Fah−/−) mouse, we found gradually increasing expression level of insulin-like growth factor 2 (IGF2) in the hepatocytes of host livers. Similarly, high levels of IGF2 were found in the livers of patients with deficient FAH activity. Recombinant IGF2 directly promotes proliferation of primary hepatocytes in vitro. Inhibition on IGF2 expression through the interruption of PI3K/Akt and MAPK pathways significantly reduced the level of liver repopulation in Fah−/− mice. Interestingly, treatment with IGF2 before hepatocyte transplantation generally improved the amount of liver repopulation seen in various mice models of liver injury. Altogether, these findings underscore the underlying mechanisms of therapeutic liver repopulation in Fah−/− mice, and indicate that IGF2 is a potential hepatocyte mitogen for liver cell transplantation therapies.Hepatocyte transplantation holds great promise as an alternative to orthotopic organ transplantation in the treatment of liver diseases. However, obtaining clinically meaningful levels of liver repopulation has not been achieved because the mechanisms regulating hepatocyte proliferation in recipient livers have not yet been well characterized. In the mouse model of Hereditary Tyrosinemia Type I, the fumarylacetoacetate hydrolase-deficient ( Hepatocyte transplantation therapy has been performed in clinical trials as an alternative to orthotopic liver transplantation for some types of genetic diseases of the liver and for acute liver failure3. However, the extent of liver engraftment and repopulation after hepatocyte transplantation was very limited. Therefore, technological improvements to improve therapeutic liver repopulation could lead to successes in cell therapy for liver diseases.Cell transplantation therapies have the potential to treat a wide variety of diseases by making up for tissue defects. Several obstacles still hinder the widespread clinical application of cell therapies. Most importantly, the difficulty in achieving sufficient donor cell engraftment into host tissues is one major technical obstacle9. Two strategies have been successfully applied. The first is to suppress the proliferative capacity of host hepatocytes through inducing cell injuries or damages7. The second is to supply or regulate hepatic mitogens as well as cell-cycle regulators to drive proliferation of the transplanted hepatocytes in recipient livers9. Among the rodent models for liver repopulation, the mouse model of Hereditary Tyrosinemia Type I (HT1), the fumarylacetoacetate hydrolase-deficient (Fah−/−) mouse, is the best example of repopulation of the liver, reaching >90% of total hepatocyte replacement by transplanted wild-type hepatocytes11.Indeed, therapeutic liver repopulation can be examined under experimental conditions in animal modelsFah−/− mouse is similar to what is seen in humans with HT110. Loss of FAH results in famarylacetoacetate (FAA) accumulation, a major toxic metabolite, which causes extensive and continuous hepatocyte injury. 2-(2-nitro-4-trifluoro-methyl-benzoyl)-1, 3-cyclohexanedione (NTBC) inhibits accumulation of toxic metabolites in hepatocytes to maintain Fah−/− mice in a healthy state. However, the underlying molecular mechanisms and factors responsible for high repopulation in Fah−/− mice still remain elusive and are not well defined. Results from previous studies found that hepatocytes in the livers of Fah−/− mice undergo DNA damage12. Furthermore, a genetic screen has been performed to reveal Foxa3 and TNFR1 as a strong promoter and suppressor of liver repopulation in Fah−/− mice13. However, it is not known whether some mitogens are expressed by injured host hepatocytes to enhance the proliferative capacity of transplanted hepatocytes in Fah−/− mice.The liver failure observed in Fah−/− mouse, which could be used to achieve therapeutic liver repopulation in clinical settings. In the present study, we analyzed the pathological changes in the liver tissues of Fah−/− mice undergoing injury due to tyrosinemia to discover potential hepatic mitogens which could promote hepatocyte proliferation. We found that the hepatocytes undergoing injury gradually upregulate IGF2 to high levels. Interestingly, IGF2 expression levels return to normal when liver repopulation is completed. Provision of exogenous IGF2 proved it to be an effective mitogen for promotion of proliferation of transplanted hepatocytes. Conversely, inhibition of IGF2 production inhibited repopulation. These findings indicate that IGF2 therapy is a potential strategy promoting liver repopulation in clinical settings.The objective of this study is to carefully elucidate the mechanism of therapeutic liver repopulation in Fah−/− mice undergo injury upon termination of NTBC administration. However, in line with previous reports14, we found that only a few scattered hepatocytes become positive for the assay of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nicked labeling (TUNEL), and only a few small necrotic foci were found in the livers of Fah−/− mice off NTBC for up to 4 weeks , which activates downstream members of PI3K/AKT and mitogen-activated protein kinase (MAPK) pathwaysion Fig. . The expion Fig. . Remarkaion Fig. .Fig. 4IGThese data indicate that IGF2 is a paracrine signal from the injured hepatocytes that enhances the proliferative capacity of repopulating donor hepatocytes through the activation of AKT and ERK1/2 signaling pathways.Fah−/− mice, we targeted the Igf2 gene using adenovirus vectors expressing shRNA targeting Igf2 (Ad-shIGF2). We used an adenovirus vector expressing GFP gene as a control (Ad-shRNA control). We designed four Ad-shIGF2 vectors, and first tested them in vitro. The Ad-shIGF2 vector with the highest knockdown efficiency was selected for in vivo studies  and circulating IGF2 (protein) were significantly decreased in the Ad-shIGF2-treated mice  plus 2/3 partial hepatectomy (PH) or CClmT/mG donor hepatocytes, which were labeled with membrane-localized tdTomato (mT) fluorescence, were transplanted into two groups of mice recipients: (1) mice treated with RS followed by 2/3 PH; (2) the mice treated with RS followed by CCl4 injection. Four weeks after transplantation, liver samples were harvested and examined for liver repopulation. The results indicated that AAV-mediated IGF2 gene delivery increased the level of liver repopulation by 21.75 ± 5.91% in mice treated with RS followed by 2/3 PH -mediated gene delivery system. ROSA PH Fig. . This oc PH Fig. . Similarion Fig. . The mToion Fig. . In addiion Fig. . Similarion Fig. .Fig. 6IGFah−/− mice. The results also imply that IGF2 could be used to improve liver repopulation in clinical application of hepatocyte transplantation.In summary, overexpression of IGF2 promoted liver repopulation in liver injury models other than 22. In some patients with acute liver failure, rapid proliferation of transplanted hepatocytes in a short time is the key step for replenishing the missing metabolic capacity. Although repeated transplantation of hepatocytes in large numbers could increase the engraftment of transplanted cells and enhance the liver repopulation, this approach has a risk of thrombosis. In addition, transplanted human hepatocytes must be isolated from unused or rejected donor organs and either used immediately or cryopreserved, therefore there is a shortage of hepatocytes of good quality and enough quantity for transplantation. If the transplanted hepatocytes could be stimulated by mitogenic factors to reach massive levels of repopulation within a short time, the utility in acute liver failure would be tremendous.Although hepatocyte transplantation is currently considered an experimental alternative to orthotopic liver transplantation in a few special circumstances, therapeutic levels of liver engraftment repopulation have been hard to reach in most clinical cases. Theoretically, successful liver repopulation will depend on an optimal combination of events, including sufficient blockage of host hepatocyte proliferation in the recipient liver, combined with stimulated proliferation of the transplanted hepatocytes to enable them to win a competition with host hepatocytes for repopulation of the liver23. Among these, HGF is regarded as a “complete mitogen”, as it can induce DNA synthesis during liver regeneration. VEGF promotes hepatocyte proliferation through stimulating HGF production. Some circulating growth factors including EGF and TGFα, produced by hepatocytes, stimulate liver regeneration in autocrine or paracrine fashion. TNF as a pro-inflammatory cytokine mediates hepatocyte apoptosis during PH induced liver regeneration. However, the mitogenic growth factors identified in PH may not apply to other types of liver “regeneration”, such as the liver repopulation that occurs in Fah−/− mice. In the present study, we examine mitogens in Fah−/− undergoing liver injury and repopulation, by examining changes in mRNA levels in Fah−/− mouse livers. Among these, IGF2 was to have high levels of induction during liver injury. Interestingly, the expression level of IGF2 increases just slightly during liver regeneration after PH19, reflecting differences between PH and liver injury in Fah−/− mice. Our study systematically analyzes IGF2 activation during liver injury in Fah−/− mice, and indicated that IGF2 is a hepatocyte mitogen which promotes liver repopulation. However, other as yet uncharacterized mitogens could also contribute to the promotion on liver repopulation, as shRNA blockade of IGF2 did not fully block the hepatocyte repopulation in Fah−/− mice. It will be necessary to characterize these as yet unknown hepatocyte mitogens in future studies.Multiple signaling pathways are known to become activated after PH, when including several mitogenic growth factors24. The findings also suggested the possible clinical application of IGF2 during hepatocyte transplantation, which was supported by our findings that overexpression of IGF2 improved liver repopulation in several models of liver injury. In both mouse and human, IGF2 is highly expressed in fetal liver, but the expression level is markedly downregulated in the quiescent adult liver26 Interesting, it is reported that supplementation of IGF2 into the hippocampus of old rats or mice enhances memory and rescues behavioral deficits28. These data suggest that supplementation of IGF2 could be safe and practical in clinical hepatocyte transplantation.The results from a recent report indicated that short-term treatment with growth hormone improves liver repopulation, which augmented the therapeutic benefit of clinical hepatocyte transplantationFah−/− mice undergoing injury upregulate IGF2, which stimulates repopulation of the liver by donor hepatocytes. Importantly, IGF2 activates PI3K/AKT and MAPK pathways in transplanted hepatocytes. Our study identifies a key mechanism driving therapeutic liver repopulation in Fah−/− mice, and suggests a practical strategy to efficiently expand hepatocytes transplanted into receipients in clinical settings.In summary, host hepatocytes of Fah−/− mice (6–8-weeks old) and wild-type donor mice were on a 129S4 background. ROSA-mTomato/mGFP (ROSAmT/mG) reporter mice and wild-type recipients were maintained on a C57BL/6 background. Fah−/− mice were maintained with or without 7.5 mg/l 2-(2-nitro-4-trifluoromethylbenzoyl)-1, 3-cyclohexanedione (NTBC) in the drinking water11. Human liver tissue was obtained from a surgical resection specimen of a patient with a hepatic hemangioma and a patient with HT1, and consent from the patients were obtained. All procedures and protocols were according to institutional guidelines.30. Isolated hepatocytes were injected intrasplenically into recipient mice as described previously29. After hepatocytes transplantation, livers were harvested and immunohistochemistry with FAH antibody or mTomato was used to examine the percentage of liver repopulation as described previously29.Mouse livers were perfused with collagenase D  solution as previously described31, C57BL/6 wild-type mice were given three injections of retrorsine , 30 mg/kg each, intraperitoneally, 3, 2, and 1 weeks before the transplantation. 1 day before the transplantation, recipient mice were subjected to a two-thirds partially hepatectomy (PH). For retrosine-based CCl4 injury32, C57BL/6 recipient mice were intraperitoneally infused with 0.6ul/g carbon tetrachloride  1 day before the transplantation.For retrosine-based 2/3 PH injury modelIsolated primary hepatocytes were plated on collagen-coated 6-well plates in Hepatic Cell Culture Medium . Six hours after seeding, non-adherent cells were removed by washing the plated with phosphate buffered saline and the attached cells were then cultured in conditional hepatic cell culture medium supplemented with 0, 20, 100, and 200 ng/ml recombinant IGF2 . For BrdU incorporation assay, cells were incubated for 24 h with BrdU regent  (Sigma-Aldrich). Then, cells were fixed with a 1:1 mixture of ice-cold methanol:glacial acetic acid after washing with PBS, and then incubated with 2 N HCl for 30 min at room temperature. Then cells were incubated with primary and second antibody as with the IHC protocols.Small hairpin RNA (shRNA) sequences for the Igf2 gene were chosen from Sigma-Aldrich and listed as follows: Igf2 shRNA-1:5′-CCGGGCTTGTTGACACGCTTCAGTTCTCGAGAACTGAAGCGTGTCAACAAGCTTTTTG-3′, 5′-AATTCAAAAAGCTTGTTGACACGCTTCAGTTCTCGAGAACTGAAGCGTGTCAACAAGC-3′; Igf2 shRNA-2: 5′-CCGGGTGGGCAAGTTCTCCAATATCTCGAGATATTGGAAGAACTTGCCCACTTTTTG-3′, 5′-AATTCAAAAAGTGGGCAAGTTCTTCCAATATCTCGAGATATTGGAAGAACTTGCCCAC-3′;Igf2 shRNA-3: 5′-CCGGCTGATCGTGTTACCACCCAAACTCGAGTTTGGGTGGTAACACGATCAGTTTTTG-3′, 5′-AATTCAAAAACTGATCGTGTTACCACCCAAACTCGAGTTTGGGTGGTAACACGATCAG-3′; Igf2 shRNA-4: 5′-CCGGCAAAGAGTTCAGAGAGGCCAACTCGAGTTGGCCTCTCTGAACTCTTTGTTTTTG-3′, 5′-AATTCAAAAACAAAGAGTTCAGAGAGGCCAACTCGAGTTGGCCTCTCTGPacI according to the manufactuer’s instructions. Viruses were purified on a discontinuous CsCl gradient. Ad-shIGF2 expression plasmids were transfected into the hepa1-6 cell line to test the knockdown efficiency of these IGF2 shRNA constructs. The Ad-shIGF2 with the highest knockdown efficiency was selected for in vivo experiments. Mice received adenovirus via tail-vein injection at a dose of 1 × 109 viral particles per gram body weight. A recombinant adenovirus vector that expresses a shRNA against GFP (Ad-shGFP) was used as a negative control.AACTCTTTG3′. The annealed oligonucleotides were ligated into the pLKO.1 vector and their sequences were verified. Then the four different Igf2 shRNAs were recombined into an adenoviral vector (AdEasy system) to generate recombinant adenovirus vector that expresses shRNA for Igf2, as previously described. Adenoviruses were generated by transfecting HEK 293A cells with vectors digested with 11) were injected through tail-vein at the volume of 100 µl 3 weeks before cell transplantation.The IGF2 overexpression and control adeno-associated virus were generated by Obio Technology . The purified virus was diluted in sterile PBS. Viruses . Statistical analyses were carried out using Graph Pad Prism 5.0c for Mac (Graph Pad Software). For parametric data, data significance was analyzed using a two-tailed unpaired Student’s Supplementary Files


Structures of social amoeba mating-type proteins suggest deep conservation of sexual differentiation processes. Dictyostelium discoideum, which encodes two pairs of small proteins that determine the three mating types of this species; none of these proteins display recognizable homology to known families. We report that the nuclear magnetic resonance structures of two of them, MatA and MatB, contain helix-turn-helix folds flanked by largely disordered amino- and carboxyl-terminal tails. This fold closely resembles that of homeodomain transcription factors, and, like those proteins, MatA and MatB each bind DNA characteristically using the third helix of their folded domains. By constructing chimeric versions containing parts of MatA and MatB, we demonstrate that the carboxyl-terminal tail, not the central DNA binding motif, confers mating specificity, providing mechanistic insight into how a third mating type might have originated. Finally, we show that these homeodomain-like proteins specify zygote function: Hemizygous diploids, formed in crosses between a wild-type strain and a mat null mutant, grow and differentiate identically to haploids. We propose that Dictyostelium MatA and MatB are divergent homeodomain proteins with a conserved function in triggering the haploid-to-diploid transition that can be traced back to the last common ancestor of eukaryotes.Homeodomain proteins control the developmental transition between the haploid and diploid phases in several eukaryotic lineages, but it is not known whether this regulatory mechanism reflects the ancestral condition or, instead, convergent evolution. We have characterized the mating-type locus of the amoebozoan  Eukaryotic organisms have a wide variety of life cycles. Many are sexual, undergoing cycling between different ploidy levels, and even asexual species change their ploidy, although with less regularity  structures that we report here reveal that MatA and MatB each have a core helix-turn-helix (HTH) motif resembling a homeodomain, with largely flexible N- and C-terminal extensions, and we further show that the C-terminal tail is the main determinant of MatA versus MatB activity. We present evidence that these proteins are transcription factors with conserved roles, controlling the transition between the D. discoideum, we set out to solve the structures of MatA and MatB, the 107–amino acid proteins encoded by the mating-type locus in type I and type II cells; the small size and hydrophilic nature of these proteins make NMR spectroscopy particularly suitable for this purpose. Both proteins are monomeric in solution, as assessed by multiangle light scattering (MALS), and maintain their structures over a broad concentration range, as assessed by circular dichroism (CD) spectroscopy (fig. S1).To uncover the mechanism by which the Mat proteins control mating-type determination in D. discoideum, namely, that it is a transcription factor that binds to DNA and acts as a master regulator of the gene expression pathways involved in sexual development.The NMR structure of MatA . The premat null background leads to mating compatibility with type II and III strains, whereas cells expressing only MatB can mate only with type III strains  experiments to assess the flexibility of these tails in solution , which is why sequence comparison alone was incapable of detecting these relationships. Certain bacterial and archaeal HTH proteins share a similar structure, for instance, the archaeal HTH-10 family, and we cannot yet confidently place MatA and MatB proteins within the homeodomain family in a phylogenetic tree (figs. S7 and S8). Nonetheless, considering the known roles for homeodomain proteins in controlling sexual cycles of other eukaryotes, we favor the hypothesis that MatA and MatB are divergent members of this family of transcription factors.MatA and MatB homologs from various scoideum . Despite homolog , middle  helices , B and DS. cerevisiae MATα2 , adding increasing amounts of MatA to a sample of double-stranded DNA (dsDNA) . Because binding , demonst binding  and MatB15N-labeled protein. In the case of MatB S71A at 1°C, addition of DNA resulted in disappearance of all signals except for the sharpest of those from the disordered tails. This was probably due to line broadening arising from intermediate rate exchange between free and bound states; the thermal instability of MatB S71A prevented the alleviation of this problem by raising the temperature. In contrast, in the case of MatA at 25°C, clear and localized effects were observed. Several peaks exhibited CSPs upon addition of DNA, and progressively larger CSPs were observed when longer DNA oligonucleotides were used, suggesting that avidity effects result in increased affinities as the DNA is lengthened  resulting from addition of various lengths of dsDNA to ngthened . A titraMany homeodomain proteins use flexible regions N- and C-terminal to the homeodomain fold to coordinate interactions with other proteins, often to increase binding affinity and sequence specificity , nong types , but theng types , demonstII cells . The cenDictyostelium Mat proteins in specifying haploid function, for example, expression of cell recognition proteins, from roles in the diploid phase of macrocyst development. A locus closely linked to mat has previously been implicated in controlling the feeding or proliferative behavior of diploid cells in a phenomenon called “vegetative incompatibility.” When parasexual diploids are selected by growth under restrictive conditions, only crosses between strains of the same mating type are routinely successful; when strains of different mating types are crossed, the very rare diploids recovered were found to have become homozygous at mat . Therefore, as in budding yeast, mat genes act in a codominant manner to induce Dictyostelium zygotic function.Mutations in genes that control the developmental processes that determine haploid- and diploid-specific functions, and most critically the switch that triggers zygote differentiation in sexual organisms, can be homeotic, best illustrated in organisms that are multicellular in both haploid and diploid phases  cleavage site between the tag and the mat sequences.MatA and MatB constructs used for expression were based on pET28a containing the coding sequence for the Escherichia coli. Cells were grown in either 2× TY for unlabeled samples or M9 minimal medium supplemented either with 15NH4Cl or with 15NH4Cl and [13C6]glucose (Sigma-Aldrich ISOTEC), depending on the desired labeling scheme, as the sole nitrogen or carbon source. Cells were grown to an optical density of approximately OD600 = 1 and cooled to 20°C, and protein expression was induced by addition of 0.5 mM isopropyl-β-d-thiogalactopyranoside. After further incubation at 20°C overnight, cells were harvested by centrifugation.Expression of MatA and MatB proteins was performed in BL21(DE3) All purification steps were carried out at 4°C or on ice. Cell pellets were resuspended in lysis buffer containing 50 mM tris-HCl (pH 8), 750 mM NaCl, 10 % (w/v) sucrose, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor mix  and lysed by sonication, and the cleared lysate was incubated with nickel–nitrilotriacetic acid (Ni-NTA) agarose resin (Qiagen) for 90 min at 4°C. The resin was washed with 150 ml of wash buffer [50 mM tris-HCl (pH 8) and 750 mM NaCl], and the bound protein was eluted with a buffer containing 50 mM tris-HCl (pH 8), 750 mM NaCl, and 350 mM imidazole. Elution fractions were pooled, concentrated, and purified by gel filtration using a Superdex 75 column , equilibrated in 50 mM tris-HCl (pH 7.4) and 500 mM NaCl. Desired fractions were pooled and cleaved with the TEV protease overnight, Ni-NTA agarose was used to remove the fusion tag and protease, and the protein was purified to homogeneity using a Superdex 75 column as before. Fractions containing the required protein were pooled and dialyzed against 4 liters of dialysis buffer  overnight at 4°C.Polyacrylamide gels for EMSAs were cast following the standard protocols; the gels used were 6% (v/v) polyacrylamide (37.5:1 acrylamide/bisacrylamide) with 1× tris-borate buffer (90 mM of each) in the gel and running buffers. Gels were prerun at 55 V for 30 min at room temperature. DNA-protein samples were incubated in 10 mM tris-HCl (pH 7.4), 1 mM EDTA, and 20 mM NaCl for 30 min at room temperature before addition of 4× loading buffer , and 5 μl of samples was loaded onto the gel. Gels were run at 80 V for 75 min and then visualized by staining with SYBR Gold DNA stain (Thermo Fisher Scientific). dsDNA fragments used in these experiments were prepared using DNA oligonucleotides produced by Integrated DNA Technologies. The following sequences were used: 5′-CCGAAACGTTTGGTGGCGCATGTAATTCATTTACACGCGCGAGGGCGTGCAAGATTC-3′ and 5′-GAATCTTGCACGCCCTCGCGCGTGTAAATGAATTACATGCGCCACCAAACGTTTCGG-3′.Dictyostelium species were identified by blastp  using the Ray assembler (http://fatcat.burnham.org) using the FATCAT-pairwise algorithm  or in association with Klebsiella pneumoniae on SM agar (Formedium). Cells were transformed by electroporation  using KOD Hot Start DNA Polymerase (Novagen) as per the manufacturer’s instructions such that the PCR products contained overlapping regions and could subsequently be combined to use as a template in a further PCR. The constructs were first cloned into bacterial vectors and then subcloned into a K. pneumoniae used in the macrocyst crosses were produced by growing up an overnight culture of the bacteria in LB medium, harvesting the cells by centrifugation, and washing three times in MSS buffer . Cells were then heated at 60°C for 20 min before cooling and storing at −20°C. Cells to be crossed were grown separately in suspension with heat-killed K. pneumoniae (diluted to OD600 = 10) in MSS buffer with tetracycline (10 μg/ml) and streptomycin (200 μg/ml) overnight and protected from light. Those strains that had grown to a density of 106 cells/ml were washed three times in fresh MSS before mixing. For crosses, 5 × 105 cells of each strain were mixed with 50 μl of an OD600 = 10 suspension of heat-killed bacteria in 500 μl of MSS per well in 24-well plates and incubated in the dark at 22°C for 7 days. Macrocysts were visualized for counting using a light microscope.Heat-killed K. pneumoniae were washed free of bacteria in MSS buffer and 106 cells of each strain were mixed in a total of 1 ml of MSS in 24-well tissue culture plates. These mixtures were shaken at 180 rpm at 22°C overnight to allow cell aggregates to form, promoting low-frequency fusion. Aggregates were broken up by repeated trituration, and then cells were cultured under selective conditions that permit diploid but not haploid cells to grow. For selection by temperature-resistant growth, haploid strains bearing mutations rendering them unable to grow at high temperature were mixed; in each mixture, a different gene is mutated so that, in diploids formed between them, each mutation is complemented by the wild-type gene. To select against temperature-sensitive haploids, cells were grown in association with K. pneumoniae on SM agar at 27°C for several days in a humid container. In double-drug selections (matA null cells transformed with mat gene (fused to GFP or mRFP) expression constructs bearing a G418 resistance cassette were crossed with tester haploid strains carrying a blasticidin resistance cassette. To select against drug-sensitive haploids, cells were plated in 10-cm tissue culture dishes in HL5 broth plus 10 μM blasticidin S and 20 μM G418. Because these experiments resulted in growth or absence of growth, we did not quantify the frequency of diploid formation in the double-drug selections; in each cross, at least 5 × 106 cells were screened in multiple independent experiments. Spore lengths were measured using the program Fiji –MALS was carried out using a Wyatt Heleos II 18-angle light scattering instrument coupled to a Wyatt Optilab rEX online refractive index detector. Samples were exchanged into 20 mM phosphate (pH 7), 50 mM NaCl, and 2 mM EDTA and loaded onto a Superdex 75 10/300 GL column  running at 0.5 ml/min and preequilibrated in a sample buffer. A bovine serum albumin standard was run before the Mat samples to generate a calibration curve, and SEC-MALS and QELS (quasi-elastic light scattering) data were analyzed using the manufacturer’s ASTRA software.CD spectra were measured using a Jasco J-810 spectrometer, scanning in the range of 260 to 190 nm at 20°C with samples of MatA or MatB in 50 mM phosphate (pH 6) and 100 mM NaCl.15N- or 15N,13C-labeled protein solutions in 50 mM 2H11 tris buffer (pH 7), 100 mM NaCl, 1 mM 2H6 dithiothreitol, and 50 μM EDTA in 95:5 H2O/2H2O; NMR samples of MatB comprised 0.3 to 1 mM 15N- or 15N,13C-labeled protein solutions in 25 mM phosphate buffer (pH 6), 100 mM NaCl, and 50 μM EDTA in 95:5 H2O/2H2O. NMR data were acquired using Bruker DMX600, DRX600, and Av-1 800 spectrometers, each equipped with a cryogenically cooled triple-resonance (1H/15N/13C) 5-mm probe. Experiments were conducted at 15°C (MatA) or 1°C (MatB), and 1H chemical shifts were calibrated using sodium 3,3,3-trimethylsilylpropionate as an external 1H reference; 15N and 13C chemical shifts were indirectly referenced to the 1H shifts using the ratio of gyromagnetic ratios NH, HNHAHB, HBHA(CO)NH, [1H-13C-1H] HCCH-TOCSY, [1H-13C-1H] HCCH-COSY, [13C-13C-1H] HCCH-TOCSY, 15N NOESY-HSQC (τm = 150 ms), and 13C NOESY-HSQC (τm = 150 ms); separate data sets were acquired for 13C aliphatic and aromatic spectral regions. All of the NOESY data sets used for structure calculations (see below) were acquired using pulse sequences modified to ensure equal RF heating in each case, for example, for 13C experiments, a period of 15N decoupling equal in length to the acquisition period was applied at the beginning of the interscan delay, and for 15N experiments, an equivalent period of 13C decoupling was similarly applied. All spectra were processed using the program TOPSPIN versions 3.1 and 3.2 (Bruker GmbH) and analyzed using the program CCPN analysis (NMR samples of MatA comprised 0.3 to 0.6 mM 1H (94.78%), 13C aliphatic (90.87%), 13C aromatic (78.95%), and 15N (81.08%). In the case of MatB, the quality of the data from scalar coupling–based backbone experiments was markedly lower, and additional use had to be made of NOE-based experiments for assignments in protein regions that suffered the most severe line broadening ; the final assignment completeness for MatB as reported by the UNIO software was 1H (82.64%), 13C aliphatic (83.82%), 13C aromatic (36.36%), and 15N (79.49%).Resonance assignments for MatA were made exclusively using the scalar coupling–based backbone experiments listed above; the resulting assignment completeness for MatA as reported by the UNIO software was 15N{1H} heteronuclear NOEs for the amide signals of MatA and MatB were determined using 250 and 120 μM solutions of 15N-labeled MatA and MatB, respectively, adjusted to 25 mM phosphate (pH 6), 100 mM NaCl, and 50 μM EDTA with 5% D2O (v/v), essentially as described by Skelton et al. (1H/15N/13C) cryoprobe. The 15N{1H} heteronuclear NOE data were collected in an interleaved manner, and 15N{1H} heteronuclear NOE values were calculated from the ratio of peak intensities of pairs of spectra acquired with and without 1H saturation; the error bars shown in et al. (15N-labeled MatA upon addition of the various fragments of unlabeled dsDNA listed below. 2D 15N-1H HSQC spectra were acquired at 25°C for samples containing 80 μM DNA and 20 μM MatA in 25 mM phosphate (pH 6), 50 mM NaCl, and 50 μM EDTA with 5% (v/v) D2O. CSPs were calculated for interaction with the 29-bp oligonucleotide, using the assignments described above according to the observed peak distributions in the 1H and 15N dimensions and applying the formula Δδ = √((Δδ(1H))2 + (Δδ (15N)/6.8)2). In the case of the 58-bp DNA, additional spectra were acquired at DNA concentrations of 40, 80, and 120 μM (with MatA concentration of 20 μM throughout), and spectra were also acquired for MatB S71A (20 μM) in the presence and absence of the 29-bp DNA (80 μM) at 1°C.Backbone amide CSP analyses were performed for samples of DNA oligonucleotide sequences were as follows: 58 bp, 5′-GAATCTTGCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTTTCGG-3′ (forward) and 5′-CCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTC-3′ (reverse); 29 bp, 5′-CCGAAACGTTTGGTGGCGGGACCAGTGAC-3′ (forward) and 5′-GTCACTGGTCCCGCCACCAAACGTTTCGG-3′ (reverse); 21 bp, 5′-CCGAAACGTTTGGTGGCGGGA-3′ (forward) and 5′-TCCCGCCACCAAACGTTTCGG-3′ (reverse); and 15 bp, 5′-CGTTTGGTGGCGGGA-3′ (forward) and 5′-TCCCGCCACCAAACG-3′ (reverse).15N NOESY-HSQC (τm = 150 ms), 13C aliphatic region NOESY-HSQC (τm = 150 ms), and 13C aromatic region NOESY-HSQC (τm = 150 ms).Initial structures of MatA and MatB were calculated using the program UNIO , all constraints involving protons within these groups were converted to group constraints (by using wildcards such as HB*). All lower bounds were set to zero (et al. (Next, structures were calculated using XPLOR-NIH (t1 noise arising from sharp signals of the N- and C-terminal tails. As a result, the NOE restraints derived from UNIO for MatB S71A included a significant proportion that corresponded to misassigned spectral artifacts, leading to degradation of the structural resolution and statistics. These artifacts were largely eliminated during multiple rounds of analysis of successive XPLOR-NIH calculations, at each stage combining analysis of violations and Ramachandran statistics with manual verification of cross peaks in the spectra.In the case of MatA, the NOESY data were of sufficient quality that the NOE restraints derived from the UNIO calculations resulted in well-converged structures with good violation and Ramachandran statistics. In contrast, MatB S71A has markedly lower thermal stability than MatA, and its NOESY spectra consequently contained broader, less intense, and more heavily overlapped signals from the structured core, also resulting in a proportionately higher contribution from the The structures calculated in XPLOR-NIH were finally subjected to a further stage of energy minimization using a full force field as implemented in the program AMBER 11 (The program CLUSTERPOSE (


S. aureus infection. However, the specific genes responsible for this susceptibility are unknown. Chromosome substitution strain 8 (CSS8) mice, which have chromosome 8 from A/J but an otherwise C57BL/6J genome, were used to identify the genetic determinants of susceptibility to S. aureus on chromosome 8. Quantitative trait loci (QTL) mapping of S. aureus-infected N2 backcross mice (F1 [C8A] × C57BL/6J) identified a locus 83180780–88103009 (GRCm38/mm10) on A/J chromosome 8 that was linked to S. aureus susceptibility. All genes on the QTL (n~ 102) were further analyzed by three different strategies: 1) different expression in susceptible (A/J) and resistant (C57BL/6J) mice only in response to S. aureus, 2) consistently different expression in both uninfected and infected states between the two strains, and 3) damaging non-synonymous SNPs in either strain. Eleven candidate genes from the QTL region were significantly differently expressed in patients with S. aureus infection vs healthy human subjects. Four of these 11 genes also exhibited significantly different expression in S. aureus-challenged human neutrophils: Ier2, Crif1, Cd97 and Lyl1. CD97 ligand binding was evaluated within peritoneal neutrophils from A/J and C57BL/6J. CD97 from A/J had stronger CD55 but weaker integrin α5β1 ligand binding as compared with C57BL/6J. Because CD55/CD97 binding regulates immune cell activation and cytokine production, and integrin α5β1 is a membrane receptor for fibronectin, which is also bound by S. aureus, strain-specific differences could contribute to susceptibility to S. aureus. Down-regulation of Crif1 with siRNA was associated with increased host cell apoptosis among both naïve and S. aureus-infected bone marrow-derived macrophages. Specific genes in A/J chromosome 8, including Cd97 and Crif1, may play important roles in host defense against S. aureus.We previously showed that chromosome 8 of A/J mice was associated with susceptibility to  An emerging body of evidence supports the concept that human genetic variation can influence host susceptibility to and outcome of infectious diseases. Examples of human genetic variation and susceptibility to specific infectious syndromes include susceptibility to severe sepsis in Chinese Han subjects with the rs1800629 variant of the TNF gene , Toll-liS. aureus remains largely unknown. We  × C57BL/6J) backcross mice, QTL mapping identified a region on chromosome 8 that was significantly linked to survival time after S. aureus infection. This region was located between 83180780–88103009 (GRCm38/mm10) and contained approximately 102 genes (Escherichia coli (Previously we demonstrated that C57BL/6J inbred mice were resistant to 02 genes . A/J andhia coli .S. aureus within the QTL region by testing three possible sources of genetic variation: 1) genes within the QTL that are differentially expressed between susceptible A/J and resistant C57BL/6J mice only in S. aureus infected state; 2) genes within the QTL that are differentially expressed between susceptible A/J and resistant C57BL/6J mice in both uninfected and S. aureus infected states; and 3) presence of damaging SNPs in genes within the QTL that result in a damaged or dysfunctional gene product ; Ier2 ; JunB ; Tbc1d9 ; and Zfp423   . These 5 p<0.05) .Crif1, Farsa, Inpp4b, Klf1, Nfix, and Tnpo2 , while other 5 genes exhibited significantly higher expression in A/J . These 5 genes comprised our candidate gene list for Strategy 2  and in C57BL/6J in 5 of the 10 genes . These 10 genes comprised our candidate gene list for Strategy 3   to generate an F1 population with heterozygous chromosome 8. To generate N2 backcross mice for QTL linkage analysis, F1 (C8A) mice were backcrossed with C57BL/6J to produce a total of 337 progeny that were used for phenotyping. Specific numbers of mice employed in experiments are provided in the tables and figures presenting the data.S. aureus clinical strain, Sanger 476 was used in the mortality and infection studies. For preparation of S. aureus for injection, overnight culture of S. aureus was diluted 100 folds with fresh tryptic soy broth (TSB) and shake at 37°C with aeration to log-phase (OD600 ≈ 0.8). S. aureus was harvested by centrifugation at 3000rpm for 10 minutes at 4°C, washed once in DPBS and re-suspended in DPBS.7CFU/g S. aureus (Sanger 476) or 2×105 CFU/g E. coli (K1H7) and observed every 6 hours for morbidity continuously for 5 days.For murine peritonitis-sepsis experiments, 8-week-old male mice (n = 8) in each strain of C57BL/6J, A/J, and CSS8 were injected via intraperitoneal route with 10http://www.informatics.jax.org/). Seventeen microsatellite markers were selected with an average inter-marker distance of 0.65 cM covering chromosome 8. A total of 337 N2 backcross mice were generated, all of which were genotyped for each microsatellite marker by PCR amplification and gel electrophoresis. J/qtl software was used to analyze phenotype and genotype data for linkage of survival time after infection with S. aureus Sanger 476 and marker location. Phenotypes were defined as either sensitive or resistant based on the dichotomization of survival data . All linkage analysis results were expressed as LOD scores. LOD score was considered “suggestive” if > = 0.43 (p = 0.63) and “significant” if > = 1.51 (p = 0.05). Threshold values for linkage were determined by a 1,000 permutation test by using J/qtl.Polymorphic microsatellite markers on chromosome 8 between C57BL/6J and A/J were chosen from a database maintained by Mouse Genomic Informatics  and multi-test correction for p-values in Partek Genomic Suite. Differentially expressed genes between susceptible A/J and resistant C57BL/6J were identified a) at all pre-infection and post-infection time points (for Strategy 2) and b) S. aureus infection stateonly in  (for Strategy 1). Student’s t-test was used to test for differential expression between 2 groups . The lists of significant differentially expressed genes were generated based on a criterion of ≥2 relative fold change at a false discovery rate (FDR) of ≤ 5% as previously described[Accession numbers for murine genes and their human orthologs were identified in NCBI and are provided in described.Blood was obtained by cardiac puncture from all mice strains. Total RNA was isolated using RNeasy kits (Qiagen) primed with random hexamer oligonucleotides and reversely transcribed using Invitrogen SuperScript II. Real-time quantitative PCR was performed using SYBR Green Mastermix (ABI). The qPCR primers for candidate genes are provided in 6 BMDMs were seeded to 6-well plate and cultured overnight. On the next day, PBS with S. aureus or same volume of PBS was added to BMDMs at MOI 10 and cultured for 1 hour. After washing twice with PBS, BMDMs were detached from plate by EDTA [Bone marrow-derived macrophages (BMDMs) were differentiated from male A/J, C57BL/6J and CSS8 as before [ by EDTA  and staiS. aureus by MOI 10 for 1 hour and further analyzed of apoptosis. In parallel experiments, cells were harvested for RNA and qPCR analysis for their level of candidate genes. A full list of gene names and siRNA ID numbers are listed in siRNAs were purchased from Invitrogen and transfected into BMDMs of C57BL/6J by Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions as before . Twenty-The 1505 version of the Sanger Mouse Genomes Project sequence variation tool was used to identify all known non-synonymous sequence variants for the genes in the QTL region for the A/J and C57BL/6NJ mouse strains. This analysis utilized the C57BL/6J mouse strain as a reference to identify variants and was carried out at1 kbp resolution. These SNPs were then processed through the S.I.F.T. (Sorting Intolerant From Tolerant) program which is a platform that can be used to predict whether a specific amino acid substitution is a functionally damaging alteration . AnalyseS. aureus challenge were harvested and incubated with a mixture of recombinant mouse CD55 protein (Thermo Fisher) plus phycoerythrin-conjugated anti-mouse CD55 (BioLegend); or recombinant mouse integrin α5β1 (R&D Systems) plus Alexa Fluo 488-conjugated anti-mouse CD49e (α5) (BioLegend) for 30 minutes at room temperature following manufacturer’s instruction. Cells were assayed using the respective fluorescence channel.BD FACSCanto was used to evaluate ligand binding. Mouse peritoneal cells with or without RNA quality and concentration were assessed with a Fragment Analyzer  and Qubit 2.0 (ThermoFisher Scientific). For each sample, two hundred nanograms of total RNA was used for library construction. poly(A) mRNA capture and construction of stranded mRNA-seq libraries from intact total RNA (RIN numbers >7) was achieved using the commercially available KAPA Stranded mRNA-Seq library preparation Kit. In brief, mRNA transcripts were first captured using magnetic oligo-dT beads, fragmented using heat and magnesium, and reverse transcribed to produce dscDNA. Illumina standard sequencing adapters were then ligated to the dscDNA fragments and amplified to produce the final RNA-seq library. Libraries were indexed using a molecular indexing approach allowing for multiple libraries to be pooled and sequenced on the same sequencing lane on a HiSeq 4000 Illumina sequencing platform. After quality check of each individual library, the indexed libraries were diluted to 10nM, pooled at equimolar ratios and sequenced on 2 lanes of HiSeq 4000 with 50bp Single Read protocol. Data was demultiplexed and Fastq files were generated using BcltoFastq 2.19 script provided by Illumina.RNA-seq data was processed using the TrimGalore toolkit, which eS. aureus-infected patients. For this reason, pairwise comparisons were made between healthy control subjects and either patients with S. aureus BSI or E. coli BSI. Differences in survival times of mice were examined with Kaplan-Meier plots and statistical differences in survival across different mouse strains were tested with the log-rank test. P-values smaller than 0.05 were considered statistically significant.Group differences in the distributions of continuous measures for candidate gene expression at times 0 and 2 hours were tested with Student’s T-test and the distributions for apoptosis were evaluated with the Mann Whitney-U test. The primary question of interest for the septic human cohort gene expression data was the presence of differences in the S1 FigMice were 8-week old males.(TIF)Click here for additional data file.S2 Fig(A) PCA-plot of A/J, C57BL/6J and F1 (CSS8 x C57BL/6J). Principal component analysis for RNA-seq data. (B) Allele specific expression of the 11 candidate genes. For the 11 candidate genes in A/J chromosome 8 QTL, an even distribution of parental origins was observed in the F1 (CSS8 x C57BL/6J). (TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S1 TableExpression data are provided for all 8 genes that were significantly differentially expressed in A/J but not C57BL/6J. No genes were significantly differentially expressed at 0 vs. 2 hours in only C57BL/6J. Multiple comparisons adjustments were applied using False Discovery Rates of ≤ 5%. 8-week old male A/J mice (n = 5 in each group) were used for experiments.(TIF)Click here for additional data file.S2 Tablehttps://doi.org/10.1371/journal.ppat.1001088.s007) [Data previously published in S Table 1 of Ahn et al (88.s007) .(TIF)Click here for additional data file.S3 Table(TIF)Click here for additional data file.S4 Table(TIF)Click here for additional data file.S5 TableA. Ct values for qPCR results of 5 candidate genes identified by Strategy 1. Male 8 week-old mice were used (n = 4 in each group). B. 18s rRNA normalized Ct values for qPCR results of 5 candidate genes identified by Strategy 1. Male 8 week-old mice were used (n = 4 in each group).(TIFF)Click here for additional data file.S6 TableA. Ct values for qPCR results of 6 candidate genes identified by Strategy 2. Male 8 week-old mice were used (n = 5 in each group). B. 18s rRNA normalized Ct values for qPCR results of 6 candidate genes identified by Strategy 2. Male 8 week-old mice were used (n = 5 in each group).(TIFF)Click here for additional data file.S7 Table(TIF)Click here for additional data file.


CSF3R, SRSF2 and SETBP1 mutational analyses in 10 CNL and 56 CMML patients. In this sample cohort, 80% of CNL patients harbored CSF3R mutations, of which the CSF3R T618I mutation was dominant. Mutations in CSF3R and SETBP1 were found in 7.1% and 5.3% CMML patients respectively, while 25% of CMML patients carried SRSF2 mutations. Strikingly, we identified that all of the CSF3R mutations detected in CMML patients were represented by a P733T mutation. The CSF3R P733T mutation represents a novel CSF3R mutation. In addition, none of the four CSF3R P733T mutated patients carried SRSF2 mutations . Both mut SRSF2 and mut SETBP1 patients had shorter overall survival (OS) and progression-free survival (PFS) compared to patients with wt SRSF2 (P < 0.001 both) and wt SETBP1 . While we found no significant differences in OS and PFS as a consequence of CSF3R mutation status, our work suggest that the CSF3R T618I mutation is a diagnostic marker with good specificity and sensitivity for CNL. In conclusion, our study highlights effective diagnostic and prognostic markers of CNL and CMML patients in the Chinese population.Chronic neutrophilic leukemia (CNL) and chronic myelomonocytic leukemia (CMML) are rare hematologic neoplasms. We performed  CSF3R and SETBP1 mutations ; 1 of the 3 patients with SETBP1 mutations also had SRSF2 mutations . Characteristics such as age, gender, WHO category, FAB category, karyotype, blood cell counts and CPSS risk stratification did not reveal any difference between mutated SRSF2 patients and wt SRSF2 patients (P > 0.05) (Table CSF3R mutation had higher HB levels (P = 0.05) compared to wt CSF3R patients. However, no statistically significant difference was identified between mut CSF3R and wt CSF3R CMML patients in terms of age, gender, WHO category, FAB category, karyotype, other blood cell counts and CPSS risk stratification. In terms of treatment for the investigated patients, we normally used hydroxyurea and Interferon-α for CNL patients, and demethylation therapy, combined chemotherapy, hydroxyurea, immunomodulation therapy and supportive treatment for CMML patients.Patients with SRSF2 and SETBP1 mutated patients showed shorter progression-free survival (PFS) and overall survival (OS) compared with wt SRSF2 (both P < 0.001) and wt SETBP1 (P = 0.02 and P < 0.001) patients. In addition, we observed that patients with CSF3R mutations had longer PFS and OS compared to wt CSF3R patients. However, the difference was not statistically significant (P > 0.05) , 14(25%) patients died, mainly due to progression to AML(10/14). Both SRSF2 mutations , SETBP1 mutations , higher BM blast counts , higher PB blast counts , higher peripheral blood mononuclear cells (PBMC)  and older age  suggested a poor prognosis, while HB  was associated with a favorable PFS. Overall, there was no statistically significant difference in OS and PFS based on sex, WHO-subtype, FAB-subtype, WBC, BPC and cytogenetics   and PFS . Likewise, SETBP1 mutations were independent predictors of poor OS   experienced shortened survival [Our study show that 80% of CNL patients carried survival . In contsurvival . BecauseCSF3R mutations in 7.1% of CMML patients. Interestingly, all of the CSF3R mutations detected in CMML patients were CSF3R P733T mutations. CSF3R P733T mutation represents a novel CSF3R point mutation not previously reported. In addition, none of the 4 CSF3R P733T mutated patients were detected with SRSF2 mutations, suggesting that CSF3R P733T mutations and SRSF2 mutations are mutually exclusive in CMML patients. Therefore, the CSF3R P733T mutation may be of diagnostic value in CMML, especially for wt SRSF2 CMML patients. In univariate analysis, we observed that CMML patients with CSF3R mutations had longer PFS and OS compared to wt CSF3R patients. However, the difference was not statistically significant. The lack of significance may be due to the small sample size in the CSF3R mutant group which resulted in low statistical power. In contrast, Kosmider and collegues identified CSF3R mutations in 3% CMML patients and showed that CSF3R mutations were associated with an reduced OS and AML-free survival in univariate analysis [CSF3R mutations were divided into membrane proximal mutations and truncation mutations based on the distribution within two distinct regions of CSF3R [CSF3R activates downstream signaling mediators-SRC family kinases (SFKs) and TNK2 and lead to increased sensitivity to Dasatinib treatment [We identified analysis . AdditioSETBP1 mutations present in 5.3% of CMML patients. In univariate analysis, SETBP1 mutations was an independent adverse predicting factor for OS (P = 0.002) and PFS (P = 0.038). Similar results were obtained for OS in multivariate analysis. These data are consistent with multiple previous studies [SETBP1 mutations occur in 4–7% CMML patients and are indicative of decreased OS and AML-free survival [Here, we demonstrate  studies , 19–21, survival .SRSF2 mutations were detected in 25% CMML patients in our study but exclusive of mutations in CSF3R. Our multivariate analysis showed SRSF2 mutation as an independent poor predictor for OS (P = 0.028) and PFS (P = 0.001), in line with previous data from Itzykson et al. and Makishima et al. [Notably, a et al. , 23.CSF3R mutations and that the CSF3R T618I mutation appears to be a diagnostic marker with good specificity and sensitivity for CNL. By contrast, CSF3R P733T mutations detected in CMML patients were completely different from CSF3R mutation types described in patients with CNL, SCN and hereditary neutrophilia, which may be a potential diagnostic marker, particularly for wt SRSF2 CMML patients. In addition, mutations in SRSF2 were commonly found in CMML patients, and represents a poor prognostic marker for CMML. Mutations in SETBP1, by contrast, were found in CNL, CMML but also other hematological malignancies, making it a rather poor isolated prognostic marker for hematological diseases. As patients with different gene mutations may have different clinical response to treatment, mutation-based personalized targeted therapy should be considered in future studies.In conclusion, we report that the majority of CNL patients studied here carried oncogenic A total of 10 CNL patients and 56 CMML patients were included in the study. 43 were male and 23 were female, at a median age of 64 (range: 24–94) years. Of the 56 CMML patients, 47 were diagnosed with CMML-1 and 9 were diagnosed with CMML-2. Of the 10 CNL patients, 1 was diagnosed with monoclonal gammopathy of undetermined significance associated with chronic neutrophilic leukemia (MGUS-CNL). 20 MDS patients, 10 chronic eosinophilic leukemia (CEL) patients and 20 healthy donors were included as controls. The diagnosis, classification and AML transformation of CMML, CNL, MDS and CEL were based on WHO 2008 criteria [CSF3R exons 14–17, SETBP1 exon 4 and SRSF2 exon 1 were amplified. Primers used were: CSF3R exon 14 F: CCACGGA GGCAGCTTTAC; CSF3R exon 14 R: AAATCAGCATC CTTTGGG TG. CSF3R exon 15 F: TGACTTTGAATCC CCTGGTC; CSF3R exon 15 R: TGAGGTTCCCTGT GGGTG; CSF3R exon 16 F: AAAATGGAAAGATC GGAG GG; CSF3R exon 16 R: CTTGGCTTCAGAAG GTGTCC; CSF3R exon 17 F: CTGTCACTTCCGGC AACAT; CSF3R exon 17 R: TGGCCCAAAGACAC AG TCGT; SETBP1 exon 4 F: ACCTGGAAGCTGTCTCC ACCCA; SETBP1 exon 4 R: CGGTGGCCATGCCGG TTCTT; SRSF2 exon 1 F: CTGTCACTTCCGGC AACAT; SRSF2 exon 1 R: TGGCCCAAAGA CACAGTCGT.We obtained DNA specimens from bone marrow mononuclear cells or peripheral-blood granulocytes from the patients, and constitutional DNA samples from matched buccal swabs as described previously . CSF3R ehttps://www.ncbi.nlm.nih.gov/projects/SNP/). The previously unreported variants were resequenced with the matched buccal swabs samples DNA from the same patients. The somatic mutation was confirmed when it associated with the bone marrow or peripheral-blood sample DNA and not the matched buccal swabs DNA.Independent validations of the detected variants were conducted using Sanger sequencing. If nonsynonymous sequence changes were detected, we determined whether the sequence variant had previously been reported as a SNP by searching in the Single Nucleotide Polymorphism database (Patient data was collected at the first diagnosis. The median follow-up period was 20 months (range: 2–60). CMML patients were divided into high, intermediate-1, intermediate-2 and low risk group according to the CMML Prognostic Scoring System (CPSS) .P-values < 0.05 (two-tailed) were considered statistically significant.The statistical analysis of data was done by using Excel and SPSS  version 17.0. Statistical analysis was performed by comparison between groups using Kruskal-Wallis test regarding quantitative nonparametric data and chi-square test regarding qualitative data. The overall survival analysis was done by Kaplan–Meier curve. Multivariate analysis of survival was carried out by Cox regression. All


Avian H9N2 and 2009 pandemic H1N1 (pH1N1) influenza viruses can infect pigs and humans, raising the concern that H9N2:pH1N1 reassortant viruses could emerge. Such reassortants demonstrated increased replication and transmissibility in pig, but were still inefficient when compared to pH1N1. Here, we evaluated if a reassortant virus containing the hemagglutinin and neuraminidase of A/quail/Hong Kong/G1/1997 (H9N2) in the A/California/04/2009 (pH1N1) backbone could become better adapted to pigs by serial passaging. The tropism of the original H9N2:pH1N1 (P0) virus was restricted to the nasal mucosa, with no virus detected in the trachea or lungs. Nevertheless, after seven passages the H9N2:pH1N1 (P7) virus replicated in the entire respiratory tract. We also compared the transmissibility of H9N2:pH1N1 (P0), H9N2:pH1N1 (P7) and pH1N1. While only 2/6 direct-contact pigs showed nasal virus excretion of H9N2:pH1N1 (P0) ≥five days, 4/6 direct-contact animals shed the H9N2:pH1N1 (P7). Interestingly, those four animals shed virus with titers similar to those of the pH1N1, which readily transmitted to all six contact animals. The broader tissue tropism and the increased post-transmission replication after seven passages were associated with the HA-D225G substitution. Our data demonstrate that the pH1N1 internal-protein genes together with the serial passages favour H9N2 virus adaptation to pigs. Since the late 1990s, H9N2 viruses have been occasionally isolated from humans and swine in China, leading to increasing concerns about their pandemic potential8. However, all reported human or swine H9N2 virus infections were dead-end events and the virus failed to spread within the human or swine population9.H9N2 avian influenza viruses are currently endemic in poultry in South East Asia and the Middle Eastα2,6-linked sialic acids  and to improve the replication of H9N2 viruses in human airway epithelial cells in vitro, supporting concerns about their pandemic potential12. The binding of the viral HA to sialyloligosaccharides, present on the host cell surface, is the first step for influenza virus infection. Because of the predominant expression of Siaα2,6Gal in human and swine upper respiratory tract14, most human and swine influenza viruses more willingly bind to receptors that contain Siaα2,6Gal15. In contrast, avian influenza viruses preferentially bind sialic acids linked to galactose by an α2,3 linkage . Therefore, a switch from Siaα2,3Gal to Siaα2,6Gal receptor-binding preference is considered an important step for avian influenza virus adaptation to mammals16. Nevertheless, enhanced Siaα2,6Gal binding is not entirely sufficient to make H9N2 viruses transmissible between mammals18. This suggests that other amino acid substitutions and/or an adequate constellation of internal genes is also required for efficient transmission of H9N2 viruses between mammals.A significant proportion of poultry H9N2 field isolates contains a leucine instead of glutamine residue at position 226 in the hemagglutinin (HA) receptor-binding site (RBS) . This mutation has been shown to enhance HA binding to terminal 19. In that sense, the sporadic isolation of avian H9N2 influenza viruses from pigs and humans in China increases the possibility of co-infections with endemic swine or human influenza viruses, which could result in the generation of novel reassortant viruses with enhanced transmissibility in mammals.Due to the segmented nature of the influenza viruses, the RNA segments of two (or more) different influenza viruses can “reassort” when co-infecting the same host cell, thereby generating progeny viruses with properties different from the parental viruses20. After its first appearance in 2009, the virus has spread around the world in both human and swine populations21. Moreover, novel reassortant viruses containing pH1N1 internal-protein and surface-protein genes from other endemic swine influenza virus have been isolated frequently from pigs worldwide25. In addition, H3N2 swine influenza viruses containing the pH1N1-derived matrix (M) protein gene (H3N2 variant) were isolated from 364 human cases in the US since 201126. This suggests that pH1N1 internal-protein genes are compatible with several surface-protein gene combinations and allow for robust replication in mammals.The 2009 pandemic H1N1 virus (pH1N1) is a swine-origin reassortant virus containing human, swine and avian influenza virus genes27. In pigs similar reassortant viruses also showed increased transmission when compared to the parental H9N2. However, their transmission was still not as efficient as that of swine-adapted viruses like pH1N128. We previously demonstrated that serial passages of A/quail/Hong Kong/G1/1997 (H9N2) virus in pigs slightly enhanced replication and transmission in swine . Although the resulting pig-adapted virus carried a number of mutations compared to the parental virus, transmission was still less efficient than that of naturally circulating viruses. In the present study, we have examined the replication and transmissibility after ten serial pig passages of a reassortant virus containing A/quail/Hong Kong/G1/1997 (H9N2) surface-protein genes and NP gene within the A/California/04/2009 (pH1N1) background to evaluate if the pH1N1 backbone confers an advantage in H9N2 adaptation to pigs. This serial passaging of the H9N2:pH1N1 reassortant resulted in a virus that replicated in and transmitted between pigs at high rates. The predominant mutation in the passaged reassortant virus was an aspartic acid to glycine at position 225 in the HA RBS. Therefore, our results showed that the combination of reassortment and mutations induced by the serial passages generated a virus with a predominant mutation at position 225 in HA RBS that replicated and transmitted at high rates in pigs.In ferret transmission studies, a reassortant H9N2 virus containing pH1N1 internal-protein genes readily transmitted to all contact animals by respiratory droplets6,5 TCID50 of H9N2:pH1N1 (P0). To determine nasal virus excretion, individual pigs were swabbed daily from 0 to four days post-inoculation (dpi). None of the pigs showed clinical signs of disease. The reassortant H9N2 virus was detected in nasal swabs of all experimentally infected pigs. The highest amount of virus was excreted during passages three and eight  virus and its descendent isolated after seven passages in pigs (H9N2:pH1N1 (P7)). The latter virus was chosen because virus titers in nasal swabs and the respiratory tract samples were highest at this passage, suggesting a substantial degree of swine-adaptation. As a positive control, we used a representative swine-adapted virus, the A/California/04/2009 (pH1N1) virus As expected, all six A/Cal/04/09 direct-contact pigs excreted high amounts of virus (AUC >17) and seroconverted with VN titers ≥192. While all six direct-contact animals in the H9N2:pH1N1 (P0) group also seroconverted and excreted virus, only two shed virus with an AUC >6. In the H9N2:pH1N1 (P7) group, in contrast, just four out of six direct-contact animals excreted virus but all of them showed an AUC >21, similar to the A/Cal/04/09 group. The remaining two H9N2:pH1N1 (P7) direct-contact pigs did not excrete any virus and failed to seroconvert. VN titers in the six H9N2:pH1N1 (P0) and four out of six H9N2:pH1N1 (P7) direct-contact pigs were <256.de novo assembled consensus sequence of the H9N2:pH1N1 (P7) stock virus are shown in Table de novo assembled consensus sequence of the H9N2:pH1N1 (P7) stock virus and the reference sequence for the A/quail/Hong Kong/G1/1997(H9N2) virus. The nonsynonymous variants detected at a frequency ≥0.5% in the H9N2:pH1N1 (P7) genome are shown in Supplementary Table We next compared the variants present in the H9N2:pH1N1 (P0) virus population and the H9N2:pH1N1 (P7) virus isolated from the nasal mucosa of a directly inoculated pig and the direct-contact pig with the highest nasal excretion in the transmission study. By comparing the virus population from the directly-inoculated and the direct-contact pigs we aimed to determine if the mutations selected during the serial passaging were maintained after transmission or if the transmission itself favored the acquirement of a different set of mutations. In addition, the H9N2:pH1N1 (P0) virus stock was included to determine whether the detected variants in the passage seven virus were already present in the virus stock before pig adaptation. Illumina MiSeq deep sequencing was performed on full genome RT-PCR products of the samples. Sequencing of H9N2:pH1N1 (P0) virus revealed contamination of the H9N2:pH1N1 virus stock with H9N2 internal-protein genes, which were most pronounced for the PB1, NS and NP gene segments (see Table i.e. the substitution of aspartic acid by glycine at position 225 of the HA RBS (HA-D225G). This mutation was absent  stock virus showed variation above 5% at positions 535 and 615 of the basic polymerase 2 protein (PB2), position 119 of the HA and position 179 of the non-structural 1 protein NS1) Table . Nonethe Table . 30. However, the continuous exposure of humans and pigs to avian H9N2 viruses might pose a real threat for public health if these viruses would acquire the genetic changes that allow them to transmit efficiently between mammals. In this study, we evaluated the efficiency of direct-contact transmission between pigs of a reassortant H9N2 virus containing pH1N1 internal-protein genes, and the effect of serial pig passages on transmission efficiency. We demonstrated, for the first time, that a pig-passaged reassortant H9N2:pH1N1 virus was excreted by more than half of the direct-contact pigs at titers similar to those of the parental pH1N1 virus.Since the late 1990s, avian H9N2 viruses have been isolated from pigs and humans, so far always as dead end events28. In fact, virus excretion from the nasal cavity and replication in the respiratory tract were present at variable rates during the ten serial pig passages. This finding contrasts with a previous study from our group using the same parental H9N2 virus (A/quail/Hong Kong/G1/1997), however without reassortment with pH1N1 internal-protein genes  virus did not show replication in the lungs during passage one, it replicated homogeneously in the entire respiratory tract during passage two. Strikingly, the virus replication in the lungs then progressively decreased during passages four, five and six to increase again at passage seven. This suggests the presence of a selection bottleneck due to the genetic pressure generated by the experimental set up31. However, a deeper analysis of the viral population would be needed to elucidate the possible cause of the decrease in the replication rates.Our data confirm the ability of the H9N2:pH1N1 reassortant viruses to infect pigs as was reported in previous studies28. In the aforementioned studies ≤50% of the direct-contact pigs excreted virus. This higher transmission rate suggests that the serial passaging in pigs contributed to the adaptation of the reassortant H9N2 virus. Yet of the six direct-contact pigs two animals that were housed in the same isolator did not excrete virus at all. This is most likely due to the low amount of virus excreted by the direct-inoculated pig (AUC 17.1), which not have reached a critical threshold to allow virus transmission. Further detailed genetic analyses of the virus excreted by the donor pig would be needed to better understand if there is, besides the low titer, also a genetic basis for the lack of transmission.We consider an influenza virus as swine-adapted when it succeeds to transmit between pigs with a similar efficiency as described for the endemic swine influenza viruses. Therefore, we compared pig-to-pig transmission of the seventh passage virus with that of the original reassortant and pH1N1 viruses. Whereas both reassortant viruses transmitted to direct-contact pigs, the seventh passage virus was clearly shed at higher titers by four out of six direct-contact pigs. This virus also showed higher transmission efficiency when compared with previous studies describing H9N2:pH1N1 reassortant viruses in pigs36, those studies focused either on the role of the PB2 and the M-coding genes or on the complete internal gene cassette. Not much is known about the exact role of pH1N1-origin NP-protein gene in the adaptation of influenza viruses to mammals. However, it has been described that the interaction between the NP and importin-α can influence the influenza virus host range. For example, the presence of the NP-N319K substitution was described as an important factor for the replication of H7N7 viruses in mice37. Nevertheless, both the H9N2 and the pH1N1 NP-protein genes lack that mutation. Thus, further analysis with reverse genetic generated reassortants would be necessary to elucidate if the combination of H9N2-origin NP-protein gene and pH1N1-origin internal genes has an effect in the replication and transmission of influenza viruses in mammals. Regarding the mutations detected during the analysis, only one of the mutations that appeared in the virus after seven passages and was maintained even after transmission, the HA-D225G substitution in the RBS. This mutation was therefore likely associated with the higher replication and transmission efficiency of the H9N2:pH1N1 (P7) virus. Interestingly, this mutation was present in all forty-seven swine and seventeen human H9N2 field isolates available in GenBank in November 2016. This mutation was also associated with enhanced replication and transmission of parental H9N2 after four passages in pigs . In the 1918 and 2009 pandemic H1N1 viruses this mutation has been associated with increased Siaα2,3Gal tropism39. In pigs and humans Siaα2,3Gal receptors are mainly found in the lungs13. The presence of the HA-D225G mutation after seven passages may therefore explain the consistent replication in the lungs during passage eight. Although specific receptor-binding tests would be necessary to elucidate the exact role of the HA-D225G mutation within the HA-RBS, our data suggest that it may be a marker for adaptation of H9N2 viruses to swine.To elucidate the effects of the serial passaging and the pig-to-pig transmission on the virus population, we compared the viral diversity in the original reassortant virus with the virus isolated after seven passages in pigs and the virus excreted after direct-contact transmission. It should be mentioned here that we detected H9N2 internal-gene segments in our original reassortant virus stock. Interestingly, the serial passaging resulted in the purification of the original virus and, after seven passages only the NP-coding gene was completely of H9N2-origin. As in the original stock the H9N2-derive NP gene segment was present at 91%, this high percentage might have been the reason for the purification towards the H9N2-origin instead of towards the pH1N1-origin as seen for the other gene segments. However, the NS-coding gene showed the same percentage of contamination and was completely selected upon passaging. Although previous studies demonstrated that pH1N1-origin internal genes conferred an advantage in replication and transmission of influenza viruses in mammals28, this report demonstrated that avian H9N2 influenza viruses can reassort with pH1N1 internal genes, resulting in enhanced virus transmission in pigs when compared to the parental, non-reassorted H9N2 virus. The present study also underscores that repeated introduction of reassortant H9N2 viruses into the swine or humans may result in the selection of virus variants with a transmission efficiency close to that of the endemic swine or human influenza viruses. However, the lack of transmission detected in one of the contact groups used for transmission of passage seven virus, emphasizes the complexity of the adaptation process of an avian virus to a mammalian species and the need for additional research to better understand this crucial step in cross-species transmission of influenza viruses.In line with previous studies17. Another passage in MDCK cells was made at the arrival of the virus to the Laboratory of Virology at Ghent University to generate a stock to be used in this study.The reassortant virus containing A/California/04/2009 (pH1N1) internal genes and A/quail/Hong Kong/G1/1997 (H9N2) HA and NA was produced by reverse genetics at the Department of Diagnostic/Pathobiology at Kansas State University. The virus was plaque purified, passaged three times in MDCK cells, characterized and experimentally validatedA/California/04/2009 (pH1N1) was kindly provided by the Centers for Disease Control and Prevention (US CDC). The virus underwent three passages in MDCK cells at the US CDC. Later one passage in eggs and one in MDCK cells were performed in the Laboratory of Virology at Ghent University before use.6.5 TCID50 of virus. Pigs were clinically monitored daily for general  and respiratory  symptoms. Nasal swabs were also collected daily from 0 to four dpi. At four dpi both pigs were euthanized with a lethal dose of pentobarbital sodium (≥100 mg/kg) in the jugular vein. The following tissue samples were collected for virus titrations: nasal mucosa respiratory part , nasal mucosa olfactory part (i.e. ethmoid labyrinth), trachea  and five different samples representative of the entire lung. The nasal mucosae (respiratory part) were pooled and a 20% (w/v) tissue homogenate was prepared and used to inoculate two pigs with this passage one virus inoculum. Ten blind  serial passages were performed in this way. Nasal swabs from all pigs, tissue samples and the inocula used for the subsequent passages were titrated on MDCK cells.Forty-seven three-week-old piglets were purchased from a commercial herd free of swine influenza and porcine reproductive and respiratory syndrome virus. Upon arrival, pigs were confirmed seronegative to influenza virus with a commercial blocking anti-influenza A nucleocapsid ELISA (ID-VET) and by virus neutralization (VN) tests. Consecutive passages started with the housing of two pigs in a biosafety level-3 (BSL-3) HEPA filtered isolator, and their intranasal inoculation with three ml of phosphate-buffered saline (PBS) containing 106.5 TCID50 of the respective virus. Two dpi, two pigs were introduced in each isolator allowing direct contact with the inoculated pig. All animals were clinically monitored and nasal swabs for virus isolation were collected daily from all pigs during eleven days after cohousing. Sixteen dpi, the animals were relocated to a BSL-2 HEPA filtered isolation unit. Serum samples were collected at 0, 16, 23 and 30 dpi and 0, 14, 21 and 28 days post-contact.For each transmission experiment nine three-week-old pigs were used. At 0 dpi three pigs were housed in three separate BSL-3 HEPA filtered isolators (one index pig per isolator) and intranasally inoculated with 10All experimental procedures were conducted in accordance with the E. U. Animal Welfare Directives, and were authorized and supervised by the Local Ethical and Animal Welfare Committee of the Faculty of Veterinary Medicine, Ghent University (EC2013/63 number).2, virus positive MDCK cells were visualized by immunoperoxidase staining. The cells were fixed with 4% paraformaldehyde for ten minutes at room temperature and subsequently incubated with mouse anti-NP monoclonal HB-65 antibody  for two hours. Incubation with horseradish peroxidase-conjugated goat anti-mouse polyclonal antibody  for one hour was followed by a development step with H2O2 as substrate and 3-amino-9-ethyl-carbazole (AEC) as precipitating agent. Virus titers were calculated by the method of Reed and Muench (1938) and expressed as log10 50% tissue culture infective doses (TCID50) per 100 milligram  or per gram (tissues).Nasal swabs from both nostrils were suspended in 1 ml PBS supplemented with 10% foetal bovine serum, 100 IU/ml penicillin and 100 μg/ml streptomycin and mixed vigorously at 4 °C for 1 hour. Tissue samples were weighed and grounded in PBS with 10 IU/ml penicillin and 10 µg/ml streptomycin to obtain 20% (w/v) tissue homogenates. Briefly, confluent monolayers of cells were inoculated with 10-fold serial dilutions of the sample. After five days of incubation at 37 °C with 5% CO50 of virus per well as previously described40. Briefly, 2-fold serum dilutions were incubated  with 100 TCID50 of MDCK cell-grown virus. MDCK cells (800.000 cells per ml) were incubated with the virus-serum mixture for 24 h, after which the virus positive cells were visualized by immunoperoxidase staining. Antibody titers were expressed as the reciprocal of the highest serum dilution that completely inhibited virus replication in MDCK cells.VN tests were performed on MDCK cells in 96-well plates, using 100 TCIDNasal virus shedding of individual pigs was quantified by calculation of the AUC. The means of the AUCs of different viruses were compared using standard two–sample Mann-Whitney U tests. Differences were considered significant when p < 0.05. GraphPad Prism Software, version 5, was used for statistical analyses.41. Two separate reactions were performed, using primers specific for the influenza A vRNAs: CommonUni12G (GCCGGAGCTCTGCAGATATCAGCGAAAGCAGG) and CommonUni12A (GCCGGAGCTCTGCAGATATCAGCAAAAGCAGG). Subsequently, all eight genomic segments were amplified in one PCR reaction containing an 1:1 mix of CommonUni12G and CommonUni12A cDNA, primer CommonUni13 (GCCGGAGCTCTGCAGATATCAGTAGAAACAAGG) (200 nM) and the Phusion High Fidelity polymerase (Thermo Scientific)43. RT-PCR was performed as described41, with the first 5 PCR cycles performed with an annealing temperature of 45 °C (instead of 72 °C). PCR products were purified using the High Pure PCR Product Purification Kit (Roche) and elution of the DNA in sterile ultrapure water.The RNA from the virus samples was isolated using the QIAamp Viral RNA Mini Kit . cDNA was synthesized with the Transcriptor First Strand Synthesis kit (Roche), already described500 ng of each purified RT-PCR sample was sheared with an M220 focused-ultrasonicator (Covaris) set to obtain peak fragment lengths of 400 bp. Next the NEBNext Ultra DNA Library Preparation kit (New England Biolabs) was used to repair the ends and to add the Illumina MiSeq-compatible barcode adapters to 100 ng of fragmented DNA. The resulting fragments were size-selected at 300 to 400 bp using Agencourt AMPure XP bead sizing (Beckman Coulter). Then, indexes were added in a limited-cycle PCR (10 cycles), followed by purification on Agencourt AMpure XP beads. The fragments were analysed on a High Sensitivity DNA Chip on the Bioanalyzer (Agilent Technologies) before loading on the sequencing chip. After the 2 × 250 bp MiSeq paired-end sequencing run, the data were base-called and converted to Illumina FASTQ files (Phred +64 encoding).41. The processed sequencing reads were subsequently mapped to the de novo assembled consensus sequence of the H9N2:pH1N1 (P7) stock virus sequence. This consensus sequence is available through GenBank accession number (KY785904-KY785911). The processed sequencing reads for the H9N2:pH1N1 (P7) stock virus were mapped to a reference genome composed of the H9N2:pH1N1 (P7) stock virus sequence and the A/quail/Hong Kong/G1/1997(H9N2) virus genome. The raw sequencing data were submitted to the NCBI Sequence Read Archive where they can be found under project number: SRP095510.Sequence data analyses were performed on the resulting Illumina FASTQ files (Phred 64+ encoding) using CLC Genomics Workbench (Version 7.0.3) following the analysis pipeline as describedSupplementary Table S1: Variants present in H9N2:pH1N1(P0) virus stock.


GDF5 and NOG were identified to be responsible for SYM1. We have previously reported on a p.Leu373Arg mutation in the GDF5 proregion present in a Chinese family with SYM1. Here, we investigated the effects of the GDF-L373R mutation. The variant caused proteolysis efficiency of GDF5 increased in ATDC5 cells. The variant also caused upregulation of SMAD1/5/8 phosphorylation and increased expression of target genes SMURF1, along with COL2A1 and SOX9 which are factors associated with chondrosis. Furthermore, we developed a human-relevant SYM1 mouse model by making a Gdf5L367R (the orthologous position for L373R in humans) knock-in mouse. Gdf5L367R/+ and Gdf5L367R/L367R mice displayed stiffness and adhesions across the proximal phalanx joint which were in complete accord with SYM1. It was also confirmed the joint formation and development was abnormal in Gdf5L367R/+ and Gdf5L367R/L367R mice, including the failure to develop the primary ossification center and be hypertrophic chondrocytes during embryonic development. This knock-in mouse model offers a tool for assessing the pathogenesis of SYM1 and the function of the GDF5 proregion.Proximal symphalangism (SYM1) is an autosomal dominant disorder, mainly characterized by bony fusions of the proximal phalanges of the hands and feet.  The disease manifests as fusions of the proximal interphalangeal joints, typically on the third to fifth digits of the hand, and also affects the thumb, making it impossible to make a fist. The bony fusions occur on metacarpophalangeal joints as well as on the auditory ossicles, leading to conductive hearing loss in some affected individuals . The disGDF5 gene, also called CDMP1, is located on 20q11.22. GDF5 plays important roles during early and later limb development, as well as in various musculoskeletal physiological processes [The rocesses . It alsorocesses . GDF5 berocesses .GDF5 are associated with various autosomal recessive and dominant syndromes related to skeletal abnormalities, such as Brachydactyly C , Acromesomelic chondrodysplasia Grebe type , Du Pan syndrome , Symphalangism proximal 1B , Multiple synostoses syndrome 2  and Brachydactyly A2  [Mutations in Gdf5L367R (the orthologous position as L373R in human) knock-in mice. The clinical phenotype of proximal symphalangism and similar histology of skeletal abnormalities were observed in the mutant mice. This offers a valuable tool for assessing the pathogenesis of SYM1 and characterizing the abnormal function of mutant GDF5.Here, we developed a human-relevant SYM1 mouse model by making a L373R mutation. Expression vectors with wildtype or L373R mutant GDF5, both lacking signal peptides, were transfected into ATDC5 cells. The percentage of mature GDF5 protein compared to total GDF5 protein (the summation of mature GDF5 and proprotein GDF5 protein) was assessed to indicate the relative hydrolysis efficiency of the GDF5 protein. The results of the Western Blotting suggested that the GDF5L373R mutation increased the proteolysis efficiency of the protein , a cartilage-specific matrix protein, and SRY-box 9 (SOX9), a high-mobility-group transcription factor required for chondrogenic differentiation of mesenchyme and chondrocyte maturation [L373R virus , which negatively regulates the expression of COL2A1 and SOX9  , homozygous (Gdf5L367R/L367R), and wild type Gdf5+/+ mice  Figure . We used) Figure . PCR ande Figure .GDF5SYM1 mice by visual and radiological inspection. The length and the appearance of limbs were not showed obviously different from new born mice of three genotypes which were observed by naked eye. While after the mice were remove from milk, they began to grab food and climb on the cage. We observed that Gdf5L367R/+ and Gdf5L367R/L367R mice would not like to climb on the cage and they often dropped from the cage because of not grabbing the cage steady. Differences became more pronounced in adult mice. Gdf5L367R/+ and Gdf5L367R/L367R mice showed “straight hands”, with the second to fifth fingers unable to curve , middle phalanges , distal phalanges , and metacarpals  13.5 and 16.5 and postnatal day (P) 0 and 7. It was clear that mesenchymal cells had already began to aggregate in the limb buds of wild type mice at E13.5, forming a narrow restricted surface area in the proximal interphalangeal joint by E16.5. In e Figure .Gdf5L367R/+, and Gdf5L367R/L367R mice. Real-time RT-PCR, western blot, and histochemistry all showed a higher expression of Sox9 and Col2a1 in wild type mice than in Gdf5L367R/+ and Gdf5L367R/L367R mice  which carries the Gdf5L367R mutation, the corresponding mutation to the human GDF5L373R mutation found in a SYM1 family previously reported on by our group. GDF5 is best known for its role in early chondrogenesis and joint formation. It has been shown to regulate the proliferation and differentiation of chondrogenic tissue during bone and cartilage development, and it is also associated with osteoarthritis [in vitro and in vivo studies have shown that GDF5 induces chondrogenesis. One study showed that cultured embryonic chick mesenchymal cells overexpressing GDF5 distinctly presented mature hypertrophic chondrocytes [L373R than wild type GDF5. The corresponding increase in the phosphorylation of SMAD1/5/8 in cells infected by GDF5L373R suggested that the GDF5L373R mutation may affect the interaction between GDF5 and bone morphogenetic protein receptors. Mature GDF5 binding to BMPR1B would induce downstream signaling cascades and SMAD-dependent signal transduction to stimulate chondrocyte proliferation [Gdf5L367R/+ and Gdf5L367R/L367R mice. Meanwhile Gdf5L367R/+ and Gdf5L367R/L367R mice showed chondrocyte proliferation, hypertrophy and calcification. We also noticed that, comparing with wild type, in Gdf5L367R/+ and Gdf5L367R/L367R mice the cells in Proximal phalanx showed endoplasmic reticulum dilatation (data was not showed). This reminded us that the abnormal unfolded mutant Gdf5 might activate endoplasmic reticulum stress which is closely interconnected with autophagy and apoptosis. According to report in recent years, autophagy could regulate the secretion of Col2a1 [Gdf5L367R/+ and Gdf5L367R/L367R mice. All the results suggested that the gain-of-function mutation GDF5L373R could cause the persistence of cartilage.In the above project, we describe a SYM1 mouse model . Except for the limited quantity of Gdf5 extracted from limb buds of the fetal mice, the most possible reason for the result might be the time points. Although the time point of joint development is E11.5-E13.5, Gdf5 plays its role much earlier than that. So more accurate monitoring need to be done during the early embryo development on the GDF5SYM1 mice.These different phenomena caused by GDF5 mutations can be associated with the position in which the mutations exists on GDF5. Similar to other BMPs, GDF5 is composed of 501 amino acids (aa), including an N terminal signal peptide domain (1-27 aa), a propeptide region, a putative polybasic processing site (28-381 aa) and a 120 amino acid polypeptide at the carboxyl terminus (382-501 aa). The GDF5 pro-form undergoes endopeptidase cleavage at the polybasic processing site (RRKKRR). Upon processing, the remaining 120 amino acids form the mature GDF5 protein , which tGdf5L367R knock-in mouse model completely replicates the human SYM1 phenotypes. Gdf5L367R/+ mice show inflexibility in their fingers and toes, similar to SYM1 patients. The results of Micro-CT scanning, Alcian blue-alizarin red staining, and histological analysis identified that the ankylosis was caused by abnormal joint fusions. After observing their skeletal development, we found that Gdf5L367R/+ mice failed to develop primary ossification centers even at E16.5, though the mesenchymal cells properly condensed at E13.5. The interphalangeal joints present high chondrocyte proliferation, hypertrophy, and calcification after birth, with high expression of chondrogenic markers. The GDF5SYM1 mouse model also provides us with viable Gdf5L367R/L367R mice to analyze. Homozygous phenotypes are limited in humans due to the mode of inheritance of SYM1. In these mice, the metacarpal joints, the proximal phalanx, and the distal phalanx are fused together, similar to the multiple synostoses syndrome (SYNS).The mouse is a convenient animal model for studying human diseases. The mouse and human GDF5 precursor proteins share 91% identity, and the mature proteins differ by only 1 amino acid. Although GDF5-deficient mice have been identified since 1994  and bracGdf5L367R/+ and Gdf5L367R/L367R mice were less alert to their environment than wild type mice. It is possible that some kind of conductive hearing loss occurs in the GDF5SYM1 mice, which makes the hearing in Gdf5L367R/+ and Gdf5L367R/L367R mice less sensitive than the wild type mice and results in an observable loss of alertness.While we did not examine the hearing of the mice to see whether they experience the progressive conductive deafness seen in some patients, we did find that both ATDC5 chondrogenic cells were cultured in DMEM/F12 medium (1:1 mixture of DMEM and Ham’s F-12 medium), with 5% fetal bovine serum .GDF5, both lacking the signal peptide, were amplified by PCR and cloned into pcDNA3.1 vectors, making pcDNA3.1-GDF5-WTdelSP and pcDNA3.1-GDF5-MTdelSP respectively. After confirming the sequencing, all plasmids were transfected into ATDC5 cells using Lipofectamine 2000 . The transfected cells were cultured and processed for Western blot assays.The coding sequences of both wild type and mutant 5 cells/well and incubated in 500 μl medium at 37°C. All lentivirus constructs were used at 20 MOI (multiplicity of infection) to infect ATDC5 cells. The original medium was replaced with DMEM/F12 medium after culturing for 1 day. The transfected cells were cultured and processed for the following Western blot assays.Lentiviruses containing wild type GDF5 (GDF5-WT), mutant GDF5 (GDF5-L373R), or negative control (NC) were produced by Genechem (China). ATDC5 cells were seeded in 24-well culture plates at a density of 2×10Total RNA was isolated from tissues using Trizol Reagent . The reverse transcription (RT) step was performed with a PrimeScript™ II Reverse Transcriptase kit .Real-time RT-PCR was performed with a total volume of 25 μl, containing 12.5 μl SYBR Premix Ex Taq , 50 ng cDNA (5 μl), and 4 x 2.5 μl primers (250 nM each) in each tube. Primer information can be found in Table All samples were run in triplicate in separate tubes. Relative expression levels were calculated using the comparative threshold cycle (CT) method, with glyceraldehyde-3-phosphate dehydrogenase (Gapdh) used as a house-keeping gene.Transfected cells or limbs of knock-in mice were lysed in ice-cold radio immunoprecipitation assay (RIPA) buffer containing 0.01% protease  for 30 min at 4°C. Cell lysis was centrifuged at 12000 rpm for 15 min at 4°C and the protein concentrations were quantified using the Bicinchoninic Acid (BCA) kit . SDS-PAGE and western blotting were performed using standard techniques. The primary antibodies of GDF5, SOX9, COL2A1, SMURF1, ID1 and phosphorylated SMAD1/5/8  were used at a dilution of 1:1000, and β-tubulin  was used at a dilution of 1:10000 in PBS-Tween 20.® In Situ – Fluorescence kit (Sigma-Aldrich) after transduction with lentivirus NC, GDF5-WT, or GDF5-L373, following the instructions of the manufacturer. Briefly, cells were incubated with primary antibodies against GDF5 and BMPR1B , then washed with wash buffer for 2×5 min. Two PLA probes (1:5 dilution) were applied and incubated for 60 min at 37°C. Ligase was diluted at 1:40 in the solution and incubated for 30 min at 37°C. The samples were then incubated for 100min with Polymerase at 37°C and mounted with Duolink In Situ Mounting Medium with DAPI for 15 min. The cells were visualized using a confocal microscope system.Interactions between GDF5 and BMPR1B were detected using the DuolinkThe L367R mutation was introduced into exon 2 using site-directed mutagenesis. The targeting vector plasmid pBR322 for the Gdf5 knock-in mice also contains a 3 kb 5’homologous arm, a Pgk-neomycin (neo) cassette flanked with two FRT sites between exon1 and exon2, a 3 kb 3’homologous arm, and an MC1-thymidine kinase cassette outside of the homology region. The homologous arms were obtained from the 129S6 Mouse BAC plasmid by homologous recombination and ampicillin screening. ES cells were electroporated with 35μg linearized targeting vector and selected in medium containing G418 (300mg/L) and ganciclovir (2 μmol/L) 24 h and 48 h after electroporation, respectively. Correctly targeted ES cell clones were screened by gDNA extraction and PCR genotyping. ES cell clones that showed the desired chromosomal integration were injected into blastocysts, which were then transplanted into pseudo pregnant female mice. Chimeric males were mated with C57BL/6 females and the resulting 5 offspring were genotyped. The Frt-flanked Neo cassette was removed by crossed with Flp mice, produced by Shanghai Nanfang Model Biology.Skeletons of newborn mice were fixed in 4% paraformaldehyde/PBS overnight at 4°C, dehydrated, then stained with Alizarin red/Alcian blue staining. For Safranin fast green staining histology, adult mouse limbs were fixed and dehydrated, then 6 μm sections were mounted on glass slides, rehydrated, and stained for 12 min separately . Living animal micro-CT imaging was performed setting micro-CT slices at 40 μm that were then reconstructed in 3D arrays.All animals were euthanized according to protocols approved by the Chinese Institutional Animal Care and Use Committee (CIACUC). Healthy newborn mice from the same brood with three genotypes were used in the research. Intact or dissected embryo limbs or skinned and decalcified newborns were sectioned using a frozen slicer. For general morphology, sections were stained with hematoxylin and eosin. After blocking with 5% normal goat serum, sections were incubated with anti-sox9  or anti-col2a1  overnight at 4°C. Sections were incubated for 30 min with secondary antibody followed by amplification with SABC , and visualization with DAB .t-test. P values of less than 0.05 were considered statistically significant.All data presented are representative of at least three independent experiments for each condition. Statistical analyses were performed using Student’s