In contrast, platinum clusters on the pristine patch have spin density distributions similar to the bare cases. The different spin density distributions come from whether underlying carbon atoms have radical characters or not. In the pristine patch, center carbon atoms do not have spin densities, and they cannot influence radical characters of the absorbed cluster. In contrast, radical characters appear on the defective sites, and thus spin density distributions of the adsorbed clusters are modulated by the Pt-C interactions. Consequently, characters of platinum clusters adsorbed on the sp Su. Su2 surractions . The DFT2 patch can modulate the catalytic activity of the supported cluster due to the onset of unpaired electrons on Pt atoms. Based on the assumption, the current study will focus on whether spin states of C96H26 support have a power to influence the properties of the adsorbed Pt clusters. Furthermore, we are interested in how introduction of vacancy-type defects on the radical C96H26 support changes the electronic properties of the surface. These changes would have an impact on determining the properties of Pt clusters adsorbed on the sp2 support. To increase our understanding of the interactions between a radical sp2 support and a Pt cluster, we performed density functional theory (DFT) calculations. The main aim in the current DFT study is to clarify how different electronic properties of C96H26 support with or without vacancy-type defects influence the interactions with Pt clusters, and concomitantly the properties of the absorbed clusters.Another interesting feature of nanometer-size graphenes is that they have radical character in the ground state, depending on their shape and size ,49,50,5196H26 patch affect the interactions with Pt clusters, we investigated how Pt6 clusters bind into the sp2 surface. Following the previous study  where Etotal(triplet state) or Etotal(singlet state) is the total energy in each state. As shown in Estate(Pt6-C96H26) values. These negative ΔEstate(Pt6-C96H26) values indicate that the triplet state of a Pt6-C96H26 configuration is energetically favorable relative to the singlet state, irrespective of the cluster shapes. Furthermore, we see from Estate values in the Pt6-C96H26 configurations than those in the bare Pt6 clusters. Thus, the Pt-C interactions influence relative stability of the triplet to singlet states of the Pt6 clusters.We estimated in 6-C96H26 structures are also displayed in 96H26, although the stable triplet state of pristine C96H26 has radical characters only on edge carbon atoms. Likewise, we see spin densities on the adsorbed Pt cluster in the configurations. Basically their spin density distributions are similar to those in the bare Pt6 clusters  of carbon atoms removed from C96H26, ranging from 1 to 3. Removed carbon atoms are colored in 96n–H26. Using the initial geometries, we obtained optimized structures for the vacancy-type defects in the triple and singlet states. Then, the energy difference between the two spin states was evaluated in each vacancy-type defect, given as ΔEstate(C96n–H26) in Estate(C96n–H26) values irrespective of the number of carbon atoms removed from C96H26. The negative ΔEstate(C96n–H26) values indicate that each C96n–H26 has energetically stable triplet state.Prior to discussing Pt clusters adsorbed on the sp95H26), while there are two five-member rings in the other vacancy-defects (C94H26 and C93H26). Besides, removing odd-numbered carbon atoms from C96H26 generates one coordinatively unsaturated carbon atom, given by red in 96H26. As displayed in 96n–H26 over the singlet state, let us first use the singlet state to increase our understanding of how a Ptk cluster interacts with a defective site on the sp2 surface. Following the previous study on interactions between a Pt6 cluster and pristine C96H26, we have a special interest on how the presence of a vacancy-type defect of the patch affects the interactions with a Pt6 cluster. In addition, we will discuss dependences of the interaction energies on size of clusters whose number of contained Pt atoms (k) being smaller than 6. 6 cluster adsorbed on the mono-, di-, and tri-vacancy-type defects, respectively.Despite the preferences of the triplet state of C6 cluster binding into the mono-vacancy-type defect in 6-C95H26 structures displayed in k-C95H26 structures have a Ptk-1 moiety contained in stable Ptk-1-C95H26 structures. Of course, there are other possibilities for Ptk binding modes. However, our computational resource is limited, reluctantly we did not obtain other optimized geometries. We evaluated the binding energy in each configuration defined as [Ebind = Etotal(Ptk-C96n–H26) – Etotal(C96n–H26) – Etotal(Ptk)], where k ranges from 1 to 6  are also displayed in Corresponding optimized structures for the smaller PtEtotal(Ptk-C96n–H26) is the total energy of an optimized C96n–H26 geometry, Etotal(C96n–H26) is that of the optimized C96n–H26 geometry, and Etotal(Ptk) is that of the optimized Ptk cluster. For the DFT calculations of the binding energies, a counterpoise correction for basis set superposition error (BSSE) was included , where E(Ptk on surface) is the total energy of Ptk cluster taken from an optimized Ptk-C96n–H26 structure and E(Ptk) is that of the optimized geometry for the bare Ptk cluster. Positive ΔE(Ptk) values in bind and ΔE(Ptk) values, the balance between the stabilization from the Pt-C bond formation and the destabilization from the cluster deformation is a key in determining the stability. From k-C93H26 and Ptk-C95H26 (Ptk-C94H26) in terms of the number of Pt atoms binding directly into orange atoms in the defective site to cleave connections between adjacent orange atoms.Compensating the weakening of the interactions, remaining Pt atoms of a clusters are additionally bound to carbon atoms of a defective site to maximize the Pt-C interactions. Then their clusters are more or less deformed from the most stable configuration in the gas-phase ,57,58. Tk-C95H26 (Ptk-C94H26), one Pt atom participates in cleaving the orange connection(s), irrespective of the cluster size. In the tri-vacancy cases containing larger ten-membered ring, two Pt atoms in a cluster bind to the defective site to split two orange connections. The accommodation of two Pt atoms cannot be seen in the Pt1-C94H26 structure, and thus the significant enhanced stabilization in Ptk-C93H26 is understandable. Moreover the acceptability of the ten-membered-ring to trap Pt atoms differentiates C93H26 from C95H26 and C94H26 in terms of their reactivity toward Ptk clusters. Due to the strong interactions between a Pt6 cluster and a vacancy-type defect, we can see unique orbital features, which cannot be seen in C96n–H26 -C94H26(B) configuration has the HOMO and LUMO consisting of 5d(Pt) orbitals. On the other hand, levels of 5d(Pt)-based orbitals in the Pt6-C95H26 strongly depend on their cluster-shape. In the Pt6(i)-C95H26(A) and Pt6(ii)-C95H26(C) configurations, such 5d(Pt)-based orbital lies larger than 1.3 eV above the LUMO, whereas the LUMO+1 consists of 5d(Pt)-based orbitals in the other Pt6-C95H26 configurations.In fact, 5d(Pt)-based orbitals, given by blue bars in state(Pt6-C96n–H26)), as tabulated in As shown in 6-C96n–H26 configurations have radical Pt6 clusters on defective graphene patches As representative cases, spin density distributions on the Pt6(i) cluster binding into C95H26 or C94H26 are shown in 6-C96n–H26 configurations (Pt6(i)-C95H26(B), and two Pt6(i)-C94H26 configurations), whereas the Pt6(i)-C95H26(A) configuration does not have such radical cluster due to the absence of 5d(Pt)-based frontier orbitals (6(i)-C95H26(B) and Pt6(i)-C94H26(B), the spin density distributions are strongly deviated from those in the bare Pt6 cluster, whereas the Pt6(i)-C94H26(B) has similar distributions. Furthermore, we found a relationship between spin densities of Pt clusters and those on the defective sp2 surface. When spin density distributions of the Pt cluster of a Pt6-C96n-H26 configuration are (not) similar to the bare case, spin densities are (not) delocalized over the carbon surface. Similar tendencies were found in spin density distributions of the stable triplet Pt13-Cn96–H26 configurations [orbitals . In the urations , because6 clusters on defective sp2 surfaces  into a nanometer-size graphene patch (C96H26) with or without vacancy-type defects. According to the DFT calculations, removing a few carbon atoms (n) from C96H26 results in the formation of five-membered rings as well as coordinatively unsaturated carbon atoms. Introduction of a vacancy-type defect on C96H26 strongly affects spin density distributions in its stable triplet state. Although spin densities appear only on edge carbon atoms of the triplet C96H26 structure, defective graphene patches have radical carbon atoms at the center where the reactive carbon atoms exist. These spin density distributions differentiate characters of Pt clusters adsorbed on defective graphene patches from those on the pristine. According to the DFT calculations, spin density maps of Pt clusters on C96H26 are similar to those of the corresponding bare clusters. In contrast, Pt clusters interact strongly with radical carbon atoms in defective graphene patches, and thus spin density distributions of the adsorbed Pt clusters are usually deviated from the bare cases. Consequently, DFT calculations propose that characters of Pt clusters adsorbed on the sp2 carbon surface can be modulated by introducing vacancy-type defects.Density functional theory (DFT) B3LYP calculations were employed to investigate the adsorption of Pt


Reoviridae). BTV serotypes 1–24  are transmitted by competent biting Culicoides midges and replicate in mammalian (BSR) and midge (KC) cells. Previously, we showed that genome segment 10 (S10) encoding NS3/NS3a protein is required for virus propagation in midges. BTV serotypes 25–27  do not replicate in KC cells. Several distinct BTV26 genome segments cause this so-called ‘differential virus replication’ in vitro.Transmission of vector-borne virus by insects is a complex mechanism consisting of many different processes; viremia in the host, uptake, infection and dissemination in the vector, and delivery of virus during blood-feeding leading to infection of the susceptible host. Bluetongue virus (BTV) is the prototype vector-borne orbivirus . BTV11 with a small in-frame deletion in S10 infected blood-fed midge midguts but virus release from the midgut into the haemolymph was blocked. BTV11 with S1[VP1] of BTV26 could be adapted to virus growth in KC cells, and contained mutations subdivided into ‘corrections’ of the chimeric genome constellation and mutations associated with adaptation to KC cells. In particular one amino acid mutation in outer shell protein VP2 overcomes differential virus replication in vitro and in vivo.A 100 nl blood meal containing ±10Culicoides midges can strongly differ for very closely related viruses.Small changes in NS3/NS3a or in the outer shell protein VP2 strongly affect virus propagation in midges and thus vector competence. Therefore, spread of disease by competent  Arthropod-borne viruses have a significant social and economic impact on both human and animal health. The majority of emerging and re-emerging infectious diseases are vector-borne and/or zoonotic , 2. VectCulicoides midges (Diptera: Ceratopogonidae). Historically, BT is caused by 24 serotypes of bluetongue virus (BTV)   of atypical BTV26 were evaluated for their ability to replicate in mammalian and Culicoides cell lines and in C. sonorensis midges. Effects of viral genetics on vector competence is discussed.To further investigate differential virus replication Culicoides (KC) cells were grown in modified Schneider’s Drosophila medium with 15% heat inactivated FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin at 28 °C . A virus stock was obtained by one passage on BSR cells at Wageningen Bioveterinary Research (WBVR) and designated BTV26. BTV11 was isolated from the spleen of a white-tailed deer from Texas in 2011, passaged once in embryonated chicken eggs, and four times in BHK21 cells before use in midge feeding/injecting. A virus stock for in vitro experiments was obtained by one passage on BSR cells at WBVR, and designated wtBTV11. All other viruses in this study were generated by reverse genetics , containing the same BTV11 as above (S111) and BTV26  sequences were designed and purchased. Each chimeric S1 contained one of three defined domains of the RdRp of BTV26 (S111/26) 26, S2[VP2]11, S4[VP4]11, and S8[NS2]11. Subsequent virus passages on BSR cells (p6) resulted in three aa mutations; one in S1[VP1]26, two in S8[NS2]11, and one incomplete aa mutation in S4[VP4]11, as well as one incomplete silent mutation in S2[VP2]11. These five nucleotide changes were also found after ‘rescue’ of BTV11(S126) in KC cells (p3r) but four aa mutations were not completely changed immediately after ‘rescue’ in BTV11(S126)kc(r). Apparently, these aa mutations were rapidly selected in both cell types and are likely associated with chimeric corrections in BTV11(S126). In addition, one incomplete E321G aa mutation in VP211 was found. After three subsequent virus passages (p3r+3p), BTV11(S126)kc contained three additional incomplete mutations, whereas the incomplete E321G aa change in VP211 was complete (Table 26 was complete after three subsequent virus passages (p3r+p6), whereas two other incomplete changes remained with mixed nucleotide mutations. The respective regions of BTV11kc and BTV11bsr were also sequenced, but no mutations were found, except for aa mutation E321G in S2[VP2]11 in BTV11kc. This indicated that the aa mutation E321G in S2[VP2]11 is associated with virus growth in KC cells.BTV11(S126) in KC cells. Seven out of nine mutations resulted in aa mutations. Five point mutations (four aa mutations and one silent mutation) are associated with corrections of chimeric interactions, since in BTV11(S126)bsr contained the same mutation. Four additional point mutations (three aa mutations and one silent mutation) seem to be associated with adaptation to KC cells, although the selection pressure was not very high since two aa mutations were still incomplete after six virus passages in KC cells. The most obvious and strongly selected change is aa mutation E321G in VP211. Even more, rgBTV11 also contained this E321G mutation after virus passages in KC cells.In summary, a total of nine mutations were found in several genome segments after rescue and passages of BTV11 or at 10 dpf to distinguish between infection, replication and dissemination of virus [5.1 TCID50/ml, M: 106.3 TCID50/ml, H: 108.2 TCID50/ml. Fully engorged midges were selected and processed at 0 dpf to confirm uptake, and at 10 dpf to study virus replication and dissemination  - is roughly calculated to a blood meal titer of ±2 × 105 TCID50/ml for wtBTV11. Thus, one MAID50 corresponds to ±20 TCID50 wtBTV11 in a blood meal estimated to be 100 nl for fully engorged competent C. sonorensis midges.Bodies of all groups were PCR-positive at 0 dpf, except for one in group L. The difference in virus dose in blood meals was clearly observed. Heads were negative at 0 dpf, except for one in groups L and M and two in group H. Likely, contamination by decapitation, virus on mouth parts, or incomplete swallowing of the blood meal can occur. At 10 dpf, positive results of bodies and heads indicated infection, replication and dissemination of wtBTV11. Bodies and heads in all three groups clearly segregated in PCR-positives and negatives. The percentage of PCR positives at 10 dpf was similar for bodies and heads in each group, and was approximately 40%, 79% and 89% for group L, M and H, respectively Fig. . The meadel) lacking a region in NS3/NS3a encompassing Late Domain, and is called ‘DISA’. Midges were blood-fed containing ±2 × 106 TCID50/ml DISA or rgBTV11. This corresponded to ±200 TCID50 which is ±10 MAID50, and thus sufficient to infect a high percentage of midges. In addition, virus replication was studied after intrathoracic inoculation with the same amount of DISA vaccine virus.BTV1 deficient for NS3/NS3a expression, named Disabled Infectious Single Animal (DISA) vaccine does not propagate in midges after intrathoracic inoculation . Here, wvia the haemolymph into the head.Virus uptake by feeding or inoculation was confirmed by 100% PCR positive bodies at 0 dpf and 0 dpi Fig. . Mean Cq50. Despite of 75% PCR-positive bodies at 10 dpf with DISA, all heads remained PCR negative. Likely, DISA initially infected gut cells but is not released into the haemolymph and was therefore not disseminated to the head. DISA-propagation in competent midges after oral uptake was unsuccessful by lack of Late Domain in NS3/NS3a protein. After intrathoracic inoculation, a small decrease in mean Cq value (more virus) in bodies and heads was observed at 10 dpi. This indicated infection and replication of DISA after intrathoracic inoculation similarly as observed in bodies after blood-feeding. Deletion of Late Domain of NS3/NS3a protein caused differential virus propagation in vivo. We conclude that functional NS3/NS3a is essential for BTV propagation in competent midges after oral uptake. These results confirmed the DISA principle of BTV lacking functional NS3/NS3a expression.At 10 dpf, 65% of the bodies and 44% of the heads of midges fed with rgBTV11 were PCR-positive, indicating infection, replication and dissemination of rgBTV11 bsr, BTV11(S126)kc, and rgBTV11 were fed to midges using a normalized virus titer of ±2 × 106 TCID50/ml bsr. As expected, rgBTV11 efficiently propagated in blood-fed midges, since bodies and heads at 10 dpf were 80% and 88% PCR-positive, respectively kc propagated in midges, as indicated by > 90% infected midges at 10 dpf. In contrast, BTV11(S126)bsr showed 36% and 16% PCR-positive bodies and heads at 10 dpf, respectively. This indicated that infection of midges by mammalian cell-adapted BTV11(S126)bsr was less efficient than by Culicoides-adapted BTV11(S126)kc and rgBTV11. In addition, mean Cq values were higher (less virus) for BTV11(S126)bsr than for BTV11(S126)kc and rgBTV11 kc propagated similar to, or even slightly better than, rgBTV11 and more importantly, much better than BTV11(S126)bsr. We conclude that infection, replication and dissemination of BTV11(S126)kc is more efficient in competent midges than of BTV11(S126)bsr. Furthermore, the increased propagation in vitro and in vitro of BTV11(S126)kc is likely caused by one aa mutation in outer shell protein VP2.BTV11 of reverse genetics derived BTV1 reassortant (corresponding to ±3 MAID50 of wtBTV11) did not infect competent midges, although intrathoracic inoculation was successful [50 but likely the used virus strain is even more important. The midge feeding model was used to study propagation of BTV11 mutants in more detail.Spread of arthropod-borne viruses depends on virus replication in the host and the competent insect vector leading to viremia in the host and virus excretion in insect saliva, respectively. wtBTV11 infects competent on 7 dpf . In thisf midges . A clearccessful . ClearlyC. sonorensis midges after blood-feeding  replicated well in BSR and KC cells  (here named DISA) lacking Late Domain of NS3/NS3a demonstrates similar characteristics  corresponds to the in-frame deletion of 77 aa codons in the experimental DISA vaccine for African Horse Sickness [in vitro characteristics. It is likely that it would also be blocked in virus release and dissemination in blood-fed midges. Taken together, a small deletion in NS3/NS3a protein encompassing Late Domain is sufficient to establish nonvirulence, the DIVA principle (Differentiating Infected from Vaccinated), and the DISA principle  .in vitro for several reassortants of typical BTV1 and atypical BTV26. In this study, we aimed to determine differential virus replication in vivo, and rescued typical BTV11 with S126. Rescue of BTV11(S126) was less efficient than BTV11, and adaptive changes after rescue were assumed to improve virus growth on BSR cells. In agreement with Pullinger et al. [26) initially showed limited virus production in KC cells  to KC cells. BTV11(S126)kc had obvious phenotypical differences as observed by increased virus growth and enlargement of immunostained foci in infected KC cell monolayers kc and rgBTV11kc. BTV11(S126) was independently rescued again, and in addition, an S126 mono-reassortant of BTV1 strain RSArrrr/01 was rescued (BTV1(S126) according to previous results [26 mono-reassortants showed hardly replication in KC cells but could be adapted to KC cells .26)  contained the same point mutation leading to the D309G aa mutation, while independently rescued BTV11(S126) had a point mutation G936A leading to aa mutation D309N. These results demonstrate the importance of mutating Asp on aa position 309. Asp-309 is mapped in the unmodelled region between NTD and PD of RdRp and has been suggested to interact with virus proteins of the replication complex [26 with proteins of BTV1 or BTV11 resulting in increased virus replication in both BSR and KC cells  is a non-conserved aa residue. The VP4, a capping enzyme, is part of the replication complex and catalyses the formation of a cap1 structure at the 5’ termini of core RNA transcripts. VP4 has distinct domains for its different activities [The aa mutation L399S in VP4tivities . The aa 11 of rescued BTV11(S126) contained aa mutations L171F and E221G. NS2 of atypical BTV25-27 also contained F-171, and E-221 in NS2 of atypical BTVs is highly conserved within a variable region. Independently rescued BTV11(S126) also contained L171F but E221G was not found, whereas S81 of rescued BTV1(S126) was not mutated. We speculate that these aa residues of NS2 are involved in interactions with VP1. NS2 recruits viral ssRNA from the cytoplasm, but its interactions with core proteins VP1, 3, 4, 6, and 7 are largely unknown (reviewed in [NS2iewed in ). Our re26, one aa mutation in VP311, and one silent mutation in S126 were found in BTV11(S126)kc  virus. More importantly, no obvious mutations in VP126 were found that are associated to adaptation to virus growth in KC cells. Surprisingly, we found that VP126 is not involved in differential virus replication in vitro.One aa mutation in VP126)kc and BTV11(S126)bsr is nucleotide mutation A981G in S211 resulting in E321G in VP211  contained also mutations in VP211; E401G and I503V, and the S126 chimeric virus based on BTV serotype 1 also contained one N229S mutation in VP21. In a previous study, BTV8/net07/e1 was also passaged in KC cells (BTV8/net07/e1kc3) leading to two nucleotide changes of which one resulted in R400G in VP28 [11 are located in or close to the externally exposed flexible tip domain of VP2 mapped to aa 191–407. A similar region of VP2 (aa residues 278–504) has been shown for AHSV [in vitro virus replication in both cell types, but the corresponding region (aa residues 284–510) in AHSV4-VP2 seems to be involved specifically in virus replication in KC cells, since virus release from KC cells of this AHSV deletion mutant was slightly delayed [in vitro.The most obvious difference between BTV11kc propagated better in competent midges than BTV11(S126)bsr  of wtBTV11 infects 50% of fully engorged midges and corresponds to a complete blood meal of 100 nl containing ±2 × 105 TCID50/ml or 20 TCID50 wtBTV11. Clearly, infection of midges by blood-feeding depends on virus uptake but the used virus strain is even more important. A small 72 amino acid in-frame deletion of NS3/NS3a protein completely blocks virus dissemination in blood-fed midges. Further, detailed knowledge of protein-protein interactions in the virion was generated by analysis of BTV reassortants. In addition, a point mutation in outer shell protein VP2 was identified in Culicoides-adapted BTV that is associated with differential virus replication in vitro and in vivo and thus with vector competence. In conclusion, two examples of small changes in BTV are shown which strongly affect virus infection, replication and dissemination of virus in competent midges. All these processes are part of the key mechanism crucial for vector competence and thus for spread of the bluetongue virus.The midge feeding model, including decapitation and separate testing of individual bodies and heads, is a suitable approach to identify viral factors involved in propagation of virus mutants in more detail. One MAID


RAG1 or RAG2 mutations are associated with defects in V(D)J recombination activity, causing severe immunodeficiency with a wide spectrum of clinical phenotypes. A R229Q mutation of RAG2 was identified in patients with severe combined immunodeficiency (SCID) or Omenn syndrome (OS). Although some factors determining the clinical features between SCID and OS were not clear, the molecular mechanism of OS was studied in a mouse model in which an EGFP tag is fused to Rag2 with the R229Q mutation. To design the human disease model mimicking severe immunodeficiency, we generated Rag2-R229Q knock-in mice without an epitope tag. Mutant mice showed impaired T and B cell differentiation with reduced V(D)J recombination activity; however, the extent to which the R229Q mutation affects severe immunodeficiency was not severe. While Rag2-R229Q mutation under some conditions may cause severe immunological and clinical phenotypes similar to human SCID or OS, R229Q mutation per se did not cause severe immunodeficiency in mice, suggesting that additional factors other than R229Q mutation are required to induce severe immunodeficiency. Thus, our report implies that the effects of genetic background and/or a tagged protein sequence may alter the mouse immune system, revealing the mechanism of phenotypic heterogeneity arising from an identical mutation. During the early stage of B and T cell development, they generate a repertoire of immunoglobulins and T cell receptors by recombining variable (V), diversity (D), and joining (J) gene segments of antigen receptor loci2. Each of the V, D, and J gene segments is flanked by recombination signal sequences (RSSs), composed of conserved heptamer and nonamer elements which are separated by a spacer of either 12 or 23 base-pairs long2. The lymphocyte-specific recombination activating gene 1 (RAG1) and 2 (RAG2) initiate the process of V(D)J recombination by introducing site-specific DNA cleavage at the junction between the RSSs and the adjacent coding segment3. Thus, the RAG genes play an essential role in the rearrangement of the genes encoding antigen-specific receptors of B and T lymphocytes, thereby facilitating the diversity of antigen recognition present in the mature lymphocyte population4.The adaptive immune response to a particular pathogen relies on B and T lymphocytes, which possess genetically rearranged and highly diverse antigen receptors5. However, patients with hypomorphic mutations in RAG1 or RAG2 that display partial V(D)J recombination activity can give rise to a wide spectrum of clinical and immunological phenotypes, ranging from Omenn syndrome (OS) to atypical SCID6. In typical OS patients, circulating B cells are mostly absent, whereas the levels of T cells are normal or elevated with a restricted T-cell receptor (TCR) repertoire7. Patients are defined to present atypical SCID when correlated conditions do not fully satisfy the criteria for OS8. While approximately 15% of the SCID-suffering infants in the United States have RAG1 mutations9, the underlying molecular mechanisms of such phenotypic heterogeneity remain unclear.RAG deficiency in humans leads to severe combined immunodeficiency (SCID), associated with a complete absence of mature B and T lymphocytes11. Based on the structure of the RAG1-RAG2 protein complex, missense mutations leading to SCID or OS can be categorized into four groups: (1) mutations destabilizing the tertiary structure of RAG1-RAG2; (2) mutations affecting polar residues involved in DNA binding; (3) mutations surrounding the active sites; and (4) mutations located at the interface of RAG1 and RAG210. Among these, R229 of RAG2 that forms salt bridges with D546 of RAG110 is regarded to be critical for the development of SCID or OS in patients12. In addition, homozygous Rag2-R229Q/Enhanced green fluorescent protein (EGFP) mutant mice (hereafter Rag2 KI/EGFP), developed by Marrella and colleagues13, presented clinical and immunological phenotypes remarkably similar to human OS with severe alopecia, erythroderma, infiltration by T lymphocytes and eosinophils into the skin and gut, and complete absence of B cells. However, this mouse model expressed the mutant protein, in which the endogenous Rag2 gene was targeted by a construct containing Rag2-R229Q with an EGFP tag at the N terminus. Furthermore, they created the mutant mice by gene targeting method using mouse 129/Sv embryo-derived stem (ES) cells and performed analysis on a mixed C57BL/6 X129/Sv genetic background13. Although the Rag2 KI/EGFP mice used as murine OS model helped us to understand the detailed pathogenesis of OS and autoimmunity, whether the R229Q mutation itself is sufficient to cause full development of OS and severely affect immunological disorder is still not clear.Recently, crystal structure analysis of RAG protein complex revealed the role of conserved residues and frequently occurring mutations in patients14. The CRISPR/Cas9 system is composed of Cas9 endonuclease and a synthetic single guide RNA (sgRNA) that contains a targeting sequence (crRNA) and a Cas9 nuclease-recruiting sequence [transactivating crRNA (tracrRNA)]14. The sgRNA includes 20 nucleotides that are complementary to a target sequence upstream of a PAM sequence (NGG) and directs the Cas9 endonuclease to a specific location in the genome. The Cas9 endonuclease, along with an sgRNA, introduces sequence specific DNA double-strand breaks which results in non-homologous end joining (NHEJ)-mediated insertions and deletions or homology-directed repair (HDR)-mediated repair in the presence of donor templates. In mice, CRISPR/Cas9 has been successfully used for generating knockout or knock-in mice that mimic human diseases15.Animal models have been providing valuable clues to the aetiology and the molecular pathogenesis of human genetic diseases caused by several types of mutations. Recently, a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9, identified in bacteria and archaea as the heritable and adaptive immune system, has become a powerful tool for genome editing in eukaryotic cells due to its accuracy, simplicity, and high efficiencyRag2-R229Q knock-in mice by CRISPR/Cas9-mediated gene editing in C57BL/6 zygotes. Since Rag2 KI/EGFP mouse model has been used to study pathogenesis of OS13 and the R229Q residue of RAG2 has been considered important for lymphocyte development in patients12, our first intention was to set them up as a potential model for gene therapy of SCID and OS. However, our Rag2-R229Q mutant mouse model exhibited a milder immunological phenotype in lymphocyte development than in the previous Rag2 KI/EGFP mouse model13. Furthermore, we could not observe clinical evidence for OS, possibly due to the absence of a reporter gene and/or the influence of genetic background. Accordingly, our results clearly demonstrated that identical mutation in RAG2 give rise to distinct in vivo phenotypes depending on external factors, while the R229Q mutation of Rag2 by itself is not sufficient to cause the full development of OS and SCID in mice.In this study, we generated Rag2 knock-in mouse carrying the R229Q mutation by CRISPR/Cas9-mediated gene editing. The R229 residue of RAG2, frequently identified in patients with SCID or OS, is considered to be critical for the development of SCID or OS12. In accordance with this, a previous work13 has shown that Rag2 KI/EGFP mice displayed immunological and clinical phenotypes similar to those of human OS; however, they generated mutant mice in which an EGFP tag is fused to endogenous Rag2 and were bred on a mixed C57BL6 X 129/Sv genetic background. To remove the effects of genetic background and an artificial tag, multiple sgRNAs and a single-stranded oligodeoxyribonucleotide (ssODN) were designed for targeting the exon3 of Rag2 to introduce the R229Q mutation on the pure C57BL/6 genetic background  mice were fertile and survived into adulthood with no obvious development defects, compared to wild-type strains . It is worthwhile to note that unlike Rag2 KI/EGFP mice, our Rag2 KI mice did not show any pathological phenotypes of human OS such as severe alopecia and skin erythroderma  analysis for thymocytes in Rag2 KI mice. During T cell development, immature T cell progenitors in the thymus progress from the CD4−CD8− double-negative (DN) to the CD4+CD8+ double-positive (DP) stage. According to previous Rag2 KI/EGFP mouse model, most thymocytes were at DN stages (3% in Rag2 WT versus 89% in Rag2 KI/EGFP)13. In contrast, only minor population of thymocytes from Rag2 KI mice was observed at DN stage , CD44+CD25+ (DN2), CD44−CD25+ (DN3), and CD44−CD25− (DN4)16. TCRβ rearrangement is initiated by Dβ-Jβ recombination during transition from the DN2 to DN3 stage and completed by Vβ-DJβ joining at the DN3 stage17. Further analysis revealed that the DN3 cell numbers from Rag2 KI mice were highly accumulated, whereas the DN4 cell numbers were reduced, indicating the arrest of thymocyte development at the DN3 stage  T cells that express TCRαβ, CD4+ and CD8+ SP T cells from the thymus of Rag2 KI mice exhibited high TCRβ expression in both Rag2 WT and KI mice, suggesting that surviving T cells were accumulated as SP T cells  of T cell subsets from lymph nodes were CD44+CD62L−, implying that SP T lymphocytes were predominantly present as effector/memory-like cells13. However, the percentage of CD44+CD62L− T cells from lymph nodes of Rag2 KI mice was slightly increased  and non-lymphoid organs (lung and liver). Consistent with the reduction in the number of T lymphocytes in the thymus, the absolute numbers of lymphocytes in peripheral lymphoid and non-lymphoid organs were mostly reduced in lls Fig. . SP T cesed Fig. . Therefo+CD4+ regulatory T (Treg) cells, which play a crucial regulatory role in the pathogenesis of autoimmune diseases22. Interestingly, OS patients generally develop autoimmune phenomena with immunodeficiency23. However, a possible explanation for this paradoxical observation is the severe reduction in the number of Treg cells in Rag2 KI/EGFP mice13. To test whether Treg cell population was changed or not in Rag2 KI mice, we analysed the expression of FoxP3 that acts as the transcription factor of regulatory T cell lineage specification on CD4+ T cells. Surprisingly, FACS analysis revealed that the percentage of CD4+ T cells expressing FoxP3 was increased in Rag2 KI mice, while the absolute numbers of FoxP3+ Treg cells were comparable between Rag2 WT and KI mice  phenotype in Rag2 KI mice  from the spleens of Rag2 WT and KI mice. As shown in Fig. Rag2 WT and KI mice. We also analysed the TCRVβ diversity by diverse indices such as Shannon-Weaver index H, Inverse Simpson’s index 1/λ, Pielou’s evenness, and Diversity Evenness score (DE50). Although the diverse indices proposed that there was a decrease in the scores representing the TCRVβ diversity in Rag2 KI mice, the difference between Rag2 WT and KI mice was not statistically significant J recombinase activity are not solely responsible for the clinical outcome. Therefore, investigating the pathogenesis of the RAG gene variant is a challenging task. Although, several algorithms have been developed to predict the pathogenicity of the RAG gene variants, these approaches have failed to elucidate the cause of the phenotypic variation23. While several studies proposed that additional factors may be required for determining distinct phenotypes from identical or similar RAG mutations8, there was no direct evidence that such factors are important contributors to the development of different phenotypes in mice. In the present study, we generated the Rag2 KI mice by the CRISPR/Cas9 system and compared the immunological phenotypes with Rag2 KI/EGFP mice as previously reported13. Intriguingly, Rag2 KI mice displayed milder immunological phenotypes than Rag2 KI/EGFP mice and there were no clues to the determinants of OS phenotypes including severe alopecia, B cell numbers, and elevated IgE levels in Rag2 KI mice. Therefore, R229Q mutation per se is not sufficient for the development of OS and SCID in mice.The relationship between genotype and phenotype in monogenic diseases is important to predict the onset, penetrance, and severity of diseases. However, accumulating evidence suggests that identical or similar RAG mutations in relatives can lead to different clinical phenotypesRag2 is well described in patients leading to severe immunodeficiency with T−B−SCID or OS12. Biochemical analysis of recombinant RAG2 proteins suggests that the R229 residue is critical for DNA binding and catalysis28. While Rag2 KI/EGFP mice recapitulate human OS13, we initially expected that our Rag2 KI mice will give rise to an immunodeficiency characterized by SCID or OS. How can identical R229Q mutations give rise to different immunological phenotypes in mice? The discrepancy may be explained by the existence of additional factors required for the development of OS. One possibility is the potential effect of EGFP on Rag2-R229Q mutation. Although the EGFP-tagged gene served as a useful reporter system to monitor its tissue specific gene expression in living animals, growing evidences suggested that EGFP itself can alter important cellular functions such as protein stability29. In RAG2-transgenic mice, it was revealed that the half-life of RAG2-GFP was 2–3 days, probably much longer than that of the endogenous RAG2 protein30. Since the expression of RAG proteins is restricted to immature lymphocytes, the long-term effect of R229Q mutation on lymphocytes of Rag2 KI/EGFP mice can lead to severe immunodeficiency. Another plausible explanation is the influence of genetic background. We established the Rag2 KI mice on a C57BL/6 background, while all experiments from Rag2 KI/EGFP were performed on a mixed 129/Sv and C57BL/6 genetic background. However, we previously reported that the Rag2 knockout mice, generated by CRISPR/Cas9-mediated gene editing on C57BL/6 and FVB/N genetic backgrounds, showed similar immunological phenotypes observed on the mixed 129/Sv and C57BL/6 genetic background31. Based on such observations, we thought that the EGFP tag might be a major cause of the discrepant phenotypes of Rag2-R229Q knock-in mice. Nevertheless, we may not exclude the potential effects of the genetic background in the mice. Therefore, our results suggest that phenotype variability in a given genotype may stem from the difference in the gene constructs and mouse strains.The R229Q mutation of in vivo23. Therefore, the development of mouse models mimicking human immunodeficiency (SCID and OS) allowed us to investigate the pathogenesis of immunological disorders. Although our data suggested that an identical mutation in RAG2 can give rise to distinct phenotypic patterns with heterogeneous clinical manifestations, there is little debate about the importance of mouse models. In parallel, hypomorphic RAG mutations were recently identified in patients with combined immunodeficiency associated with granulomas and/or autoimmunity (CID-G/AI)32. However, the lack of appropriate mouse models recapitulating the CID–G/AI is a stumbling block to characterize the pathophysiology of this phenotype23. Overall, our data propose that generating mouse models for heterogeneous conditions should consider the pathogenic mutations and mouse strains with the complexity of factors affecting the in vivo phenotypes.Since the RAG proteins are not expressed in mature lymphocytes, the steps required for isolation of immature lymphocytes from patients should be preceded by the appropriate method. For this reason, it was difficult to study the pathogenesis of the RAG variants 33 with slight modifications. In brief, we used the mMESSAGE mMACHINE T7 Ultra kit  to synthesize Cas9 mRNA. The resulting products were diluted into diethyl pyrocarbonate -treated injection buffer (0.25 mM EDTA pH 8.0 and 10 mM Tris pH 7.4). The sgRNAs targeting exon3 of Rag2 were obtained from ToolGen, Inc. . To avoid potential risk of off-target effects, each sgRNA was designed to recognize a specific target sequence that does not share homology with any sequence of mouse genome. After screening the sgRNA cleavage efficiency in vitro, two sgRNAs were selected for zygote injection. The sequences of Rag2 sgRNAs used in injection are listed as follows: sgRNA #1 (5′- GTTAGCAGGGCGTATATTACTGG-3′) and sgRNA #2 (5′-CTTATTCTATACAAGTTAGCAGG-3′).Cas9 mRNA was synthesized as described previouslyRag2 gene, and blocking silent mutations to prevent re-cutting by Cas9 to aid PCR-based genotype analysis. Polyacrylamide gel electrophoresis (PAGE)-purified ssODN, oriented in the antisense direction, was synthesized from Integrated DNA Technologies  and dissolved in the injection buffer (0.25 mM EDTA pH 8.0 and 10 mM Tris pH 7.4). The sequence of the ssODN was as follows: 5′-CACTGGAGACAGAGATTCCTCCTGGCAAGACTGTGCAATTCACTGCTGGGGTACCCAGGGGAAGGTCCACGCGAATGCGGTAAAGATTTGCTGGCTGGATGTTACTGGCAAGTGAGTGTCCTCCCAAAATATAAACGGTATCGTTTCTGGCAATAGAAACATGAAAAGACAGC-3′The ssODN donor template was designed to contain 173 nucleotides with homology arms, a R229Q mutation at Rag2-sgRNAs (100 ng/μl in each), ssODN (200 ng/μl), and Scr7 (1 mM) were mixed in injection buffer  and injected into the cytoplasm using a piezo-driven manipulator (Prime Tech). The manipulated embryos were transferred into the oviducts of foster mothers to produce mutant mice. All animal care and experiments were performed in accordance with the guidelines for Korean Food and Drug Administration (KFDA) and were approved by the Institutional Animal Care and Use Committees (IACUC) of the Laboratory Animal Research Center at Yonsei University (Permit Number: 201507-390-01). All mice were maintained in the specific pathogen–free (SPF) facility of the Yonsei Laboratory Animal Research Center.C57BL/6JBomTac mice were purchased from Taconic Biosciences . To obtain mouse zygotes, female C57BL/6J mice (6‒8 weeks) were superovulated by intra-peritoneal injections of 5 IU pregnant mare serum gonadoptropin  and 5 IU human chorionic gonadotropin  at a 48-h interval. Mouse zygotes were collected by mating the superovulated females and wild-type C57BL/6J males. Prior to microinjection, Cas9 mRNA (20 ng/μl), two 34. PCR amplification was performed with the primers encompassing the sgRNA target sites. Following PAGE-based PCR genotyping, the resulting PCR products were isolated using the DNA purification kit  and then cloned into the TA cloning vector . The mutant sequence of cloned PCR products was confirmed by DNA sequencing. For routine genotyping, the following primers were used for detecting both wild-type and mutant alleles: WT-F (5′-TGT CCCTGAACCCAGATACG-3′), MT-F (5′-AACATCCAGCCAGCT-3′), WT-R (5′-TTAGCAGGGCGTATATTACT-3′), and MT-R (5′-AGGCTGCAGACCATCCTTTT-3′).Founder mice were identified by PAGE-based PCR genotyping from tail biopsies35. The prepared cells were stained with the following antibodies: anti-CD4-BV605 , anti-PD-1-BV421 , anti-CD2-PE-Cy7 , anti-CD23-PerCP-Cy5.5 , anti-CD21/35-BV421 , anti-CD43-PE , anti-IgM-APC , anti-IgD-BV605 , anti-CD8-PerCP-Cy5.5 , anti-CD25-FITC , anti-CD3-PE-Cy7 , anti-CD44-V450 , anti-CD45R/B220-FITC , anti-CD62L-FITC , anti-CD69-PE , and anti-TCR-β-FITC . For the staining of transcription factor FoxP3, the cells were stained using anti-FoxP3-APC  after permeabilization/fixation step. In all experiments, cells were labelled with the Live/Dead fixable Stain Kit (eBioscience) to discriminate the live and dead cells. The data were acquired by FACS CANTO II (BD Biosciences) and analysed by FlowJo software (Tree Star).To obtain single-cell suspensions, lymphocytes were isolated from the thymus, spleen, inguinal lymph nodes, lungs, and livers as described beforeSerum immunoglobulin levels  of mice were analysed by a multiplex bead-based assay panel  using fluorescence-encoded beads. The samples were prepared according to manufacturer’s instructions and analysed by FACS CANTO II (BD Biosciences). The serum immunoglobulin level of IgE was measured using a Mouse IgE ELISA MAX kit (BioLegend).+ cells were isolated from the spleen using positive selection (Miltenyi Biotec). The isolated cells were stored in RNAprotect® cell reagent (QIAGEN), and quantitative TCRVβ repertoire was determined by Repertoire Genesis, Inc. based on next generation sequencing (NGS) analysis. The diversity of TCRVβ repertoire analysis is described by Shannon-Weaver index H’, Inverse Simpson’s index 1/λ, Pielou’s evenness, and Diversity Evenness score (DE50), which are provided by Repertoire Genesis, Inc.CD90.2t-test using GraphPad Prism software. A p value less than 0.05 was considered statistically significant.Data were analysed by two-tailed unpaired Student’s Supplementary Information


On the other hand several studies suggested that NLRP3 inflammasome contributes to maintenance of gastrointestinal immune homeostasis and that activation of NLRP3 is regulated by protein tyrosine phosphatase non-receptor 22 (PTPN22). PTPN22 polymorphism was implicated in the risk for various autoimmune diseases including type 1 diabetes (T1D) but not for celiac disease (CD). The aim of our study was to evaluate the role of inflammasome related polymorphisms in subjects with either T1D or CD as well as in subjects affected by both diseases. We examined PTPN22 rs2476601 (p.Arg620Trp), NLRP3 rs35829419 (p.Gln705Lys), and CARD8 rs2043211 (p.Cys10Ter) in 66 subjects with coexisting T1D and CD, 65 subjects with T1D who did not develop CD, 67 subjects diagnosed only with CD and 127 healthy unrelated Slovenian individuals. All results were adjusted for clinical characteristic and human leukocyte antigen (HLA) risk. PTPN22 rs2476601 allele was significantly more frequent among subjects with T1D (Padj = 0.001) and less frequent in subjects with CD (Padj = 0.039) when compared to controls. In patients with coexisting T1D and CD this variant was significantly less frequent compared to T1D group (Padj = 0.010). Protective effect on CD development in individuals with T1D was observed only within the low risk HLA group. On the other hand, we found no association of NLRP3 rs35829419 and CARD8 rs2043211 with the development of T1D, CD or both diseases together. In conclusion PTPN22 rs2476601polymorphism was significantly associated with the risk of developing T1D in Slovenian population, while no associations of proinflammatory NLRP3 and CARD8 polymorphisms with T1D and CD were observed. Interestingly, the same PTPN22 variant protected from CD. We hypothesize that this effect may be mediated through the NLRP3 inflammasome activation.Genetic polymorphisms in genes coding for inflammasome components  Type 1 diabetes (T1D) is characterized by chronic autoimmune mediated destruction of pancreatic β-cells, leading to symptomatic partial, or in most cases, absolute insulin deficiency requiring lifelong administration of exogenous insulin . Both muDR3-DQ2/DR4-DQ8 and DR3-DQ2/DR3-DQ2 genotypes were found to be highly predisposing for developing both diseases together (NLRP3 rs35829419 (p.Gln705Lys) is a gain-of-function polymorphism associated with pro-inflammatory phenotype (CARD8 rs2043211 (p.Cys10Ter) results in non-functional protein and leads to loss of CARD-8 inhibition of caspase-1  region encoding HLA-DQ2 and DQ8 molecules which account for much of the etiology of T1D and CD contribute to the coexistence of both diseases. The risk is further modified by the HLA genotype, which is the combination of HLA alleles inherited from both parents . In our together . Howevertogether . Cumulattogether . Among ntogether . It is htogether , 15. Thetogether . The bestogether . NLRP3 rhenotype , 19. Casaspase-1 , 23. It aspase-1 . It seemaspase-1 .PTPN22 rs2476601 (p.Arg620Trp) polymorphism has been linked to several autoimmune diseases  interacts with and dephosphorylates NLRP3 upon proinflammatory insults, allowing robust NLRP3 activation and IL-1β secretion . The PTPdiseases . The assulations . Interes with CD , 30 and  with CD .NLRP3 and PTPN22 polymorphisms in either T1D or CD onset. However, T1D and CD frequently occur in the same individual, pointing to shared mechanisms. The aim of our study was to investigate the role of PTPN22 rs2476601 (p.Arg620Trp), NLRP3 rs35829419 (p.Gln705Lys), and CARD8 rs2043211 (p.Cys10Ter) in subjects with T1D who did not develop CD, in subjects with CD who did not develop T1D and in subjects who developed both diseases. Furthermore, the role of these polymorphisms was assessed while also taking into account the presence of high or low risk HLA genotype.Previous studies have shown conflicting and heterogeneous findings with regard to the role of In total 325 individuals, who were previously HLA typed , were inPTPN22 rs2476601 (c.1858T>C) polymorphism we used TaqMan SNP Genotyping assay . NLRP3 rs35829419  and CARD8 rs2043211  were genotyped using a fluorescence-based competitive allele-specific (KASPar) assay according to the manufacturer's instructions . Genotyping was repeated in 20% of samples to check for genotyping reliability.For the detection of DRB1, DQB1, and DQA1 genotyping was performed as previously described (The escribed . We perfDRB1-DQA1-DQB1) as described earlier  were grouped into two HLA-risk categories encoded as low-risk (LR) and high-risk (HR) categorical variables by means of the known risk associated with T1D and CD in the Slovenian population. The heterozygous genotype DR3-DQ2/DR4-DQ8 and genotype DR4-DQ8/ DR4-DQ8 were defined as HR for T1D  haplotype combinations were considered as low risk.HLA alleles were deduced into three loci haplotypes  and 95% confidence interval (CI), as well as for the analysis of interaction between polymorphisms. Two-tailed Fisher's exact test was used for comparisons of the frequencies of HLA deduced genotypes. The level of statistical significance was set at 0.05. All statistical analyses were performed using IBM SPSS Statistics 19.0 .P = 0.072, Clinical characteristics of subjects with T1D, CD, or both including HLA-risk categories are shown in p < 0.001) and DR3-DQ2/DR3-DQ2 (p < 0.001) were associated with an increased risk for developing the combination of both diseases. On the other hand low risk genotypes had neutral effect on co-occurrence. When individuals with T1D who later developed CD were compared to those with T1D alone, genotype DR3-DQ2/DR3-DQ2  conferred an increased risk for CD and DR4-DQ8/X  was negatively associated. Comparisons of individuals in whom CD diagnosis proceeded T1D with CD only group revealed that DR3-DQ2/DR4-DQ8 genotype was associated with an increased risk for developing T1D .The distribution of HLA genotypes differed significantly among the groups . Two higP = 0.413 for PTPN22 rs2476601, P = 0.461 for NLRP3 rs35829419, and P = 0.616 for CARD8 rs2043211) as well as in the entire study group . Minor allele frequencies were 14.2% for PTPN22 rs35829419, 38.2% for CARD8 rs2043211, and 6.6% for NLRP3 rs2476601  and the difference remained statistically significant after adjustment for gender . On the other hand, polymorphic PTPN22 rs2476601allele was significantly less common in subjects with CD when compared to controls , the difference remained significant after adjustment for gender . There were no significant differences in genotype frequencies for NLRP3 rs35829419 and CARD8 rs2043211 between cases and controls, not even after adjustment for clinical parameters  and the difference remained significant after adjustment for age at diagnosis, gender and HLA genotype . Proportion of carriers of at least one polymorphic PTPN22 rs2476601 allele in subjects with coexisting T1D and CD was significantly higher compared to CD , however the difference was no longer significant after adjustment for age at diagnosis, gender and HLA genotype . NLRP3 rs35829419 and CARD8 rs2043211 genotype frequencies did not differ significantly among subjects with T1D, CD, or coexisting diseases  , but not in the HLA HR group. Among carriers of at least one polymorphic PTPN22 allele, T1D first were more likely HLA HR than T1D (P = 0.014). A similar trend was observed when comparing group of subjects with both T1D and CD (T1D first + CD first) to subjects with only T1D or CD, but it did not reach statistical significance. No multiplicative interaction was observed between NLRP3 rs35829419 and CARD8 rs2043211 (When comparing subjects that were first diagnosed with T1D and then developed CD (T1D first) to patients with only T1D, we observed a multiplicative interaction between = 0.047) . Subjects2043211 .PTPN22 and inflammasome polymorphisms in the risk for development of T1D and CD as separate or concurrent diseases. Our results suggest a dual role of the PTPN22 polymorphism in the development of autoimmune diseases: while rs2476601 polymorphism was associated with increased risk for T1D, it was protective against development of CD. On the other hand, NLRP3 and CARD8 polymorphisms were not associated with the risk of either T1D or CD.The present study investigated the role of PTPN22 and NLRP3 polymorphisms with several autoimmune diseases suggesting their general role in the etiology of autoimmunity, however these risk alleles are not shared among all autoimmune disease  showed association pattern with target tissue specificity being less involved in pathogenesis of autoimmune diseases triggered by environmental factors primarily acting on target tissues such as gut mucosa  were compared to individuals with T1D who did not develop CD. Protective effect on CD development was observed within the low risk HLA category but no significant difference was observed between groups within the HLA high risk category. More than half of patients who had coexisting T1D and CD carried the high risk HLA-DR3-DQ2/DR3-DQ2 or HLA-DR3-DQ2/DR4-DQ8 genotype. On the other hand, DR4-DQ8/X was protective for developing CD in individuals with T1D. If the results could be confirmed on a larger number of patients of different ethnic origin, assessing the presence of HLA-DQ2 and DQ8 genotypes in individuals negative for celiac disease-specific antibodies at the time of T1D diagnosis could be considered as an algorithm tool to optimize further screening frequency. Yearly screening for CD may be considered in T1D individuals with high risk HLA-DR3-DQ2/DR3-DQ2 or HLA-DR3-DQ2/DR4-DQ8 genotypes. On the other hand, individuals carrying DR4-DQ8/X genotype could be further genotyped for PTPN22 rs2476601 (c.1858T>C) polymorphism and if positive, less frequent screening for CD may be suggested.Since HLA remains, by far, the strongest predictor of T1D and CD risk all results in our study were adjusted for HLA risk and only odds ratios adjusted for given HLA risk category were reported. Previous studies have shown that the strength of association between PTPN22 variant on T1D and CD susceptibility and provide interesting information on incidence of this variant in subjects diagnosed with both autoimmune diseases together. To better understand the complex effect of genetic factors and their regulatory role in the pathogenesis of autoimmune diseases larger studies and functional studies are warranted. Due to the low prevalence of subjects with concurrently diagnosed T1D and CD the main advantage of our study was the use of longitudinal data  at the 43The study protocol was approved by the Republic of Slovenia National Medical Ethics Committee (No. 98/09/12) and written informed consent was obtained from all participating individuals and/or their parents, when appropriate, prior to the enrolment.NLRP3 and CARD molecular genetic studies, statistical analysis, and edited the manuscript. TH carried out the PTPN22 molecular genetic studies. AM and BV-J performed analysis of the HLA genetic studies. NB and JB were responsible for the clinical data and biological sample collection used in this study. VD and TB contributed to the study design and conception and edited the manuscript. All the authors collaborated in revising the manuscript and accepted the final version of the manuscript.DS collected clinical data, interpreted data, and drafted the manuscript. KG carried out the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Avian influenza virus H9N2 has been endemic in birds in the Middle East, in particular in Egypt with multiple cases of human infections since 1998. Despite concerns about the pandemic threat posed by H9N2, little is known about the biological properties of H9N2 in this epicentre of infection. Here, we investigated the evolutionary dynamics of H9N2 in the Middle East and identified phylogeny-associated PB2 mutations that acted cooperatively to increase H9N2 replication/transcription in human cells. The accumulation of PB2 mutations also correlated with an increase in H9N2 virus growth in the upper and lower airways of mice and in virulence. These mutations clustered on a solvent-exposed region in the PB2-627 domain in proximity to potential interfaces with host factors. These PB2 mutations have been found at high prevalence during evolution of H9N2 in the field, indicating that they have provided a selective advantage for viral adaptation to infect poultry. Therefore, continuous prevalence of H9N2 virus in the Middle East has generated a far more fit or optimized replication phenotype, leading to an expanded viral host range, including to mammals, which may pose public health risks beyond the current outbreaks. The G1-like clade of H9N2 influenza viruses can undergo genetic reassortment with other influenza virus subtypes to produce novel zoonotic viruses, such as the Gs/GD lineage H5N1, H7N9, H10N8, and H5N8 viruses. Since 1998, the G1-like subclade of H9N2 influenza virus has been widely circulating in birds in Central Asia and the Middle East and a number of human cases have been reported. However, little is known about the biological properties of H9N2 viruses in this epicentre of infection. Our data showed that, during about two decades of evolution in nature, G1-like subclade strains evolved to produce strains with appreciably higher replication phenotypes in Central Asia and the Middle East, which led to their expanded host range, including to humans. Therefore, G1-like subclade strains in these areas may accumulate mutations to produce novel viruses and the large gene pool in these areas would enable reassortment with other influenza viruses. This study indicated the need for studies of H9N2 viruses in such areas to monitor their evolutionary dynamics and possible genetic changes. The avian influenza (AI) virus H9N2 subtype was first identified in South China in 1994 . The intH9N2 virus strains are classified in Eurasian and American lineages. The Eurasian lineage is divided into several clades, with the majority of strains classified in G1-like and Y280-like clades. G1-like clade viruses have mainly circulated in South China, Central Asia and the Middle East and are represented by the prototype A/quail/Hong Kong/G1/1997 virus. The G1-like clade in Central Asia and the Middle East has diversified further into four co-circulating subclades (designated A-D), with occasional intra-subclade reassortments [It has been reported that the G1-like subclades were introduced into Central Asia and the Middle East from East Asia after 1998  and spreThe evolutionary dynamics of the H9N2 and H5N1 viruses that co-circulate in the Middle East, centered on Egypt, needs to be understood to facilitate strategies for the control of these viruses. Several studies have reported on the infectivity and transmissibility of the Middle Eastern H5N1 viruses , 15 and We previously reported that the H9N2 G1-A/B reassortant isolated in Egypt replicates to markedly high titers in human cells and in mice , indicatIn our previous study, we found that a representative strain of the G1-A/B reassortant, A/chicken/Egypt/CL42/2013 (H9N2) (referred to here as EG/2013), replicated at a high level in human cells and in mice. EG/2013 replication was higher than that of a H5N1 clade 2.2.1 strain carrying the representative human adaptation PB2-E627K mutation [To elucidate the replication capacity of the G1-A/B reassortant in human cells, the polymerase activity of the EG/2013 (H9N2) virus in human 293T cells was compared to that of three other zoonotic AI viruses; i.e., A/quail/Hong Kong/G1/1997 (H9N2), A/duck/Egypt/D1Br/2007 (H5N1) and A/Shanghai/1/2013 (H7N9). A/quail/Hong Kong/G1/1997 (G1/1997) (H9N2) is the prototype virus of the G1-like clade. A/duck/Egypt/D1Br/2007 (EG/2007) (H5N1) is an ancestral strain of clade 2.2.1 that is unique to Egypt: most clade 2.2.1 field isolates, including EG/2007, have the PB2-E627K mutation . A/ShangMinigenome assays showed that the EG/2013 (H9N2) polymerase complex  had significantly higher polymerase activity in 293T cells than that of the three other AI viruses in this study . To deteG1-A/B reassortants, including EG/2013, generally carry the PB2-E627V mutation . HoweverTo investigate the different amount of the effects mediated by V627E and K627E, the effect of K627E was investigated in the EG/2013 background. As with V627E, K627E caused only 1.5-fold and 5.4-fold reductions in polymerase activity , indicatHowever, the apparently distinct antigenicity of the different virus subtypes prevented our direct comparison of the PB2 expression levels of EG/2007 (H5N1), sh/2013 (H7N9) and EG/2013 (H9N2). Therefore, the expression of PB2-flag, which was not affected by PB2 antigenic properties was analyzed further. The PB2-flag expression levels of EG/2007 (H5N1) and EG/2013 (H9N2) were similar, although they were higher than sh/2013 (H7N9) . Wild-tyTo trace evolution of the PB2 gene in the G1-A/B reassortant, we analyzed the phylogeny of PB2 genes, including the PB2 genes of the G1-like prototype A/quail/Hong Kong/G1/1997 and the descendant G1-like subclades that were isolated in Central Asia and the Middle East from 1998 to 2015, including EG/2013. The PB2 genes in viruses isolated in Central Asia and the Middle East clustered in several groups . These lWe then searched for the PB2 mutations that the G1-A/B reassortant had accumulated during its evolution in the field, with reference to the ancestral G1/1997 virus. A total of 32 amino acid substitutions were identified and grouped in four mutation categories, designated a-d . CategorTo determine the effect of each mutation on viral polymerase activity, we introduced a reverse mutation of each mutation that had been identified into the EG/2013 background, both with and without PB2-V627E, and measured the polymerase activities of the revertants by minigenome assays in avian DF-1 cells at 37°C and in 293T cells at 33°C. As a reference, a reassorted polymerase complex composed of PB2 from G1/1997 and PB1, PA and NP from EG/2013 (referred to here as EG/2013-G1/PB2) was included in these assays. In avian cells at 37°C, none of the single reverse mutations had a significant effect on EG/2013 polymerase activity . In contBased on the effects of the reverse mutations in the EG/2013 background, we selected three PB2 mutations  for further study and introduced them as forward mutations into the ancestral G1/1997 background to assess their effects on polymerase activity. The E627V and K526R mutations significantly increased G1/1997 polymerase activity in human cells . But, thIn subsequent experiments, we introduced the E543D forward mutation together with the A655V compensatory mutation into the G1/1997 background. In addition, the D543E reverse mutation was introduced into the EG/2013 background, either singly or together with V655A: EG/2013 carried PB2-655V and, therefore, D543E did not necessarily require V655A in this background. In addition, the phylogenetically oldest G1-A strain (A/chicken/Saudi Arabia/532/1999) carried E627V, E543D and A655V , which pThe cooperative effects of E627V, E543D/A655V and K526R on polymerase activity were then investigated. These mutations acted synergistically to increase G1/1997 polymerase activity by > 2 logs in human cells : the E62in vitro transcribed RNA templates , had the lowest progeny virus yield in both cell types by > 3 logs in human cells at early times post-infection: EG/2013-wt > R526K = V627E and D543E > R526K/V627E > R526K/V627E/D543E(/V655A).We investigated the effect of the amino acids at PB2 residues 627, 543, 655 and 526 on the replication kinetics of the G1-A/B reassortant using recombinant viruses in the EG/2013 background. DF-1 cells and human airway epithelial Calu-3 cells were infected with EG/2013-wt and the revertants and the replication kinetics of the viruses were compared. In both cell types, the revertants produced significantly lower progeny virus titers than EG/2013-wt , with coE627V/E543D/A655V/K526R, had the greatest progeny virus yield in both cell types: G1(3P+NP)-wt < E627V < E627V/E543D/A655V < E627V/E543D/A655V/K526R. Together, these results suggested that, during about two decades of evolution in the Middle East, the ability of G1-like clade viruses to replicate in avian and human cells gradually increased and the E627V/E543D/A655V/K526R quadruple mutations acted cooperatively in evolution of the G1-A/B reassortant, with the most fit phenotype able to replicate more successfully in human cells at the lower temperature.The role of the PB2 amino acid at residues 627, 543, 655 and 526 in the G1/1997 background on virus replication was studied next. For these experiments, we used a reassortant virus carrying polymerase-associated genes  from G1/1997 and the other genes from EG/2013 to assess polymerase-specific effects on virus replication. The reassortant virus was designated G1(3P+NP). The G1(3P+NP) mutants produced significantly higher virus titers than G1(3P+NP)-wt in both DF-1 cells and Calu-3 cells , with moin vitro to in vivo infections, BALB/c mice were inoculated intranasally with serial dilutions of two wild-type and four representative mutant strains; i.e., EG/2013-wt, G1(3P+NP)-wt, EG/2013V627E, EG/2013V627E/D543E/V655A/R526K, G1(3P+NP)E627V and G1(3P+NP)E627V/E543D/A655V/K526R. These viruses were categorized into three PB2 groups; i.e., ancestral genotype, G1(3P+NP)-wt and EG/2013V627E/D543E/V655A/R526K; intermediate genotype, G1(3P+NP)E627V and EG/2013V627E; and current genotype, G1(3P+NP)E627V/E543D/A655V/K526R and EG/2013-wt. The body weight and survival of each infected mouse was monitored.To assess the relevance of the cooperative effects of the E627V/E543D/A655V/K526R mutation on 5 and 106 FFU of ancestral genotype viruses G1(3P+NP)-wt and EG/2013V627E/ D543E/V655A/R526K caused little body weight change and no mortality (50) was > 106 FFU for both viruses. In contrast, the same doses of current genotype viruses EG/2013-wt and G1(3P+NP)E627V/E543D/A655V/K526R caused drastic weight loss and 60–100% mortality, resulting in MLD50 values of 3.2 x 104 FFU and 6.8 x 104 FFU, respectively (V627E and G1(3P+NP)E627V had intermediate weight loss and virulence in mice  and virus titers in the organs were measured by focus-forming assays. Virus titers in the infected mice were in agreement with their polymerase activity and replication kinetics in human cells in vitro. Current genotype EG/2013-wt and G1(3P+NP)E627V/E543D/A655V/K526R viruses replicated most efficiently in the lungs and nasal turbinates E627V viruses showed intermediate replication in the mice.To determine virus titers in the infected mice, groups of five mice were inoculated intranasally with 10rbinates , with tiV627E/D543E/V655A/R526K viruses caused a weak inflammatory response in the lungs of the infected mice E627V viruses caused minimal to mild bronchitis or bronchiolitis in infected mice, indicating an intermediate phenotype. The degree of histopathological changes generally corresponded to the amount of H5 antigen detected by immunohistochemistry in the bronchial and bronchiolar areas of the lungs  and EG/2013ted mice . Howeverhe lungs . CollectThe influenza virus polymerase complex is intrinsically flexible and can adopt multiple conformations depending on the presence or absence of specific RNA –27. Our In the apo form of the polymerase complex, our model indicated that PB2 residues 543 and 655 were located close together . This waThe influenza virus H9N2 G1-like clade continues to evolve and expand its geographic host range in Central Asia and the Middle East. These areas include most predominantly Islamic nations, which have significant poultry population but minimal swine production. In contrast, East Asia, centered on China, has large swine production . RegionaHere we have elucidated the phylogeny of the G1-like clade in Central Asia and the Middle East and investigated its host range-associated mutations. Our results showed that the G1-like clade strains in these areas accumulated multiple PB2 mutations during their evolution in poultry. Four of the conserved mutations acted cooperatively in a G1-A/B reassortant that had an increased replication capacity in mammalian species. To our knowledge, this study presents the first comprehensive data analyzing the relationship between the phylogeny of the H9N2 virus in the Middle East and its host range expansion to mammals.In this study, the PB2 E627V, E543D, A655V and K526R mutations acted cooperatively and produced increased replication of the G1-A/B reassortant in human cells and mice. However, the PB2-R190K and S643T mutations only had marginal effects. This was consistent with the hypothesis that the H9N2 virus PB2 gene was a key determinant in the host adaptation of AI viruses to mammals . The G1-Several of the mutations identified in this study have been previously described in other AI viruses. PB2-E627K is the best characterized human-adaptation mutation in AI viruses, particularly H5N1 viruses , 32, andin vitro studies showing higher polymerase activity and viral RNA synthesis and virus titers in the lungs and nasal turbinates. This indicated that increased replication fitness contributed to high influenza virus virulence in mice. However, several studies reported that influenza A virus polymerase can produce additional viral RNA products, often called aberrant RNAs, which trigger an innate immune response [The exact mechanism of the effects of the PB2 mutations identified in this study remains unknown. These mutations clustered on the surface of the PB2 627 domain, which is a solvent-exposed region of the polymerase complex in both its transcription pre-initiation form and apo form. This suggested the possibility that the mutations may affect the interactions between the PB2-627 domain and host factor(s), such as the ANP32A proteins that have been shown to be involved in host species specificity mediated by the influenza virus PB2-627 domain , 38–40. response . Thus, tThe H9N2 virus and the H5N1 clade 2.2.1 viruses have been prevalent in poultry in Egypt, which has provided the opportunity for reassortment between the two viruses. Fortunately, the emergence of H9N2/H5N1 reassortants has not been reported in Egypt thus far. However, we recently reported that Egyptian H9N2 and H5N1 viruses have a high genetic compatibility and a substantial potential to generate reassortants with greater fitness in mammalian species than the parental viruses . The resIn conclusion, this study showed that the influenza virus G1-A/B reassortant, that has been prevalent in Egypt, has diversified phylogenetically in the Middle East during its natural evolution and generated a distinct phenotype with an expanded host range to mammals, including humans. Our results highlighted the need for studies of H9N2 viruses to trace their evolutionary dynamics in this area and closely monitor possible genetic changes in these viruses. In addition, implementation of effective control measures may be needed to reduce the risk of a future pandemic.Ten-day-old embryonated chicken eggs were purchased from Shimizu Laboratory Supplies, Japan. All animal experiments were conducted in compliance with Japanese legislation  and guidelines under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology in Japan. Animal care, housing, feeding, sampling, observation, and environmental enrichment were approved by the Animal Experiment Committee of the Kyoto Prefectural University of Medicine .All experiments with live H9N2 viruses were performed at enhanced Biosafety Level 3+ (BSL3+) in Kyoto Prefectural University of Medicine. All studies with recombinant DNA were conducted under the relevant laws in Japan and approved by the Biological Safety Committee of Kyoto Prefectural University of Medicine  after risk assessments were conducted by the Living Modified Organisms Committee of Kyoto Prefectural University of Medicine and, when required, by the Ministry of Education, Culture, Sports, Science and Technology of Japan.Human bronchial epithelial  cells and chicken fibroblast (DF-1) cells were obtained from the American Type Culture Collection, and MDCK cells and 293T cells were obtained from the RIKEN BioResource Center Cell Bank. The cells were maintained in Dulbecco’s Modified Eagle’s Medium with 10% FCS. Influenza virus A/chicken/Egypt/CL42/2013 (EG/2013) (H9N2) is a representative strain of the G1-A/B reassortant that has been prevalent in Egypt and has been described previously [As described previously , 48, recRenilla luciferase was also co-transfected to monitor transfection efficiencies. The transfected cells were incubated at 33°C or 37°C and lysed at 24 h post-transfection. The firefly luciferase activities were normalized relative to Renilla luciferase activity.293T and DF-1 cells were transfected with pCAGGS plasmids encoding the PB2, PB1, PA or NP genes of EG/2013 (H9N2), G1/1997 (H9N2), EG/2007 (H5N1) and Sh/2013 (H7N9) and a human or chicken polymerase-I driven plasmid expressing firefly luciferase as described previously , 47. A phttp://www.gisaid.org). Phylogenetic analysis was performed using MEGA6 software for the neighbor-joining method, with the nucleotide sequences of representative strains of the H9N2 G1-like clade from Central Asia and the Middle East, including prototype G1/1997 and EG/2013 viruses, and of A/duck/Hong Kong/Y280/1997 as the prototype of the H9N2 Y280-like clade virus. Although a few human and swine H9N2 virus infections have been reported in Central Asia and/or the Middle East, the human- and swine-derived H9N2 sequences were not included in these analyses, because they have not yet been deposited in a pubic database. Confidence levels for phylogenetic trees were calculated by performing 1,000 bootstrap replicates.The sequences of the PB2 and HA gene segments from reference H9N2 strains were obtained from the GISAID database  and G1/1997 (H9N2). Cells were harvested at 48 h post-transfection in cold PBS and lysed in 700 μl Tris lysis buffer  with Complete Mini EDTA-free protease inhibitor cocktail (Promega) at 4°C for 40 min. The lysates were cleared by centrifugation at 800 g for 5 min. Anti-Flag M2 Agarose Affinity Gel (Sigma-Aldrich) was added to the cleared lysates and they were mixed at 4°C for 2 h. The mixtures were washed 3 times with the Tris lysis buffer and purified PB2-flag, and PB1 and PA were analyzed by western blotting.For expression analyses, 293T cells were transfected with equal amounts of plasmids encoding the non-tagged or flag-tagged PB2 gene of EG/2007 (H5N1), Sh/2013 (H7N9) or EG/2013 (H9N2). At 16 h post-transfection, the cells were harvested with sample buffer. For the PB2-flag immunoprecipitation assays described above, final immunoprecipitates were boiled in sample buffer and analyzed by western blotting. The samples were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore). Immunoblot analysis was performed using anti-influenza PB2, PB1 and PA antibody (GeneTex), anti-flag antibody (Sigma-Aldrich) and HRP-conjugated secondary antibody. The bands were visualized with Amersham ECL Select Western Blotting Detection Reagent and the Amersham Imager 600 . The band intensities were quantified by Amersham Imager 600 Analysis Software .9, 108, 107, 106, 105, 104, 103 and 102 copies) of in vitro transcribed viral RNA standards were used to generate a standard curve. The specificity of strand-specific qRT-PCR was tested using an NA gene that was amplified by PCR using a human polymerase-I driven plasmid encoding a full-length EG/2013 NA vRNA template. The primers, listed in in vitro using the RiboMax Large-Scale RNA Production System-T7 (Promega). Reverse transcription was performed with 109 copies of each template RNA  using the tagged primers described above. The cDNA products were quantified by qPCR reactions using the strand-specific primer sets described above.293T cells and DF-1 cells were transfected with pCAGGS plasmids encoding the wild-type or mutant PB2, PB1, PA or NP genes of EG/2013 and G1/1997 and a human or chicken polymerase-I driven plasmid encoding a full-length NA vRNA or cRNA template to reconstitute the NA RNP. At 24 h post-transfection, viral RNAs were extracted from the cells with an RNeasy Mini Kit (Qiagen) and treated with RNase-free DNase Set (Qiagen) to eliminate any residual transfected plasmid DNA. cDNAs derived from NA vRNA, cRNA and mRNA were reverse transcribed with the tagged primers listed in DF-1 and Calu-3 cells (90% confluent in collagen-coated 24-well plates) were infected in triplicate with the indicated viruses at an MOI of 0.005 and 0.001, respectively. After 1 h at 37°C, the cells were washed with PBS and maintained in DMEM-F12 containing 0.2% BSA and 2 μg acetylated trypsin/ml (Sigma-Aldrich) at 33°C or 37°C. The supernatants were collected at the indicated times post-infection and viral titers were determined by assays of focus-forming units (FFU) as described previously .4 FFU virus were collected 3 dpi and virus titers were assayed by FFU assays. For histopathological analysis, lungs of mice infected with 104 FFU were collected at 3 dpi, fixed in 4% buffered paraformaldehyde, embedded in paraffin, cut into 5 μm sections, stained with hematoxylin and eosin, and examined by light microscopy. Immunohistochemical straining of viral antigen was performed as described previously [Four to five-week-old female BALB/c mice (Japan SLC), under mixed anesthesia (medetomidine-butorphanol-midazolam), were inoculated intranasally with 50 μl samples of 10-fold serial dilutions of viruses in PBS. The body weight and survival of each mouse was monitored daily for 14 d. Mice that lost more than 30% of their original weight within a few days were humanely euthanized. Lungs and nasal turbinates of mice infected with 10eviously , using aThe transcription pre-initiation form and the apo form of the G1/1997 trimeric polymerase complex of the indicated G1/1997 mutants were modeled using published crystal structures of the influenza virus polymerase complexes . MOE was used for software and most of the modeling conditions were described previously , 47, 50.Statistical analysis was carried out using GraphPad Prism Version 6 software (GraphPad Software Inc.). Statistically significant differences of virus pairs were determined by ANOVA with Tukey’s multiple comparison test.S1 FigThe cells were transfected with plasmids expressing the indicated flag-tagged PB2, and cell lysates were analyzed by western blotting using anti-flag antibody. Representative images are shown. Each data point is the mean ± SD of five independent experiments. NS indicates no statistically significant difference.(TIF)Click here for additional data file.S2 FigThe phylogenetic tree was reconstructed from nucleotide sequences of the HA genes of the Middle Eastern and Central Asian reference strains in the GISAID database, the G1-like clade prototype strain G1/1997, the G1-A/B reassortant strain EG/2013, and the Y280-like clade prototype strain A/duck/Hong Kong/Y280/1997. CK, DK, QL and TK in virus strain names denote chicken, duck, quail and turkey hosts. Virus particles are shown as ovals with horizontal bars representing the eight gene segments . Gene segments in the descendent viruses are colored according to their corresponding source viruses to illustrate gene ancestry through reassortment. Yellow in segment 1 (PB2) indicates phylogeny-associated mutations that contribute to expansion of the viral host range to humans.(TIF)Click here for additional data file.S3 FigPolymerase activity of EG/2013 and the PB2 revertants carrying single mutations was measured in human 293T cells at 37°C. The viruses were either not carrying PB2-V627E (A) or were carrying PB2-V627E (B). The data are expressed relative to the results for EG/2013-627V (wt) (A) and EG/2013-V627E (B). Colors of the vertical bars indicate the four mutation categories based on the time periods when the mutations were identified, as shown in (TIF)Click here for additional data file.S4 FigHuman 293T cells were transfected with PB2 expression plasmids carrying the indicated mutations and either not carrying V627E (A and B) or carrying V627E (C and D). At 16 h post-transfection, the cells were harvested and analyzed by western blotting using anti-PB2 antibody. Representative images are shown. (A and C). After quantification of the band intensities, the amount of expression of each PB2 construct was calculated relative to that for EG/2013-627V (wt). Each data point is the mean ± SD of five independent experiments. (B and D) Representative results of western blotting for EG/2013-627V (wt), the PB2 mutants and the reference EG/2013-G1/PB2. NS indicates no statistically significant difference.(TIF)Click here for additional data file.S5 Figin vitro transcription as described in Materials and Methods. The specificity of primers for EG/2013 vRNA, cRNA and mRNA is shown with respect to the percent of the corresponding RNA template. Each data point is the mean ± SD of three independent experiments. These data indicated that the qRT-PCR primers were highly specific for distinguishing viral vRNA, cRNA and mRNA, as previously reported [The strand specificity of the primers was verified using EG/2013 NA vRNA, cRNA and mRNA templates prepared by reported .(TIF)Click here for additional data file.S6 FigAvian DF-1 cells (A) and human Calu-3 cells (B and C) were infected with G1(3P+NP) virus and PB2 mutants carrying E543D alone or in combination with the indicated mutations at an MOI of 0.005 and 0.001, respectively, and incubated at 37°C (A and B) or 33°C (C). Virus titers at the indicated times post-infection were determined by FFU assays. Each data point is the mean ± SD of three independent experiments. The data indicated that the E543E/A655V double mutation, but not the E543 single mutation, produced efficient G1 replication in avian and human cells, in agreement with the minigenome assay data in (TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


This study analyzed the expression of membrane OX40 and OX40L (mOX40 and mOX40L) and levels of soluble OX40 and OX40L (sOX40 and sOX40L) in T1D patients to determine their clinical significance. Peripheral blood (PB) was collected from patients with T1D and healthy control participants. Expression of mOX40 and mOX40L on immune cells was detected by flow cytometry. Levels of sOX40 and sOX40L in sera were measured by ELISA. We demonstrated for the first time enhanced sOX40 and sOX40L expression and reduced mOX40 and mOX40L levels in T1D patients which correlated with the clinical characteristics and inflammatory factors. These results suggest that OX40/OX40L signal may be promising biomarkers and associated with the pathogenesis of T1D. Type 1 diabetes (T1D) is a chronic and organ-specific autoimmune disease, is influenced by inherited or environmental factors, is increasing in all age groups, especially examined in children, and leads to the loss of insulin production in beta cells when immune cells invade the pancreatic islets . The actOX40/OX40L is a pair of important positive costimulatory signal molecules in the second signal system of T cells. OX40 CD134) and OX40L (CD252), members of the TNF receptor superfamily (TNFRSF) and TNF superfamily (TNFSF), play important roles in T cell expansion in tumors, during infectious inflammation, and in autoimmune diseases , 11. OX44 and OX4The level of sOX40 and sOX40L is elevated in some tumors and infectious diseases. This suggests that sOX40 and sOX40L act as costimulators to induce immune responses. However, to the best of our knowledge, the significance of membrane and soluble forms of OX40 or OX40L expression and their correlation with clinical parameters in T1D have not been investigated. To characterize OX40 and OX40L expression and determine the role of OX40/OX40L signal in the development and pathogenesis of T1D, we evaluated the expression of sOX40 and sOX40L in the PBMC and sera of patients with T1D and healthy controls (HCs) and analyzed the correlation with clinical and inflammatory indicators.n = 119) at the Endocrinology Department in the Second Affiliated Hospital of Soochow University, Suzhou, China, between 2015 and 2017. The diagnostic criteria for T1D were according to “The T1D Exchange Clinic Registry” [n = 108) were matched to T1D patients with age, gender, and race. In addition, HCs were tested for glucose levels, all T1D autoantibody negative. The study participants were excluded if they had one of the following conditions: acute or chronic inflammatory diseases, infectious diseases, and cancer. For the detection of membrane forms of OX40 and OX40L on immune cells, 46 patients and 44 HCs were enrolled. The approval from the Ethics Review Board of the Second Affiliated Hospital of Soochow University was granted to the patients.In our study, blood samples were collected after overnight fasting from patients  was isolated from T1D patients or healthy donors. Flow cytometry was performed on PBMC incubated with fluorochrome-labeled monoclonal antibodies (mAbs) for 30 min. Anti-CD3-FITC (clone: UCHT1), anti-CD4-FITC (clone: RPA-T4), anti-CD8-FITC (clone: SFCI21), anti-CD14-FITC (clone: RMO52), and anti-CD19-FITC (clone: J3-119) mAbs were from Beckman Coulter. Anti-OX40-PE (clone: ACT35) and anti-OX40L-PE (clone: RM134L) were from eBioscience. Cells were washed and then immediately measured by flow cytometry . Data were analyzed using FlowJo software .To assess the expression of mOX40 in CD3The samples were centrifuged at 800xg for 8 min, and the sera was stored at -80°C for ELISA. To determine the relationship of sOX40 or sOX40L with the disease activity, serum levels of sOX40 and sOX40L were determined by ELISA using kits obtained from Kalang (Shanghai) and Cusabio (Wuhan), respectively. Sera samples were plated into the 96-well microplates, and ELISA was conducted following the manufacturer's instructions. Plates were read in a microplate reader  for the absorbance at 450 nm.γ), tumor necrosis factor-alpha (TNF-α), and IL-17a were quantified in patients using a cytometric bead array system (CBA)  according to the instructions of the manufacturer. For each reaction, 50 μl sera were mixed with 50 μl beads to each assay tube. 50 μl PE detection reagent was then added, and the samples were incubated for 3 hours at room temperature. Samples were washed with wash buffer, centrifuged, and then run on flow cytometer; data were analyzed using BD™ CBA Software.Cytokines interleukin IL-2, IL-4, IL-6, and IL-10, interferon gamma  and IBM SPSS statistic 22  were used for graph creation.Statistical analysis was performed using the IBM SPSS statistic 22 . All the quantitative data was presented as the mean ± standard deviation (SD). For statistical analysis, differences in continuous variables between two independent samples were evaluated by the Mann-Whitney +, CD4+, and CD8+ T cells in PBMC samples was less frequent in patients with T1D than HCs  . However, there was no significant difference in mOX40L on CD19+ B cells between T1D patients and HC   and CD19+OX40L+ B cells . A significant negative correlation between CD3+OX40+ T cells , CD4+OX40+ T cells , and creatinine (Cr) was observed; we also found a significant negative correlation between CD3+OX40+ T cells  and CD4+OX40+ T cells  with uric acid (UA). However, we did not found any correlation between CD8+OX40+ T cells and Cr or UA . Meanwhile, we found that the concentration of sOX40L was also significantly higher than that in HCs   and hemoglobin A1c (HbA1c) , that is, T1D patients with higher sOX40 expression levels had higher UA and HbA1c  was also positive but was not significant with UA  , IL-6 , IL-10 , and IFN-γ   . To expl Figures . The cor Figures . These dytokines . However 0.0056) .+ T cells  and CD4+ T cells  in T1D patients   .Costimulatory molecules play important roles in regulation of the immune response in autoimmune diseases. Therefore, they may be useful as treatment indicators for the disease .+ B cells in T1D patients and HCs. In addition, sOX40 levels in patients were positively correlated with the disease activity, as indicated by T1D disease activity, and sOX40L levels in the patients were positively correlated with inflammatory cytokines. Our findings suggest that sOX40 and sOX40L might counteract the aberrant immune response and potentially serve as monitoring indicators of disease progression and therapeutic targets in T1D treatment.The TNF/TNFR family members of costimulatory molecules are critical in regulating T cell responses . Here, wCostimulatory molecules can induce the activation of T cells, promote cell survival, support the formation of potential memory T cells, and produce the release of the cytokines , 22. TheAutoantibodies in T1D are risk indicators for the diagnosis and prediction of the disease and surrogate markers for autoimmune diabetes –37. The +CD25highOX40+ cells than healthy subjects [+, CD4+, and CD8+ T cells and a reduction of OX40L on CD14+ monocytes in T1D patients correlated with clinical parameters. Bresson et al. have found that OX40 agonist therapy can slow down T1D progression [The detailed functions of some costimulatory molecules are still controversial. Until now, one of the most attractive approaches to prevent T1D is using islet antigen-specific regulatory T cells (Tregs). Luczynski et al. discovered that the mRNA level of OX40 was lower in Treg cells of children with T1D when compared to the reference patients . Howeversubjects . In our gression . We specTaken together, our study revealed the dissociation between mOX40 and mOX40L expression and sOX40 and sOX40L levels in T1D patients for the first time. We provided evidence for the diagnostic value of OX40 and OX40L in T1D patients. The abnormal expression of OX40 and OX40L molecules appears to be connected with the severity of the disease. Thus, OX40 and OX40L may be promising biomarkers for diagnosis and prognosis of T1D. The in-depth mechanisms of membrane and soluble OX40 and OX40L in T1D remain to be elucidated, and the role of OX40 and OX40L in immune pathogenesis of T1D requires further research.


Fibroblast growth factor receptor 2 (FGFR2) play a vital role in skeletogenesis. However, the molecular mechanisms triggered by FGFR2 in osteoblasts are still not fully understood. In this study, proteomics and bioinformatics analysis were performed to investigate changes in the protein profiles regulated by FGFR2, with the goal of characterizing the molecular mechanisms of FGFR2 function in osteoblasts.P-value of < 0.05) were selected to determine differentially expressed proteins (DEPs). The bioinformatics analysis including GO and pathway analysis were done to identify the key pathways underlying the molecular mechanism.In this study, FGFR2-overexpression cell line was established using the lentivirus-packaging vector in human osteoblasts (hFOB1.19). Next, the isobaric tags for relative and absolute quantitation (iTRAQ) in combination with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used to compare the proteomic changes between control and FGFR2-overexpression cells. Thresholds  contains supplementary material, which is available to authorized users. A complex network that regulates the differentiation of mesenchymal stromal cells into osteoblasts and terminal differentiation into osteocytes under appropriate stimulation controls bone development. Although the development occurs via two types of processes known as intramembranous ossification and endochondral ossification, osteoblasts are its major bone forming cells for bone deposition in both processes . The funFibroblast growth factor receptor 2 (FGFR2), a member of the FGF receptor family, controls cell growth, differentiation and survival in multiply tissues . Among tBased on this background, we subsequently established stable osteoblasts overexpressing FGFR2 mediated by lentivirus and systematically analyzed altered signaling triggered by FGFR2 in human osteoblasts. Finally, we demonstrated intracellular signaling pathways in osteoblasts with FGFR2 overexpression are indicative of a less proliferative profile as compared with profiles from control.The human fetal osteoblastic 1.19 cell line (hFOB) was obtained from American Type Culture Collection  and were cultured as previously described , 8.The full length FGFR2 (NM_000141) was synthesized from human cDNA library using the following primers: forward primer 5’- CCAACTTTGTGCCAACCGGTCGCCACCATGGTCAGCTGGGGTCGTTTCATC-3’, reverse primer 5’- AATGCCAACTCTGAGCTTTGTTTTAACACTGCCGTTTATG -3’. The sequences were cloned into lentiviral vector GV341  with IRES-GFP. Lentivirus particles were produced by co-transfecting 293T cells with lentivirus vectors and pHelper 1.0, pHelper 2.0 packaging plasmids . Supernatants containing virus particles were collected, filtered and concentrated. The hFOB cells were transduced the FGFR2 or vector virus particles to generate the FGFR2 and control (NC) sublines. To generate cells stably expressing the vector control or FGFR2 constructs, the transfected cell were selected with 1 μg/ml puromycin  and pools of selected cells were subjected to proteomic experiments . Each fraction was concentrated by vacuum centrifugation and reconstituted in 40 μL of 0.1% (v/v) trifluoroacetic acid for liquid chromatography-tandem mass spectrometry (LC-MS/MS) .The LC fractions were analyzed using a Q Exactive MS (Thermo Finnigan) equipped with Easy nLC. A 10 mL aliquot of each fraction was injected for nanoLC-MS/MS analysis. The peptide mixture was loaded onto the C18-reversed phase column  in Buffer A (0.1% formic acid) and separated with a linear gradient of Buffer B (80% acetonitrile and 0.1% formic acid) at a flow rate of 250 nL/min controlled by IntelliFlow technology over 60 min. The gradient included 0 to 40% (v/v) for 55 min, 40% to 100% (v/v) for 58 min, and 100% (v/v) for 60 min. MS data acquisition was performed using the 10 most abundant precursor ions from the survey scan (300–1800 m/z) for high-energy collisional dissociation (HCD) fragmentation. The target value for Automatic Gain Control (AGC) was 3e6. Dynamic exclusion for selected precursor ions was 60 s. The resolution was set as follows: 70,000 atm/z 200 for MS scan and 17,500 at m/z 200 for HCD spectra. The normalized collision energy was 30 eV and the under fill ratio was defined as 0.1%.p-value<0.05 [MS/MS spectra were searched using MASCOT engine  embedded into Proteome Discoverer 1.4  against uniprot human database . For protein identification, the Mascot search parameters were set as follows: Peptide mass tolerance: 20 ppm; MS/MS tolerance: 0.1 Da; Enzyme: Trypsin; Missed cleavage: 2; Fixed modification: Carbamidomethyl (C), iTRAQ8plex(K), iTRAQ8plex(N-term), Variable modification:Oxidation(M), False discovery rate (FDR)≤0.01 . The avelue<0.05 .http://www.geneontology.org/). To assess functional associations between proteins, differentially expressed in FGFR2-overexpressing osteoblasts, the online tool STRING 10 was applied. Pathways enrichment of proteins clusters were performed according to KEGG pathway database.Differentially expressed proteins (DEPs) were analyzed according to GO terms for biological process, cellular component and molecular function in the database  was used for statistical analysis. Significances of difference between groups were determined by a non-paired Student’s t-test.To character the proteomic profile regulated by FGFR2, we firstly establish the hFOB11.9 cell line with stable FGFR2 overexpression. The hFOB cells were infected with the lentivirus carrying the humanFGFR2 gene or the empty vector. And the FGFR2-overexpressing (FGFR2) and control (NC) cell sublines were generated by puromycin selection. The efficiency of infection in the two cell sublines was determined by the detection of GFP signals by fluorescence microscopy. As shown in Fig. In this study, iTRAQ was used to assess proteome changes induced by FGFR2 overexpression. Based on data acquisition, 249, 427 spectra, 14967 unique peptides and 3395 proteins were identified  with a FDR of less than 0.01% Fig. . Among 1Among 149 DEPs, 78 and 71 proteins were significantly up-regulated and down-regulated in the FGFR2-overexpressed osteoblasts, respectively. The top 15 up-regulated proteins included ubiquitin-conjugating enzyme E3, ADNP homeobox protein 2 and Homo sapiens heat shock 70kDa protein 6  The basic statistics of the iTRAQ data. (B) Peptide length distribution. X-axis showed the peptide length, while Y-axis showed the corresponding peptide count. (C) Molecular weight distribution of the identified proteins. X-axis showed molecular weight (kDa), while y-axis showed number of proteins. Table S1. Protein list identified in biological replicate 1 and 2 with an FDR of 0.01% at the peptide level. Table S2. The categories enriched molecular function (MF) by GO annotation. Table S3. The categories enriched biological process (BP) by GO annotation. Table S4. The categories enriched cellular component (CC) by GO annotation. Figure S2. Real time PCR analyses of TNNI3 and UBE2E3 in hFOB cells overexpressing FGFR2 and control cells. Statistics analysis were performed using non-paired Student’s t-test, with *** representing p < 0.01. 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