which is well tolerated by IR-A.The comparison of D11-bound structures of IGF-II, [S39_PQ]IGF-II and IGF-II, revealed that both analogs differ from IGF-II in the orientation and structuring of their C-loops  and 4. TQ]IGF-II B and 5. st Tyr27 C. A comp1GZR) . Asn26 is at the C terminus of the IGF-I B-domain, which is structurally altered upon binding to IGF-1R or IR (cyan). Analogous structural events are observed upon insulin binding to IR , Asn26 is the last IGF-I B-domain residue resolved in the complex with the hybrid IGF-IR/IR where it has been captured in the binding site formed from the IGF-IR α-CT and IR L1 domains . However, the structure of the complex did not reveal any specific contacts between IGF-I Asn26 and IR L1 domain or IGF-IR α-CT. However, it cannot be ruled out that Ser29 within the IGF-II molecule  may be involved in direct contacts to IGF-1R, and this hypothesis could be supported by a positive effect of S29N mutation on IGF-1R binding affinity of IGF-II. Hence, Ser29 may represent an important site for engineering of the IGF-1R binding specificity in IGF-II analogs.In the crystal structure of human IGF-I (Protein Data Bank code 1GZR) , the Asnd C-loop A. Asn26 1R or IR  Fig. 6,,1GZR . An additional N-terminal Gly (−1) was incorporated to facilitate TEV cleavage. Mutation S29N, Gly-Ser insertion following Arg34, Pro-Gln insertion following Ser39, and a combination of both insertions were obtained by site-directed mutagenesis  performed with appropriate mutagenic primers of the IGF-II sequence subcloned into the pBluescript vector. After sequence verification, the mutant fragments were reintroduced into the full-length IGF-II in the expression vector. Constructs were transformed into E. coli BL21(λDE3) and cultivated using LB medium or minimal medium containing [15N]ammonium sulfate and d-[13C]glucose. The bacterial culture was grown at 37 °C to an optical density (550 nm) of ∼1, induced with 1 mm isopropyl β-d-1-thiogalactopyranoside, and further cultured for 4–5 h. Cells were harvested by centrifugation for 20 min at 4,000 × g, and cell pellets were stored at −20 °C prior to further processing.The human IGF-II sequence was cloned into a modified pRSFDuet-1 expression vector fused with an N-terminal Hism Tris-HCl, pH 8.0, 50 mm NaCl, 5 mm EDTA, 50 μm PMSF) using 10 ml of buffer/1 g of biomass and homogenized by three passes through an Avestin EmulsiFlex-C3® apparatus at 4 °C and homogenization pressure of 1,200 megapascals. Inclusion bodies from the cell lysate were obtained by centrifugation at 20,000 × g at 4 °C for 20 min and further washed as a suspension in a wash buffer  with 0.1% (v/v) Triton X-100, sonicated in an ice bath, and centrifuged . The wash procedure was repeated in the absence of 0.1% (v/v) Triton X-100, and wet paste consisting of inclusion bodies was stored at −20 °C.Cells pellets were resuspended in lysis buffer  after the following dilution step. The suspension was gently diluted into 50 mm Tris-HCl, pH 8.0 buffer with 300 mm NaCl and 8 m urea to a final concentration of ∼1 g (wet weight of inclusion bodies)/50 ml and incubated for 2–3 h at room temperature with moderate stirring. The solution of the denatured fusion protein was then loaded onto an equilibrated HisTrap HP (5 ml) column connected to an ÄKTA FPLC® system , and after washing with 50 mm Tris-HCl, pH 8.0 buffer with 300 mm NaCl, the retained protein was eluted using a 0–500 mm imidazole gradient in 50 mm Tris-HCl, pH 8.0 buffer with 300 mm NaCl within 10 column volumes. The presence of the fusion protein in collected fractions was verified by SDS-PAGE and anti-His6 Western blotting, and the pooled fractions were dialyzed at 6 °C against 50 mm Tris-HCl, pH 8.0, 300 mm NaCl. The fusion partner was subsequently cleaved by an overnight TEV digestion in the presence of reduced and oxidized glutathione (1.5 mm GSH and 0.15 mm GSSG) at room temperature. Cleaved IGF-II was separated from the fusion protein by a gravity flow nickel chelating chromatography  and further desalted on a Chromabond C4 column (Macherey-Nagel) using 80% CH3CN (v/v) with 0.1% TFA (v/v) for elution. The collected protein fraction was lyophilized; resuspended in 7% (v/v) acetic acid, 27% (v/v) CH3CN, 0.03% TFA (v/v); and purified on a semipreparative RP-HPLC column  using a CH3CN/H2O gradient supplemented with 0.1% TFA (v/v). The separated fractions were lyophilized, the purity of products was analyzed by analytical RP-HPLC, and the identity of the products was verified by high resolution electrospray ionization mass spectrometry .The inclusion bodies were resuspended in a minimum volume (2 ml/g of wet paste) of 50 m1H/13C/15N cryoprobes. To confirm the correct fold of IGF-II analogs, 1D 1H spectra (unlabeled samples) and 2D 1H-15N HSQC spectra were acquired. The NMR spectra were collected using 350-μl samples of protein (75–380 μm) dissolved in 50 mmd4-acetic acid (pH 3.0), 5% D2O (v/v), 0.01% (w/v) NaN3. Data for IGF-II and analogs bound to a high affinity Domain 11 variant of IGF-2R (D11) , 0.01% (w/v) NaN3) at 35 °C.All NMR data for free IGF-II and analogs were acquired at 25 °C using 600- and 850-MHz Bruker Avance II spectrometers, both of which were equipped with 2R (D11) , 72 were13C/15N uniformly labeled IGF-II or analogs to determine essentially complete sequence-specific resonance backbone and side chain assignments. Constraints for 1H-1H distances were derived from 3D 15N-1H NOESY-HSQC and 13C-1H NOESY-HMQC, which were acquired using an NOE mixing time of 100 ms.To determine the structure of either free or bound IGF-IIs, a series of double and triple resonance spectra , 80 weresupplemental Table S1.The family of converged structures was initially calculated using Cyana 2.1 . The comm).CD spectra were measured in a quartz cuvette with an optical path length of 0.5 mm (Starna Cells) using a J-815 spectropolarimeter  at room temperature. The far- and near-UV CD spectra were used to identify changes in protein secondary and tertiary structures. The spectral regions were 200–300 nm. The final spectra were obtained as an average of five accumulations. The spectra were corrected for the baseline by subtracting the spectra of the corresponding polypeptide-free solution. Analogs or IGF-II was dissolved and measured in 5% aqueous acetic acid  of an analog was calculated from at least three (n ≥ 3) independently determined binding curves. The dissociation constant of human 125I-insulin was set to 0.3 nm.Receptor binding studies with the insulin receptor in membranes of human IM-9 lymphocytes (containing only human IR-A isoform) were carried out, and recently . BindingdK) was determined with GraphPad Prism 5 software using a method of non-linear regression and a one-site fitting program and taking into account potential depletion of free ligand. dK values of analogs were determined and calculated by the same procedure as for IR-A.Receptor binding studies with the insulin receptor in membranes of mouse embryonic fibroblasts derived from IGF-I receptor knock-out mice that solely expressed the human IR-B isoform were performed as described in detail previously , 87. Bin125I-IGF-I was set to 0.2 nm. Mouse embryonic fibroblasts expressing human IR-B or IGF-1R were a kind gift from Prof. Antonino Belfiore  and Prof. Renato Baserga . Here we should note that the use of bovine serum albumin (e.g. Sigma-Aldrich A6003) void of “IGF-binding-like” proteins, which interfere with these binding assays, is essential for the preparation of the binding buffer (Receptor binding studies with the IGF-I receptor in membranes of mouse embryonic fibroblasts derived from IGF-1R knock-out mice and transfected with human IGF-1R were performed as described previously , 87. Bing buffer .R. H. and K. Křížková contributed equally to the paper. R. H., K. Křížková, K. Kedrová, and I. S. carried out protein expression and purification. R. H., P. S., Z. T., and V. V. carried out NMR experiments and structure refinement. K. Křížková, K. Kedrová, M. C., and L. Ž. tested the analogs. M. F. and C. W. carried out DNA cloning. P. U. measured CD spectra. J. J. and L. Ž. conceived the study, designed experiments, and analyzed data. R. H., K. Křížková, M. P. C., J. J., V. V., and L.Ž. wrote the paper. All authors discussed the results and commented on the manuscript.