Using HEK cells transfected with recombinant forms of PR65A with phosphomimetic (P-PR65A) and non-phosphorylated (N-PR65A) amino acid substitutions at these sites, these phosphorylations were shown to inhibit the interaction of PR65A with PP2Ac and PP2A holoenzyme signaling. Forty-seven phospho-proteins were increased in abundance in HEK cells transfected with P-PR65A versus N-PR65A by phospho-protein profiling using 2D-DIGE analysis on phospho-enriched whole cell protein extracts. Among these proteins were elongation factor 1α (EF1A), elongation factor 2, heat shock protein 60 (HSP60), NADPH-dehydrogenase 1 alpha sub complex, annexin A, and PR65A. Compared to controls, failing hearts from the Dahl rat had less phosphorylated PR65A protein abundance and increased PP2A activity. Thus, PR65A phosphorylation is an in vivo mechanism for regulation of the PP2A signaling complex and increased PP2A activity in heart failure.Serine-threonine Protein phosphatase 2 A (PP2A), a member of the PPP family of phosphatases, regulates a variety of essential cellular processes, including cell-cycling, DNA replication, transcription, translation, and secondary signaling pathways. In the heart, increased PP2A activity/signaling has been linked to cardiac remodeling, contractile dysfunction and, in failure, arrythmogenicity. The core PP2A complex is a hetero-trimeric holoenzyme consisting of a 36 kDa catalytic subunit (PP2Ac); a regulatory scaffold subunit of 65 kDa (PR65A or PP2Aa); and one of at least 18 associated variable regulatory proteins (B subunits) classified into 3 families. In the present study, three  The PR65A phospho-peptides were identified by MALDI-MS mass spectrometry and phosphorylated amino acids identified by MALDI-TOF. Three phosphorylated amino acids were identified, S303, T268, and S314. Each of these phosphorylation sites is conserved across the mouse, rat, and human proteins.Phosphorylated amino acids in PR65A were identified in non-failing  hearts from Dahl salt-resistant (R) rats. Phospho-enriched proteins extracted were separated by 2D-DIGE. The protein spot corresponding to PR65A, identified by its MW 65.28 and PI 5.0, was excised from the gel, subjected to tryptic digestion and phospho-peptides were enriched employing TiOIn order to study the effects of the identified PR65A serine/threonine phosphorylations on signaling, PP2A activity and PR65A interaction with PP2Ac were compared in HEK cells transfected with recombinant PR65A with phosphorylated amino acids mutated to alanines to eliminate phosphorylations (N-PR65A) and recombinant PR65A with the amino acids mutated to glutamines to mimic phosphorylation (P-PR65A). PP2A activity was less (P<0.01) in cells transfected with P-PR65A versus N-PR65A . The effIn order to proximally link PR65A phosphorylation with cell protein phosphorylations and targets, phosphoproteins in HEK cells transfected with P-PR65A versus N-PR65A were profiled using 2D-DIGE analysis. Approximately 200 total phosphoprotein spots were resolved. Of these #60 and #37 were more and less, respectively, than 1.5-fold intense in cells transfected with P-PR65A versus N-PR65A. Eight proteins that were at least 2.2 fold more intense in cells transfected with recombinant P-PR65A versus N-PR65A were reasoned to be the most likely targets of PP2A and identified by MALDI MS . Among tPhosphorylated PR65A, measured in control (Dahl R) and failing Dahl (Dahl S) rat hearts by immunoprecipitation, was greater in Dahl R versus Dahl S hearts  P<0.05. in vivo by the phosphorylation of PR65A at S303, T268, and S314, via modulation of formation of the PP2A holoenzyme complex. Thr286, Ser303, and Ser314 are located at the concave surface and the bottom ridge of the A subunit and within HEAT repeats 7 and 8  and ethics committee approved this study.HEK cells were purchased from ATCC. Antibodies against PR65a and PP2A catalytic subunit were from Santa Cruz Inc and Millipore Inc. Antibody against EF1A was from Cell signaling Inc. PP2A activity kit was purchased from Millipore Inc. QuickChange™ mutagenesis kit was from Stratagene.Male 250–300 g Dahl salt-sensitive (S) or Dahl salt-resistant (R) rats were purchased from Harlan Sprague Dawley. All animal studies were done in accordance with University of Illinois at Chicago Institutional Animal Care and Use Committee (IACUC). Rats were maintained on a 8% NaCl diet ad lib.PR65A phosphoprotein was resolved from phospho-enriched protein homogenates of left ventricle tissue by 2Dl gel electrophoresis, the corresponding protein spot was excised from the gel and subjected to tryptic digestion followed by tandem mass spectrometry analysis.α-cyano-4-hydroxycinnamic acid ) and spotted on the matrix-associated laser desorption/ionization (MALDI) plate.The PR65A spot was picked up by an Ettan Spot Picker  based on the in-gel analysis. The gel spot was washed 2–3 times then digested in-gel with modified porcine trypsin protease . The digested tryptic peptides were subjected to Supel-Tips  to enrich phosphopeptides, following the manufacturer's protocol. The phosphopeptides were desalted by Zip-tip C18 . Peptides were eluted from the Zip-tip with 0.5 µl of matrix solution (http://prospector.ucsf.edu/prospector/mshome.htm) to run a virtual digest. All precursors containing predicted phosphorylation sites were manually entered into the inclusion list for MSMS analysis. MALDI-TOF MS and TOF/TOF tandem MS/MS were performed on an AB SCIEX TOF/TOF™ 5800 System . MALDI-TOF mass spectra were acquired in reflectron positive ion mode, averaging 4000 laser shots per spectrum. TOF/TOF tandem MS/MS fragmentation spectra were acquired on all precursors containing predicted phosphorylation sites, plus the 5 most abundant ions present in each sample (excluding trypsin autolytic peptides and other known background ions).The protein accession number was submitted to UCSF Protein Prospector  and S/T to E mutations  by site-directed mutagenesis using the QuikChange™ mutagenesis kit  according to the manufacturer's protocols. All constructs were sequenced by the DNA sequencing core facility at University of Illinois at Chicago prior to use in the experiments.Full-length PR65α DNA construct (Origene Inc) was used to introduce S/T to A (HEK cells were transfected with recombinant PR65α constructs for 48 hrs using lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After 48 hrs, protein extracts were prepared for immunoprecipitation and PP2A activity assay studies.Cell lysates from either heart tissue or HEK cells transfected with PR65A and sds22 constructs with S/T to A and S/T to E substitutions were incubated overnight at 4°C with PR65A regulatory subunit antibodies  with constant shaking. Fifty µl of protein A/G agarose beads were added to each reaction and incubated with constant shaking at 4°C for 2 hrs. The samples were spun down for 1 min and the pelleted beads were washed sequentially with PBS with 1% Triton-X-100, PBS with 0.5% Triton-X-100, and PBS with 0.1% Triton-X-100. The pellets were re-suspended in SDS sample buffer and subjected to Western blotting with monoclonal antibodies against catalytic subunits of PR65A .PP2A was immunoprecipitated with monoclonal antibodies against the catalytic subunit  followed by incubation with a phosphorylated peptide substrate for 15 min. Then malachite green solution was added and the phosphate released measured by the color development at 650 nM. Assay kits available from Millipore, Inc. PP2A activity was measured as the difference between phosphatase activity in the presence and absence of okadaic acid (5 nM added 15 min prior to substrate).Phosphoprotein analysis was performed using 2-D DIGE using CyDye staining and MALDI-MS for identification as previously described Based on the fold changes in the phosphoprotein abundance, protein spots chosen for analysis were excised by Ettan Spot Picker  and washed multiple times to remove staining dye and other inhibitory chemicals. Gel spots were dried and then rehydrated in digestion buffer containing sequencing grade modified trypsin. Proteins were digested in-gel at 37°C and digested peptides were extracted from the gel with TFA extraction buffer and shaking. The digested tryptic peptides were desalted using C-18 Zip-tips (Millipore) and then mixed with CHCA matrix  and spotted into the wells of a MALDI plate. Mass spectra (MS) of the peptides in each sample were obtained using an ABSciex 4700 Proteomics Analyzer and ten to twenty of the most abundant peptides in each sample were further subjected to fragmentation and tandem mass spectrometry (MS/MS) analysis. The combined MS and MS/MS spectra were submitted for database search using GPS Explorer software equipped with the MASCOT search engine to identify proteins from the NCBI non-redundant protein database. Proteins that were identified with a confidence interval (C.I.) greater than 99% are reported.The structure of the A subunit (PR65A) in the B′γ1 holoenzyme (PDB code: 2NPP) was used as the starting model for the phosphorylated PR65A. The phosphate groups were added to residues Thr286, Ser303, and S314 of the A subunit (B′γ1 holoenzyme), and changes required for accommodating the phosphate groups were modeled. Structural modeling was performed and energy minimized using Sybyl (Tripos). The model with minimum changes was used for structural comparison with the A subunit from different PP2A holoenzymes (B′γ1 and PR70 holoenzymes).Methods for control and failing rat heart models used have previously been described Values are expressed as the mean ± SD. Differences among different treatments were determined by one way ANOVA for repeated measurements. Results are considered significant at P<0.05.


Transcription Activator-Like (TAL) effectors are DNA-binding proteins secreted by phytopathogenic bacteria that interfere with native cellular functions by binding to plant DNA promoters. The key element of their architecture is a domain of tandem-repeats with almost identical sequences. Most of the polymorphism is located at two consecutive amino acids termed Repeat Variable Diresidue (RVD). The discovery of a direct link between the RVD composition and the targeted nucleotide allowed the design of TAL-derived DNA-binding tools with programmable specificities that revolutionized the field of genome engineering. Despite structural data, the molecular origins of this specificity as well as the recognition mechanism have remained unclear. Molecular simulations of the recent crystal structures suggest that most of the protein-DNA binding energy originates from non-specific interactions between the DNA backbone and non-variable residues, while RVDs contributions are negligible. Based on dynamical and energetic considerations we postulate that, while the first RVD residue promotes helix breaks – allowing folding of TAL as a DNA-wrapping super-helix – the second provides specificity through a negative discrimination of matches. Furthermore, we propose a simple pharmacophore-like model for the rationalization of RVD-DNA interactions and the interpretation of experimental findings concerning shared affinities and binding efficiencies. The explanatory paradigm presented herein provides a better comprehension of this elegant architecture and we hope will allow for improved designs of TAL-derived biotechnological tools. Xanthomonas genus via Type III Secretion System (T3SS) and, after localization to the nucleus, specifically bind to DNA sequences, thus interfering with native cellular functions and supporting infection TAL (Transcription Activator-Like) effectors are proteins secreted by phytopathogenic Gram-negative bacteria from the cassette insertion TAL systems recently gained wider attention after a direct relationship between the RVD type and the targeted DNA base was established by both experimental et al. obtained the structure of PthXo1, a naturally-occurring 23.5 repeats TAL bound to its corresponding DNA target, at 3 Å resolution et al. solved the structure of a designed 11.5 repeats TAL system in both bound and unbound states with resolutions of 1.85 Å and 2.5 Å respectively The understanding of the structural features of TAL effectors in general, and their relationships to DNA-binding in particular, are essential steps towards effective protein engineering for tailored biotechnological applications. Following the first structural NMR data of a single TAL repeat Although those structures have provided a much clearer understanding of DNA recognition by TALs, some aspects of this unique mechanism still remain elusive. In particular, the source of high specificity in RVD-DNA base recognition – despite limited interactions – and the grounds for both RVD compositions and occurrences are still unclear. Moreover, the structural roles of the other residues and their importance to protein function have not been addressed yet. Furthermore, although the RVD-to-DNA code has been shown to work well in most cases, some combinations have proven sub- or non-functional, raising the issue of context dependence i.e. not H) of the protein and DNA. Productions were run in the NPT ensemble  with all atoms free. Langevin dynamics was used to enforce ensemble parameters. A 12 Å cutoff distance was defined for short-range interactions, while the Particle Mesh Ewald summation method was used to compute long-range interactions. The RATTLE algorithm was used to treat covalent bonds involving hydrogen atoms in order to allow an integration step of 2 fs. Ions (Mg2+ and Cl−) were added to the systems to neutralize charges and obtain a final magnesium concentration mimicking physiological conditions (∼50 mM). The structural ensembles generated by MD simulations were used to establish statistics on the conformational states accessible to the systems. This information was used to highlight the structural importance of certain residues in the protein architecture and for the DNA recognition mechanism. Full details about system set-ups and MD simulation protocols are reported in The available crystal structures of both free (PDB code: 3V6P) cf.To further investigate TAL specificity, energy decompositions of pairwise interactions were performed using MM/GBSA  and MM/PBSA (Molecular Mechanics/Poisson Boltzmann Surface Area) implicit solvent calculations in order to identify the hot spots at the protein-DNA interface and the stabilizing interactions within TAL Quantum mechanical calculations were performed to obtain the molecular electrostatic potential (MEP) of DNA bases. The Gaussian 09 suite of program cf.a, b and c, e, f). All systems are stable with conservation of secondary structure within the simulated time-scales (i.e. recognised by TAL) and anti-sense strands of DNA . Those data are in accordance with the B-factors of the crystallographic structures.All simulated TAL-DNA systems showed comparable root mean square deviations and positional fluctuations; between 4.0 and 4.8 Å for the large systems, and between 1.9 and 2.5 Å for the small systems. Differences among system categories (TAL[22.5] or TAL[11.5]) are not statistically significant  and the RVD loops fluctuate as much as the outer region  – 2.1 +/− 0.5 Å when unbound vs 0.6 +/− 0.2 Å when bound; values are for the RVD loops of the small system – showing an overall stabilizing effect of the DNA double helix on TAL architecture. Moreover, when DNA is removed from the DNA-bound crystal structure 3V6T prior to simulation (TAL[11.5]/P1-apo), the protein rapidly stretches and the pitch approaches that of the unbound TAL 3V6P  globally, as template in the structural rearrangement of TAL systems from a stretched to a packed structure and ii) locally, to stabilize and reduce the mobility of the RVD loops.In the absence of DNA, TAL proteins are more flexible -S3d, g r region 5 d, g – TAL 3V6P . This device versa; an essential feature to attain the DNA-wrapping super-helix architecture characteristic of TAL proteins. Interactions of repeat i with its neighbours i-1 and i+1 are mainly constituted by K16(i)-Q17(i-1) and E20(i)-R24(i-1), forming a stable H-bond network linking together the TAL repeats in a regular and stable structure . Much less important are the interaction of H33(i) with Q23(i+1)/L26(i+1)/P27(i+1), all located at the kink generated by P27 . This property is consistent with the large helical content of the protein and the requirements for secretion through the T3SS injectisome. According to recent studies on T3SS d by P27 . The preDetailed information about TAL-DNA interactions at the single-residue level can be extracted from the decomposition of pairwise interaction energies as calculated at the molecular mechanics level a, b and c, e, f and S11). The second position of each RVD (X13) accounts for a small, although significant, fraction of the remaining DNA-binding energy .The first RVD position (X12) is almost always either H or N, the side-chain of either of them forming a stable interaction with the backbone carbonyl of I9; this promotes the helix break between α1 and α2 and allows the formation of the RVD loop in between. The low intrinsic α-helix propensities of N and H oxyanion clip constituted by G14, K16 and Q17  share with H and N (at X12) a very low intrinsic α-helix propensity  GGKQ in . These ddynamical interactions between the nucleobase and the X13 side-chain over the course of our simulations  The geometrical constraints on the backbone of the RVD loop and the restrictions on both size and conformational freedom of the X13 side-chain are the key elements to understand nucleobase discrimination and amino acid occurrences in RVDs. A simple unified pharmacophore-like model for both purines and pyrimidines has been constructed to highlight the nature and extent of the ulations . Since oe.g. in the NN RVD) clearly favours purine bases over pyrimidine bases owing to the lack of steric clashes with groups on α and β  can avoid this unfavourable interaction. Finally, even though scarcely present in the crystal structures, the specificity of the NS RVD can also be rationalized with the aid of the pharmacophore-like model and shown to be consistent with experimental findings. Indeed, as for the other amino acids present at position X13, the side-chain of serine is too small to allow a complete sampling of all loci and is therefore only able to properly discriminate thymine on a steric basis, thus agreeing with early observations about serine specificity Negative discrimination provides a rationale for D13 (in HD and ND RVDs) exclusively targeting cytosine; the carboxylate moiety on D13 forms a stabilizing interaction with the amino group on C4 (β) of cytosine, but would clash with the methyl group on C5 (α) of thymine, and would provide destabilizing interactions with the lone pair of the pyridine-like nitrogen N7 (γ) of either purines. Another illustration of negative discrimination is thymine targeted by NG, HG or N* RVDs. Here, any side-chain would clash with the methyl group at the α locus and thus only absence of side-chain  of each repeat to TAL binding are likely on a similar scale; any small RVD mismatch would have an energetic cost that would turn protein-DNA binding into an unfavourable process  has even greater specificity for guanine in silico, which we hope will retain the stronger selectivity of K13 for guanine without overall loss in binding efficiency  and a small contribution from X13. This suggests a mechanism of negative discrimination between X13 and the nucleobase. The low structural fluctuation of the RVD-loop imposed by the oxyanion clip in the bound state allowed us to devise a pharmacophore-like model for rationalization of the RVD-to-DNA code. The structurally-imposed incomplete molecular recognition of nucleobases by X13 explains the selectivity of the different RVDs and their shared affinities. The observation of a significant interaction between X13 and the preceding DNA base might provide an explication for the context-dependence of TAL activities. We hope that the recognition mechanism proposed herein will allow for more efficient rational designs, deepening alongside our knowledge of the intricate, yet very elegant architecture of TAL proteins.The dynamical study described in this report suggests that the composition of each RVD can be deconvoluted into its constituent parts and their contributions treated separately. X12 only interacts with the protein at the helix-RVD-loop transition. Together with the low α-helix propensities of both H and N, it suggests a purely structural role for X12, associated with the disruption of the α1 helix in order to allow proper folding of the RVD-loop and dense wrapping of DNA. By extension, X13 seems to be the sole player in DNA sequence recognition. However, the energetic pattern of TAL-DNA binding shows a strong dominance of the Figure S1Root Mean Square Deviations (RMSD). Calculations performed on Cα and P atoms for different portions of the systems (labels in the upper-right box of each graph). The overall RMSD for each portion as well as the corresponding standard deviation (in brackets) are reported next to each label. (a) TAL[22.5]/P1, (b) TAL[22.5]/P2, (c) TAL[11.5]/P1, (d) TAL[11.5]/P1-apo, (e) TAL[11.5]/P3, (f) TAL[11.5]/P4 and (g) TAL(TIF)Click here for additional data file.Figure S2Projection of the first four normal modes onto the trajectory. Normal modes obtained by Essential Dynamics Analysis (protein Cα atoms) of the MD trajectories. Labels are reported in the upper-right box of each graph, together with the statistical weight of each normal mode (in brackets). (a) TAL[22.5]/P1, (b) TAL[22.5]/P2, (c) TAL[11.5]/P1, (d) TAL[11.5]/P1-apo, (e) TAL[11.5]/P3, (f) TAL[11.5]/P4 and (g)TAL(TIF)Click here for additional data file.Figure S3Root Mean Square Fluctuation (RMSF). Calculations performed on protein Cα atoms. (a) TAL[22.5]/P1, (b) TAL[22.5]/P2, (c) TAL[11.5]/P1, (d) TAL[11.5]/P1-apo, (e) TAL[11.5]/P3, (f) TAL[11.5]/P4 and (g)TAL(TIF)Click here for additional data file.Figure S4Secondary structure time evolution. The colours represent the different secondary structure elements . (a) TAL[22.5]/P1, (b) TAL[22.5]/P2, (c) TAL[11.5]/P1, (d) TAL[11.5]/P1-apo, (e) TAL[11.5]/P3, (f) TAL[11.5]/P4 and (g)TAL(TIF)Click here for additional data file.Figure S5Average Root Mean Square Fluctuation (RMSF) per TAL repeat. Calculations performed on protein Cα atoms. (a) TAL[22.5]/P1, (b) TAL[22.5]/P2, (c) TAL[11.5]/P1, (d) TAL[11.5]/P1-apo, (e) TAL[11.5]/P3, (f) TAL[11.5]/P4 and (g)TAL(TIF)Click here for additional data file.Figure S6Per-residue decomposition of the intra-protein total interaction energy. Calculations performed on the TAL[22.5]/P1 system using the MM/GBSA (single-trajectory) approach. Graph obtained by taking the average per repeat and displaying a three-repeat window. For clarity, only values below −0.5 kcal/mol are reported.(TIF)Click here for additional data file.Figure S7Per-residue decomposition of the intra-protein interaction energy (only backbone contributions). Calculations performed on the TAL[22.5]/P1 system using the MM/GBSA (single-trajectory) approach. Graph obtained by taking the average per repeat and displaying a three-repeat window. For clarity, only values below −0.5 kcal/mol are reported.(TIF)Click here for additional data file.Figure S8Per-residue decomposition of the intra-protein interaction energy (only side-chain contributions). Calculations performed on the TAL[22.5]/P1 system using the MM/GBSA (single-trajectory) approach. Graph obtained by taking the average per repeat and displaying a three-repeat window. For clarity, only values below −0.5 kcal/mol are reported.(TIF)Click here for additional data file.Figure S9Decomposition of the protein-DNA total interaction energy of TAL[22.5]/P1. Calculations performed on the model system TAL[22.5]/P1 using the MM/GBSA (single-trajectory) approach. For clarity, only values below −0.5 kcal/mol are reported.(TIF)Click here for additional data file.Figure S10Decomposition of the protein-DNA total interaction energy of TAL[11.5]/P1. Calculations performed on the model system TAL[11.5]/P1 using the MM/GBSA (single-trajectory) approach. For clarity, only values below −0.5 kcal/mol are reported.(TIF)Click here for additional data file.Figure S11Contributions from different sections of TAL to the total protein-DNA binding energy (subdivided by type). Calculation performed using the MM/GBSA (single-trajectory) approach. (a) TAL[22.5]/P1, (b) TAL[22.5]/P2, (c) TAL[11.5]/P3, (d) TAL[11.5]/P4 and (e) TAL[11.5]/P1. Colour-code of the repeat-specific bars; red = G13, yellow = N13, blue = D13 and green = I13 (a and b) or green = S13 .(TIF)Click here for additional data file.Figure S12Contributions from an increasing number of TAL repeats to the total protein-DNA binding energy. Calculations performed using the MM/GBSA and MM/PBSA (single-trajectory) approaches on model system TAL[22.5]/P1 (cf. Methods S1 for details).(TIF)Click here for additional data file.Methods S1Supporting methods and references.(PDF)Click here for additional data file.


GAD65Abs are a sensitive and specific marker for type 1 diabetes (T1D). These autoantibodies can also be found in 6-10% of patients classified with type 2 diabetes (T2D), as well as in 1-2% of the healthy population. The latter individuals are at low risk of developing T1D because the prevalence rate of GAD65Abs is only about 0.3%. It has, therefore, been suggested that the antibody binding to GAD65 in these three different GAD65Ab-positive phenotypes differ with respect to epitope specificity. The specificity of reactive oxygen species modified GAD65 (ROS-GAD65) is already well established in the T1D. However, its association in secondary complications of T1D has not yet been ascertained. Hence this study focuses on identification of autoantibodies against ROS-GAD65 (ROS-GAD65Abs) and quantitative assays in T1D associated complications.Autoantibodies against glutamate decarboxylase-65 (GAD65 as compared to native GAD65 (N-GAD65). Uncomplicated T1D subjects also exhibited reactivity towards ROS-GAD65. However, this was found to be less as compared to the binding recorded from complicated subjects. These results were further proven by competitive ELISA estimations. The apparent association constants (AAC) indicate greater affinity of IgG from retinopathic T1D patients (1.90 × 10-6 M) followed by nephropathic (1.81 × 10-6 M) and uncomplicated (3.11 × 10-7 M) T1D patients for ROS-GAD65 compared to N-GAD65.From the cohort of samples, serum autoantibodies from T1D retinopathic and nephropathic patients showed high recognition of ROS-GAD65 that induce increased production of ROS-GAD65Abs. Hence regulation of ROS-GAD65Abs could offer novel tools for analysing and possibly treating T1D complications.Increased oxidative stress and blood glucose levels with extended duration of disease in complicated T1D could be responsible for the gradual formation and/or exposing cryptic epitopes on GAD Previous studies suggest that they are involved in antigen processing and presentation and thus modulate the immune response  or ROS-GAD65 . Conversely, serum autoantibodies of uncomplicated T1D patients showed significant binding with ROS-GAD65  as compared to N-GAD65 . Moreover, sera from diabetic nephropathic  and diabetic retinopathic  patients exhibited statistically higher significant differences in the binding pattern of serum autoantibodies with ROS-GAD65 as compared to N-GAD65 .In a pilot study serum samples from uncomplicated and complicated T1D patients were screened for autoantibodies against N-GAD65 as compared to N-GAD65. Whereas, nephropathic and retinopathic T1D complicated subjects showed an increase of 118.4% and 100% respectively, in the reactivity with modified antigen when compared with unmodified antigen.OD was considered as a direct measure of the concentrations of autoantibodies in the serum samples. In all the study groups there were higher levels of reactivity of modified antigen as compared to its native form. NH samples showed 14% increased and uncomplicated serum samples exhibited 65.7% increased reactivity towards ROS-GAD65 and ROS-GAD65 as inhibitors. Significantly higher recognition of modified antigen was observed by the serum autoantibodies from nephropathic [70.3 ± 8.2 mean maximum percent inhibition (MMPI)] and retinopathic patients (74.5 ± 6.5 MMPI] as compared to uncomplicated T1D serum samples (50.6 ± 7.2 MMPI). N-GAD65 exhibited no marked difference in recognition of serum IgG from both complicated [nephropathy (39.2 ± 5.4 MMPI) and retinopathy (41.1 ± 5.3 MMPI)] and uncomplicated (35.2 ± 5.9 MMPI) subjects of T1D, Table 65 (7.3 ± 3.6 MMPI) and M-GAD65 (7.2 ± 3.2 MMPI)].The binding specificities of serum autoantibodies from uncomplicated and complicated (Nephropathic and retinopathic) T1D patients were evaluated by competitive ELISA using N-GADp < 0.001) in complicated subjects as compared to uncomplicated T1D patients. Complicated subjects, T1D who had retinopathy (3.9 ± 0.31 nmoles/mg protein) exhibited higher amounts of protein bound carbonyl content as compared to nephropathic (3.4 ± 0.28 nmoles/mg protein) T1D patients.In vivo carbonyl content was considered a biomarker of oxidative stress. Oxidative stress levels were estimated for every patient of each group of T1D Table . Data sh65 (0-40 μg) were mixed with 100 μg of patient's IgG and incubated for 2 h at 37°C and overnight at 4°C. Microsurface adsorption-spectral correction (MSASC) technique showed the interaction of IgG with modified protein. Langmuir equation was used to estimate AAC of complicated and uncomplicated T1D samples for ROS-GAD65  and uncomplicated T1D patients (2.63 × 10-7 M) as shown in Figure The amount of antigen bound to antibody was also evaluated by quantitative precipitin titration curve. IgG of uncomplicated (serum no. 11) and complicated [nephropathic (serum no. 3) and retinopathic (serum no. 6)] subjects was purified by affinity chromatography on Protein A-Agarose column. The purified IgGs were found to elute in a single symmetrical peak. Varying amounts of modified GAD+ by NAD(P)H oxidase and glycation reactions [65  [The levels of ROS increase many folds during T1D via formation of sorbitol by polyol pathways, regeneration of cofactors NADPH and NADeactions ,13. Longeactions . This st65 by serum autoantibodies of complicated T1D patients was estimated when compared with autoantibodies from uncomplicated T1D subjects. N-GAD65 did not show any marked differences in recognitions of circulatory autoantibodies from complicated and uncomplicated T1D subjects. The affinity of GAD65Abs was found to be higher in subjects who had developed T1D with neuropathy [Significant recognition of ROS-GADuropathy  or autoiuropathy . In cont65 and ROS-GAD65 were ascertained by competitive ELISA. Twenty serum samples from normal humans were taken under the same experimental condition as controls. A characteristic difference was observed in the pattern of inhibition ELISA assays obtained from complicated and uncomplicated T1D subjects in respect to ROS-GAD65 antigen. This indicates that the ROS-modified GAD65 is an effective inhibitor showing substantial higher titres of circulating autoantibodies in complicated T1D subjects as compared to uncomplicated T1D. Moreover; amongst the complicated patients, retinopathic showed highest recognition for ROS GAD65 as compared to nephropathic and uncomplicated patients. Further the study also elucidates that with increased duration of disease and poor glycemic control leads to increased oxidative stress and hence the complications. The oxidative stress was further ascertained by the levels of protein bound carbonyl content in patients which is a biomarker of protein oxidation [65 of complicated T1D patients presents more number of epitopes that resemble in vitro ROS modified GAD65 and conceivably generates significant number of autoantibodies. GAD65Ab titers are higher and more prevalent in patients with other associated autoimmune diseases such as thyroiditis [65Ab epitopes.From the cohort, twenty serum samples each from complicated (nephropathic and retinopathic) and uncomplicated T1D patients were selected and binding affinities of circulating autoantibodies with N-GADxidation . Type 1 roiditis . The strroiditis  and recoroiditis ,20 has b65. So, the avidity of GAD65 became more complex and gain increased strength of binding because of interdependency of epitopes. Figure 65 than N-GAD65 by IgG isolated from retinopathic T1D subjects followed by nephropathic and uncomplicated T1D patient. The enhanced recognition of ROS-GAD65 by retinopathic T1D patient IgG showed the possible participation of oxidative stress and long duration of disease as given in Table 65 inducing the molecule to express its cryptic epitopes.During immune-pathophysiology significant amounts of circulating immune complexes are formed and deposited in kidneys leading to diabetic nephropathy, retinopathy and other tissues causing severe injury . As we d65Abs were detected in complicated (retinopathic and nephropathic) as compared to uncomplicated T1D patients. This risk of the disease may be exemplified due to acceleration in the formation of free radicals with gradual increase in duration of disease. This leads to conformational alterations in N-GAD65 protein which could increase or expose cryptic epitopes. Dynamic changes in the GAD65Abs binding pattern suggest subsequent epitopes spreading with disease progression. This could be one of the etiologies of increased GAD65Ab immunogenicity that implicated in T1D complications. Measurement of these autoantibodies could be shown to be useful in assisting the prediction for the development of T1D progression/or complications. Reduction in the levels of ROS may lead to decrease in in vivo GAD65 molecules modification thus, leads to delay in the progression of complications. Hence antioxidants may play important role in the treatment.In conclusion, significantly high levels of circulating ROS-GADIn the present study 100 T1D (60 uncomplicated and 40 complicated) and 50 control normal human (NH) subjects were investigated. All the patients were on the insulin treatment with suitable doses depending on the clinical examinations. All the serum samples of patients were collected from the laboratory of Endocrinology, Department of Medicine  and their clinical features are shown in Table These patients (n = 60) have had T1D and remained free from any complications (retinopathy and nephropathy). These patients are negative proteinuria.These patients (n = 20) had T1D and all were proteinuria positive (urinary protein excretion rates ≥300 mg/24 h) in the absence of hematuria or infection in midstream urine samples.These patients (n = 20) had retinopathy defined as having more than five dots or blots per eye; hard or soft exudates and vitreous hemorrhage.65  was modified with hydroxyl radicals. Briefly, solution  of N-GAD65 (100 mg/ml) in 50 mM sodium phosphate buffer, pH 7.4, was irradiated with 254 nm UV light for 30 min at room temperature in the presence of 10 mM hydrogen peroxide . After modification, extensive dialysis has been was done with 50 mM sodium phosphate buffer to remove excess hydrogen peroxide and hydroxyl radicals. Protein concentration determined by Bradford's method [Human-GADs method .et al. [379 = 22,000 M-1.cm-1. Protein concentration of the samples was determined by Bradford's method [Protein bound carbonyl groups from sera of different diabetic groups and NH subjects were analyzed according to Levine et al.  and the Direct binding ELISA was performed on polystyrene microtitre flat bottom plates , as described previously ,24. Brie65 was determined by competitive ELISA [The antigenic specificity of modified GADve ELISA . VaryingThe percent inhibition was calculated using the formula:inhibited is the absorbance at 20 μg/ml of inhibitor concentration and A uninhibited the absorbance at zero inhibitor concentration.Where A Immunoglobulin G was isolated from uncomplicated and complicated T1D sera on Protein A-Sepharose CL-4B column  . The hom65 antigen in an assay volume of 500 μl. The mixture was incubated for 2 h at room temperature and overnight at 4°C. The immune complexes were pelleted, washed twice with PBS and dissolved in 250 μl of 1 N NaCl. Protein concentrations were measured by colorimetric method [One hundred micrograms of IgG was incubated with varying amounts of ROS-GADc method . The binc method .The values are given as arithmetic mean ± SD wherever indicated. Multiple comparisons were analyzed by student t test using SPSS16 software program and p < 0.05 was considered to be statistically significant.MWAK designed and carried out all of the experiments and analysed the results. KB participated in data analyses. SNM helped in drafting the manuscript. WAK helped to carry out some ELISA experiments and in writing the discussion. All authors read and approved the final manuscript.


Using DNA microarray analysis, we previously identified that integrin α6 (ITGA6) was upregulated over 10-fold in EVI1high leukemia cells. In this study, we determined whether the increased expression of ITGA6 is associated with drug-resistance and increased cell adhesion, resulting in poor prognosis. To this end, we first confirmed the expression pattern of a series of integrin genes using semi-quantitative PCR and fluorescence-activated cell sorter (FACS) analysis and determined the cell adhesion ability in EVI1high leukemia cells. We found that the adhesion ability of EVI1high leukemia cells to laminin increased with the increased expression of ITGA6 and integrin β4 (ITGB4). The introduction of small-hairpin RNA against EVI1 (shEVI1) into EVI1high leukemia cells reduced the cell adhesion ability and downregulated the expression of ITGA6 and ITGB4. In addition, the overexpression of EVI1 in EVI1low leukemia cells enhanced their cell adhesion ability and increased the expression of ITGA6 and ITGB4. In a subsequent experiment, the introduction of shRNA against ITGA6 or ITGB4 into EVI1high AML cells downregulated their cell adhesion ability; however, the EVI1high AML cells transfected with shRNA against ITGA6 could not be maintained in culture. Moreover, treating EVI1high leukemia cells with neutralizing antibodies against ITGA6 or ITGB4 resulted in an enhanced responsiveness to anti-cancer drugs and a reduction of their cell adhesion ability. The expression of ITGA6 is significantly elevated in cells from relapsed and EVI1high AML cases; therefore, ITGA6 might represent an important therapeutic target for both refractory and EVI1high AML.Ecotropic viral integration site-1 (EVI1) is one of the candidate oncogenes for human acute myeloid leukemia (AML) with chromosomal alterations at 3q26. High EVI1 expression (EVI1 Individual HSCs are typically anchored to the stroma via a network of adhesion molecules high AML cells was higher than that of the EVI1low cells. The increased adhesion of EVI1high AML cells was dependent on the expression of the integrins α6 (ITGA6) and β4 (ITGB4) in complex with laminin, particularly laminin-332. Because the increased adhesion ability of the AML cells increased their resistance to chemotherapy, and the expression of ITGA6 was significantly higher in AML cases in relapse, ITGA6 might be a novel molecular target in EVI1high leukemia cells.In this study, we observed that the cell adhesion ability of the EVI1high leukemia cells (p<0.01). To confirm the results of the microarray analysis, the expression levels of a series of integrin genes were determined in three EVI1low and EVI1high cell lines using semi-quantitative RT-PCR. We determined the levels of the eight different β integrin genes (1 through 8), fifteen different α integrin genes  and two cadherin molecules (N and VE). As shown in Fig. S1A, ITGB1, ITGB3, ITGA4, and ITGA5 were expressed in five of the six leukemia cell lines, and ITGB4 and ITGA6 were specifically expressed in the three EVI1high leukemia cell lines. To confirm the results of the expression patterns of the integrin genes, we used the six cell lines and two primary leukemia cell lines from patients with the inv(3) (q21q26) subtype (PT9 and PT11) high leukemia cell lines  containing 38,500 genes ll lines Fig. 1A.high and EVI1low leukemia cell lines. The three human EVI1low myeloid cell lines  showed a reduced binding affinity to laminin and matrigel but an increased binding affinity to fibronectin. In contrast, the three EVI1high cell lines  exhibited an increased affinity for laminin, matrigel and fibronectin, with significant differences  were attached to the MC3T3-E1 cells compared with the three EVI1low leukemia cell lines , and this difference was statistically significant (p<0.01). Because the EVI1high leukemia cell lines exhibited an enhanced ability to adhere to the laminin complex , and a control cell line expressing an shRNA for firefly luciferase (shLuc) (AML1/shLuc) were established. Because the EVI1 expression was significantly downregulated in the AML1/shEVI1-1 to 3 cell lines . Taken together, the results demonstrated that ITGB4, ITGA6, ITGA9, and VE-cadherin are the candidate integrin genes with increased adhesion ability in EVI1high leukemia cells. Moreover, we determined the cell adhesion ability of the AML1/shEVI1 cell lines and found that the adhesion of AML1/shEVI1-1 to 3 cells to matrigel was significantly reduced compared with that of the parental UCSD/AML1 and AML1/shLuc cells  were introduced into UCSD/AML1 cells with EVI1ll lines Fig. 2A,low U937 cells and determined the resulting levels of expression of the various integrin genes. The adhesion of U937/EVI1 cells to matrigel was increased three-fold, and the expression of ITGB3, ITGB4 and ITGA6 was also increased in U937/EVI1 cells relative to their adhesion ability and expression in the U937/parental and U937/GFP cells . Because ITGB4 and ITGA6 form a heterodimer that binds to the laminin receptor and treatment with a neutralizing antibody to ITGB3 did not suppress the cell adhesion ability of EVI1high leukemia cells   and two primary human AML (PT9 and PT11) cells lines were cultured on matrigel and treated with or without anti-ITGA6, ITGB2, ITGB3, or ITGB4 antibodies. Two days after treatment, the binding activity of all four cell lines was significantly reduced by the treatment with anti-ITGA6 or anti-ITGB4 antibodies; however, isotype IgG, anti-ITGB2 or anti-ITGB3 antibodies did not inhibit the binding of EVI1high leukemia cells to matrigel  and three EVI1high  leukemia cell lines were co-cultured with MC3T3-E1 cells, and the mixed cultures were subsequently treated with anti-ITGA6 or anti-ITGB4 antibody or the control isotype anti-rabbit IgG. The ability of the three EVI1high leukemia cells to bind to MC3T3-E1 cells was significantly reduced upon treatment with anti-ITGA6 or anti-ITGB4 antibody, whereas the binding ability of the two EVI1low leukemia cells did not change . We subsequently introduced shRNA against the a3 chain of laminin-332 (shLN332) into MC3T3-E1 cells and measured the ability of the MC3T3-E1 cells to adhere to various types of myeloid leukemia cells. After the introduction of shLN332 or shLuc into MC3T3-E1 cells , the expression of the a3 chain of laminin-332 was significantly reduced  or laminin-511 (a5b1g1)  reduced Fig. 4A,high leukemia cells with a neutralizing antibody against integrin improves the drug sensitivity of the cells in the adhered state. Two EVI1high leukemia cell lines, UCSC/AML1 and MOLM1, and two primary leukemia cell lines, PT9 and PT11, were used in this experiment; the IC50 of Ara-C for these cells cultured on the matrigel coated plates was approximately 10−6 M . The four cell lines were seeded on matrigel-coated culture plates ith or without anti-ITGA6 or anti-ITGB4 antibodies in the presence of 10−6 M Ara-C. After four days of culture, the percentage of surviving cells in the cultures treated with anti-ITGA6 or anti-ITGB4 antibodies were significantly reduced in the two EVI1 and primary AML cell lines compared with the control cells , the treatment with anti-ITGA6 or anti-ITGB4 antibodies or anti-cancer drugs might recover the drug-sensitivity of EVI1high leukemia cells.We next determined whether treating EVI1high leukemia cells is highly dependent on EVI1 expression and partially dependent on ITGB4 expression.To determine whether the effect of Ara-C was dependent on the cell adhesion ability, AML1/shITGB4, AML1/shEVI1 and control AML1/shLuc cells were cultured on matrigel-coated or control BSA-coated plates and treated with Ara-C for three days; we could not use AML1/shITGA6 cells in this experiment. After three days of culture, the number of viable AML1/shLuc cells was significantly higher when the cells were cultured on matrigel-coated plates compared with conventional culture; however, the percentage of surviving AML1/shITGB4 cells was reduced at each time point.Fig. 5E.high/low, which were cultured on matrigel or BSA-coated plates. As shown in high expression (AML1/shLuc) on the matrigel-coated plates grew more faster rapidly on the matrigel-coated plates than that on the BSA-coated plates, although the growth rate of the AML1/shLuc cells was faster than that of the AML1/shEVI1 cells. The cell cycle analysis represented revealed that, relative to AML1/shLuc cells cultured on the BSA-coated plates, the population of EVI1high AML1 cells in G0/G1 and G2/M phase increased on the matrigel-coated plates, while the S-phase populations were decreased; however, each cell cycle population of AML1/shEVI1 cells did not change under either culture condition , the increased quiescence of adhered EVI1high AML cells might be one of the reasons for resistance to anti-cancer drugs.To confirm the advantages of cell adhesion in drug-responsiveness, we determined the growth and cell cycle status of AML cells with EVI1ondition Fig. 6D.high leukemia cells because the increased expression of EVI1 in AML is a well-known prognostic factor for poor outcome and is associated with drug resistance high leukemia cells with increased ITGA6 and ITGB4 expression exhibited stronger adhesion to laminin complexes than the EVI1low leukemia cells; although, the EVI1low leukemia cells exhibited increased adhesion to fibronectin. Moreover, the interaction between EVI1high leukemia and MC3T3-E1 osteoblastic cells was partially dependent on laminin-332. The downregulation of EVI1 or ITGB4 in UCSD/AML1 leukemia cells restored chemo-sensitivity. The knockdown of ITGA6 expression in EVI1high leukemia cells greatly reduced their survival in culture; thus, we conclude that the increased cell adhesion ability of EVI1high leukemia cells is primarily dependent on the expression of ITGA6, and that signaling from IGTA6 is crucial for the survival of EVI1high leukemia cells.Resistance to treatment with anti-cancer drugs is determined by a variety of factors, including somatic cell genetic differences in tumors and the phenotypes of leukemia stem cells (LSC). LSCs can infiltrate bone marrow niches, leading to enhanced self-renewal and proliferation, and enforced quiescence; the resistance to chemotherapeutic agents and adhesion molecules, such as VCAM-1 and VLA-4, has been described in the localization and retention of normal HSCs and/or leukemia cells within the bone marrow niche in vivohigh AML cells was significantly decreased in the bone marrow after the intravenous injection of anti-human ITGA6/ITGB4 complex antibodies into NOG immune deficient mice (data not shown). As a corollary, the increased expression of the ITGA6/ITGB4 complex in EVI1high leukemia might be an important factor in maintaining leukemia stem cells in the bone marrow.The expression of the ITGA6/ITGB4 complex could promote tumor progression and the metastasis of various cancer cells, including breast, colorectal, and thyroid carcinomas. Moreover, the laminin receptor for ITGA6 is ubiquitously expressed in human and mouse hematopoietic stem and progenitor cells high AML cells became more resistance to anti-cancer drug therapy, and the population of cells in the G0-phase was increased. Because the increased population of G0-phase cells is likely dependent on the expression of EVI1, future research should focus on the characterization of the mechanism underlying the increased quiescent of cells through EVI1 expression subsequent to their adhesion to matrigel. Moreover, the induction of PI-3K/AKT/Bcl-2 signaling through the VLA-4-fibronectin interaction results in resistance to anoikis and drug-induced apoptosis high AML cells to matrigel induced expression of BCL and phosphorylation of AKT (data not shown). Therefore, the adhesion of AML cells may provide advantages to the surviving cells, and the mechanism of drug-resistance is potentially much more complicated; however, we should characterize the survival mechanism of leukemia cells in the bone marrow niche.The adhesion of EVI1high and EVI1low AML deposited in Gene Expression Omnibus  Fig. S6, the expression of ITGA6 was significantly higher in the 12 cases of EVI1high AML than in the 10 cases of EVI1low AML (p<0.05); however, the differences in expression of ITGB4, ITGA4 and ITGB1 did not account for the differences between the two groups. We also determined the expression patterns of four integrin genes from the expression profiles of AML cases in remission and relapse that were deposited in GEO at NCBI (GDS1059) high and relapsed AML, which suggests that ITGA6 might be an important target for the molecular-targeted treatment of refractory leukemia, including EVI1high AML.To support our data, we examined the expression patterns of ITGA6/ITGB4 and VLA-4 (ITGA4/ITGB1) heterodimers by comparing the gene expression profiles of EVI12. The UCSD/AML1, PT9 and PT11 cells 2. The MOLM1 cells were purchased from the Hayashibara Institute, and the MC3T3-E1 and HNT34 cells were purchased from the RIKEN cell bank. The 293T cells were cultured in high-glucose DMEM supplemented with 10% fetal calf serum, and the MC3T3-E1 cells were cultured in MEM alpha supplemented with 10% fetal calf serum.The MOLM1, HNT34, U937, K562, and HL60 cells were cultured in RPMI1640 medium supplemented with 10% fetal calf serum at 37°C and 5% COThis experimental method was previously described −3 to 10−7 M cytosine-arabinoside (Ara-C) for 48 h. The number of viable cells was determined using trypan blue exclusion.The UCSD/AML1, MOLM1, PT9 and PT11 cells were plated onto tissue culture plates with BSA or matrigel-coated wells and subsequently incubated with 10The neutralizing antibodies used included rat anti-ITGA6 (Santa Cruz) and mouse anti-ITGB2, ITGB3 and ITGB4 (Millipore).http://lpgws.nci.nih.gov/cgi-bin/PrimerViewer). The primers for the laminin family genes were produced based on Primer Bank (http://pga.mgh.harvard.edu/primerbank/).Total cellular RNA was isolated using Trizol (Invitrogen), and the cDNA was prepared using oligo(dT) primer and reverse transcriptase . The polymerase chain reaction (PCR) was performed using Ex Taq (Takara-Bio) on a GeneAmp 2400 machine (Applied Biosystems). The primers for all integrin family members, EVI1 and b-actin were produced based on methods used in previous studies 5′-GATCGCTCTAAGGCTGAACTAGCAGTTCAAGAGACTGCTAGTTCAGCCTTAGATTTTTTG-3′; human ITGA6, 5′-GATCTCCTTTCAGGTTCAGTAGTTATTTCAAGAGAATAGTTACTGAATCTGAGAGGTTTTTG-3′; human ITGB1, 5′-GATCTGGAGGGTTGTTTCGGGTTTCATTCAAGAGATGAAGTCCGAAGTAATCCTCCTTTTTG -3′; and human ITGB4, 5′-GATCGCCAGCGACTACACTATTGGATCTCGAGATCCAATAGTGTAGTCGCTGGTTTTTG . We used the pSIREN-retroQ-ZsGreen-shLuc plasmid containing an shRNA against firefly luciferase (Takara-Bio) as a control. The retroviral particles were generated using the p10A1 packaging vector (Takara-Bio) via the transient transfection of 293T cells. The transfection was conducted using Hilymax liposome transfection reagent (Dojin). For retroviral transduction, 1×106 cells were plated onto a 6-cm dish containing 5 ml retroviral supernatant containing 100 ng/ml of polybrene for 24 h. After two weeks, the ZsGreen-positive cells were sorted using a JSAN cell sorter (Bay Bioscience). The reduction of EVI1 or ITGB4 expression was confirmed using RT-PCR.The pSIREN-retroQ-ZsGreen plasmid (Takara-Bio) was used to inhibit EVI1 expression, and expression of ZsGreen (green fluorescent protein) was used as a marker. The following sequences were cloned into the BamHI-EcoRI sites of the pSIREN-retroQ-ZsGreen plasmid to create shRNAs against human EVI1 4 precipitation. For retroviral transduction, 1×105 U937 cells were plated onto 96-well flat-bottomed plates and infected for 24 h with either an EVI1 retroviral supernatant (pGCDNsam-EVI1-IRES-EGFP) or a control retroviral supernatant (pGCDNsam-IRES-EGFP) containing 100 ng/ml of polybrene. After 7 days, the green fluorescent protein (GFP)-positive U937 cells were sorted using a JSAN cell sorter .The pGCDNsam-EVI1-IRES-EGFP construct was kindly provided by Dr. A. Iwama . To produce recombinant retrovirus, the plasmid DNA was transfected into 293GP cells along with the vesicular stomatitis virus G (VSV-G) expression plasmid via CaPO5′-GATCTGAACTCCTTAAATGATTATGAATTCAAGAGATTCATAATCATTTAAGGAGTCTTTTTTACGCGTG-3′. The target sequence for the knockdown was determined using the BLOCK-iT RNAi Designer (http://www.invitrogen.com/rnai). We used pSIREN-retroQ-ZsGreen-shLuc containing shRNA against firefly luciferase (Takara-Bio) as a control. We transiently introduced the laminin-332 or luciferase shRNA expression vector into MC3T3-E1 cells using an Amaxa Nucleofector (Lonza) and determined the expression levels of the a3 and g2 subunits of laminin-332.The pSIREN-retroQ-ZsGreen plasmid (Takara-Bio) was used to inhibit the expression of the a3 chain of laminin-332, and the expression of ZsGreen (GFP) was used as a marker. The following sequence was cloned into the BamHI-EcoRI sites of the plasmid to create an shRNA against the a3 chain of murine laminin-332: 3 cells/well were plated onto a 96-well culture plate and incubated for 1–3 days. Approximately 10 ml of Cell Counting Kit-8 reagent was added to the plate, and the absorbance at 450 and 620 nm was measured using an Immunomini NJ2300 plate reader (Nunc). A cell viability assay was performed using the Cell Counting Kit-8 reagent according to the manufacturer's protocol. For this assay, 5×103 cells were plated onto a 96-well culture plate containing Ara-C (1×10−6 M), rat anti-ITGA6 (20 mg/ml) or mouse anti-ITGB4 (20 mg/ml). The cells were incubated for 1–2 days and 10 ml of Cell Counting kit-8 reagent was added. The absorbance at 450 and 620 nm was measured. The cell viability was calculated by dividing the value obtained for the treatment well by the average value obtained for the control wells. For cell viability assay, cells were seeded in 5×104 cells/ml to matrigel coated culture plate. Cells were co-cultured with Ara-C (1×10−6 M) and anti-ITGA6 antibody (20 mg/ml) or anti-ITGB4 (20 mg/ml) or isotype control IgG (20 mg/ml). After incubation for 1 to 4 days, living cell numbers were counted by trypan blue exclusion. The cell viability was calculated by dividing the value for the treatment well by the value for the control wells. Each experiment was performed in triplicate. Data are shown as mean ± S.E. Statistical analysis was performed using Student's t-test.A cell proliferation assay was performed using a Cell Counting Kit-8 (Dojin) according to the manufacturer's protocol. Briefly, 5×102 of fibronectin , 5 mg/cm2 of laminin , 0.3 mg/ml of collagen (Nitta Gelatin) or 5% w/v BSA . After coating with ECM, 1×105 cells/ml were transferred to each pre-coated well and incubated for 24 h at 37°C. After washing two times with phosphate buffered saline (PBS), the floating and adherent cells were counted using trypan blue, and the number of adherent cells was divided by the number of floating cells to calculate the cell adherence ratios. In the neutralization experiments, the cells were pre-incubated with 20 mg/ml of anti-ITGA6, ITGB2, ITGB3 or ITGB4 antibodies prior to plating on the matrigel Four types of extracellular matrices  and BSA (as a control) were used for the cell adhesion assay. A culture plate was coated overnight with a 1/40 dilution of Growth Factor Reduced-Matrigel , 5 mg/cmThe intensity of ITGB3, ITGB4 and ITGA6 expression in UCSD/AML1 and U937 cells was analyzed using FACSCalibur (BD Bioscience). The cells were stained with phycoerythrin (PE)-labeled anti-human ITGA6 (Clone: GoH3), ITGB4 (Clone: 58XB4) or ITGB3 (BI-PL2) antibodies purchased from Biolegend, Inc.6 cells from each culture were fixed in BD Cytofix/Cytoperm buffer for 20 min., followed by a 300-mg/ml DNase treatment for 30 min. The cells were subsequently washed twice in BD Perm/Wash Buffer and stained with anti BrdU-APC  for 15 minutes, followed by staining with 7-AAD . The cells were harvested and analyzed using FACSCalibur with FL-4 (for BrdU-APC) and FL-3 (for 7-AAD) settings.To assess the percentage of cells in sub G0/G1, G0/G1, and G2/M and S phase after 48 h of culture on matrigel or BSA-coated plates, 1×106 cells from each culture were fixed in 70% ethanol for 2 h, followed by a 2.5 µg/ml RNAse treatment for 30 minutes. The cells were subsequently washed twice in PBS/1% fetal bovine serum (FBS) and stained with mouse anti-human Ki67  and Alexa647-conjugated anti-mouse IgG (Invitrogen) for 30 minutes, followed by staining with 7-AAD . The cells were harvested and analyzed using FACSCalibur with FL-4 (for Ki67-Alexa647) and FL-3 (for 7-AAD) settings.To assess the percentage of cells in G0 phase after 48 h of culture on matrigel or BSA-coated plates, 1×10Student's t-test was used to compare differences between the two groups.Figure S1Expression of integrin genes and cell binding ability of various myeloid leukemia cells.A. The expression of 25 members of the integrin family was detected using semi-quantitative RT-PCR in HL60, U937, and K562 (cell lines with EVI1low expression) and in UCSD/AML1, MOLM1, and HNT34 (cell lines with EVI1high expression). The expression of EVI1 and b-actin in these cells is also shown. B. The expression of 18 genes in the integrin family was detected using RT-PCR in AML1/shLuc and AML1/shEVI1 cell lines. C and D. The adhesion ability of various HNT-34 cell lines  to bind to matrigel is shown in C, and the expression of integrin genes in conjunction with EVI1 and b-actin is shown in D. E and F. The ability of PT9-related (PT9/shLuc and PT9/shEVI1) and PT11-related (PT11/shLuc and PT11/shEVI1) cell lines to bind to matrigel was measured (E), and the expression of various integrin genes in these four cell lines was determined using RT-PCR (F) G. The expression patterns of integrin genes were determined in U937/GFP and U937/EVI1 cell lines in conjunction with b-actin and EVI1.(DOC)Click here for additional data file.Figure S2Expression of various laminin chains in MC3T3-E1 cells. The relative expression levels of laminin a chains 1 to 5, laminin b chains 1 to 3, and laminin g chains 1 to 3 in MC3T3-E1 cells were determined using semi-quantitative RT-PCR.(DOC)Click here for additional data file.Figure S3Dose-response functions for AraC against four EVI1high AML cells. UCSD/AML1, MOLM1, PT9 and PT11 cells were incubated in BSA- or matrigel-coated wells of tissue culture plates and subsequently incubated with 10-3 to 10-7 M cytosine-arabinoside (Ara-C) for 48 h. Relative cell viability is calculated as a percentage relative to standard controls. Data are shown as mean ± S.E. Statistical analysis was performed using Student's t-test. A star (*) indicates p<0.05 and double stars (**) indicate p<0.01.(DOC)Click here for additional data file.Figure S4high and EVI1low leukemia cells cultured with or without MC3T3-E1 cells determined by treatment with VP-16 or Ara-C.Drug sensitivity of EVI1 UCSD/AML1 (EVI1high) leukemia cells and HL60 (EVI1low) leukemia cells were treated with Ara-C or VP-16 for three days under plastic flasks (open diamonds) or co-cultured with MC3T3-E1 cells (closed squares); the viable cells were counted at each indicated time point. The percent cell viability compared to the number of untreated cells is shown for each indicated day. A star (*) indicates p<0.05.(DOC)Click here for additional data file.Figure S5Decreased cell growth with increased cell population in G0-phase of U937 cells with EVI1 expression cultured on the matrigel-coated plates. A. U937 cells were introduced by EVI1 expression vector (U937/EVI1) or control vector (U937/GFP) to determine their cell growths with culture condition on BSA or matrigel-coated plates. B and C. Cell cycle of U937/GFP as a control (B) and U937/EVI1 (C) were analyzed by BD FACSCalibur after double-stained by BrdU-APC and 7-AAD. Percentages of each cell cycles were shown by white bars (BSA-coated) and black bars (matrigel-coated). D. The percentage of cells in G0 phase in U937/EVI1 (black bars) and U937/GFP cells (white bars) cultured on matrigel- or BSA-coated plates were analyzed by BD FACSCalibur after double-stained by Ki67-Alexa647 and 7-AAD. Each experiment was performed in triplicate, and experiments were independently repeated at least three times. Results are shown as mean ± S.E. Statistical analysis was performed using Student's t-test .(DOC)Click here for additional data file.Figure S6Expression profiles of ITGA6 and ITGB4 in AML patients. A and B. The expression patterns of ITGA6 (A) and ITGB4 (B) are shown as gene expression profiles for ten AML patients with EVI1low and ten AML patients with EVI1high expression . C and D. The expression patterns of ITGA6 (C) and ITGB4 (D) are shown as gene expression profiles for patients in remission and for AML patients who had relapsed .(DOC)Click here for additional data file.


Influenza A viruses are a threat to poultry and human health. We investigated evolution of influenza A virus H7 and N9 subtypes in wild and domestic birds. Influenza A(H7N9) virus probably emerged after a long silent circulation in live poultry markets in eastern Asia. The purpose of this study was to investigate the recent evolutionary history of H7 and N9 virus subtypes in eastern Asia and identify the most recent wild bird ancestor of influenza A(H7N9) virus hemagglutinin and neuraminidase.,To assess global phylogeny of influenza A virus H7 and N9 subtypes, we analyzed 715 hemagglutinin and 309 neuraminidase nucleotide sequences of viruses isolated during 1927–2012 worldwide . BayesiaPhylogenetic analyses showed maintenance of influenza A virus H7 subtypes in wild birds in eastern Asia since 1999 (clade A) , panel AThis pattern suggests that the old genetic lineage of influenza A virus H7 subtypes that circulated in eastern Asia since 1999 (clade A) may have been progressively replaced by a more recent lineage (clade B). Clustering of clade B viruses with those isolated in Europe suggest that gene flow has recently occurred in Eurasia, probably as the result of waterfowl migrations and poultry trade. However, the European origin of viruses from Asia was not supported on the basis of phylogeographic analysis .,,,Consistent with results of previous studies ,,,, panel A,Limited epidemiologic and genetic information about influenza A virus H7 subtype circulating in eastern China during 2004–2011 precludes more precise conclusions on origins of human influenza A(H7N9) virus and relatedness to influenza A virus circulating in wild birds. However, on the basis of genetic analyses of recently isolated viruses from chickens, pigeons, and the environment, maintenance and genetic reassortment of emerging influenza A(H7N9) virus might have occurred in live poultry markets in Shanghai, China  virus might have originated from influenza A(H11N9) viruses that circulated in eastern China, although limited information about influenza A virus N9 subtypes circulating in wild birds in this region represents a major challenge to identifying the donor of influenza A(H7N9) virus neuraminidase. Reassortments between influenza A(H11N9) viruses from Asia and influenza A(H1N3 and H7N3) viruses circulating in live-poultry markets in Zhejiang Province were documented in domestic duck in 2011 in nearby Jiangsu Province (A/duck/Jiangsu/10-d4/2011(H1N3) . Hemagglutinin of influenza A(H7N9) virus belongs to clade A. Genetic data indicate that the most recent ancestral wild bird–origin virus for A/Hangzhou/1/2013(H7N9) virus and Zhejiang domestic duck viruses can be dated to 2004. The influenza A(H11N9) virus that circulated in eastern Asia for ≈10 years, with associated intercontinental gene flows and reassortments, may be the donor of influenza A(H7N9) virus neuraminidase. Hosts and areas in which ancestral viruses have been maintained are unknown. However, influenza A(H7N9) virus probably emerged after a long silent circulation in live poultry markets in eastern Asia.Global phylogeny of influenza A virus H7 and N9 subtypes and phylogeography of recent H7 and N9 subtype genetic lineages in eastern Asia.


Arabidopsis thaliana mutants, as a factor critical to gamete fusion in flowering plants. Interestingly, GCS1 is highly conserved among various eukaryotes covering plants, protists and invertebrates. Of these organisms, Chlamydomonas  and Plasmodium  GCS1s similarly show male gametic expression and gamete fusion function. Since it is generally believed that protein factors controlling gamete fusion have rapidly evolved and different organisms utilize species-specific gamete fusion factors, GCS1 may be an ancient fertilization factor derived from the common ancestor of those organisms above. And therefore, its molecular structure and function are important to understanding the common molecular mechanics of eukaryotic fertilization. In this study, we tried to detect the central functional domain(s) of GCS1, using complementation assay of ArabidopsisGCS1 mutant lines expressing modified GCS1. As a result, the positively-charged C-terminal sequence of this protein is dispensable for gamete fusion, while the highly conserved N-terminal domain is critical to GCS1 function. In addition, in vitro fertilization assay of Plasmodium berghei  knock-in lines expressing partly truncated GCS1 showed similar results. Those findings above indicate that the extracellular N-terminus alone is sufficient for GCS1-based gamete fusion.Fertilization is one of the most important processes in all organisms utilizing sexual reproduction. In a previous study, we succeeded in identifying a novel male gametic transmembrane protein GCS1 (GENERATIVE CELL SPECIFIC 1), also called HAP2 (HAPLESS 2) in the male-sterile  Angiosperm fertilization is comprised of certain processes, from pollination to gamete fusion Lilium longiflorum pollen Arabidopsis GCS1 is identical to HAP2, which was previously identified as a pollen tube related factor from the hap2 phenotypes Lilium and Arabidopsis GCS1s were demonstrated to be expressed exclusively in male gametes (generative and sperm cells) and localized to the cell surface Arabidopsis GCS1 mutant pollen exhibits serious male sterility in which none of the sperm cells are able to fuse with female gametes, suggesting that GCS1 is an indispensable factor for gamete fusion In our previous study, we succeeded in identifying the novel protein GCS1 in male generative cells isolated from Plasmodium berghei  and Chlamydomonas reinhardtii , it has been shown that their GCS1 is similarly expressed in the male gamete and functions in gamete fusion GCS1-knockout Chlamydomonas male cannot perform gamete fusion, but does achieve attachment based on FUS1, which is a transmembrane protein expressed exclusively in the female gamete GCS1 expression in the hydra (a cnidarian), implying that animal GCS1s function in a similar manner Surprisingly, GCS1 is highly conserved and putative orthologs have been identified in various eukaryotes, e.g., protists, amoebae and invertebrates Chlamydomonas GCS1 revealed GCS1 to be a glycoprotein in which two types of N-glycosylation occur, and a rapid degradation of GCS1 molecules is triggered by gamete membrane fusion so as to prevent polygamy et al. investigated the molecular importance of N- and C-terminal sequences for the GCS1 transmembrane domain, using partially-modified AtGCS1 constructs AtGCS1 mutation, roughly suggesting that both termini are required for the GCS1 function of gamete fusion. In addition, they indicated that the positively-charged histidine rich domains are indispensable for normal double fertilization, since reduction of the positive charge causes reduced fertility and an occasional single fertilization, where only one sperm cell fuses with the egg or central cell in an ovule Since GCS1 possesses no known functional protein functional domains, the molecular structure and central domain(s) for gamete fusion are important issues AtGCS1 constructs, which are based on green fluorescence protein (GFP) insertion targeted to certain characteristic AtGCS1 sequence regions. To ensure the conservation of these GCS1 characteristics, similar constructs were also produced in PbGCS1. We report that the gamete-fusion functional GCS1 domain(s) is generally in the N-terminus and the function is drastically impaired even when the N-terminus is split from the gamete membrane.In the present study, an effort was made to obtain more detailed characteristics of GCS1 structure and function using partially-modified AtGCS1 domains described above with the GFP cDNA insertion at appropriate restriction-enzyme-recognizing sites. As a result, 6 modified AtGCS1 (mAtGCS1) constructs were obtained . The products expected from these constructs are shown in AtGCS1 clone, which was successfully used in GCS1 knockout rescue in a previous study Arabidopsis GCS1 mutant (+/gcs1) plants to assess which of the constructs maintains or loses function, namely which domain(s) is critical to gamete fusion. The +/gcs1 line was used in our previous study and the gcs1 pollen is completely male-sterile As previously described, AtGCS1 is composed of an N-terminus signal sequence (SS), which probably leads to its cell membrane localization, a long body sequence containing the HAP2-GCS1 domain that is highly conserved among GCS1-possessing organisms, a hydrophobic transmembrane (TM) domain and a highly basic C-terminal histidine-rich (HR) sequence  [2]–[3],mAtGCS1 construct is hemizygously introduced into +/gcs1 plants, the pollen from the transformants are categorizable into 4 genotype groups, namely wild type pollen (I), gcs1 pollen (II), mAtGCS1-possessing pollen (III) and mAtGCS1-possessing gcs1 pollen (IV) (gcs1 pollen is linked with the kanamycin-resistance gene (RKan), while all of the mAtGCS1 constructs are linked with the gentamycin-resistance gene (RGen). Of these groups, the pollen from groups I and III is evidently fertile because of normal GCS1 expression. On the other hand, the group II pollen is infertile because of the GCS1 mutation, and therefore, RKan is never inherited by the offspring plants paternally. However, if an mAtGCS1 construct complements the GCS1 mutation, both RKan and RGen in group IV should be inherited by the offspring plants paternally. In this case, one-third (∼33.3%) of the offspring should survive in the Kan- and Gen-containing media, while the remaining ones (∼66.7%) should not be alive, because they are from groups I and III in which the wild-type plants were pollinated with the transformant pollen. Based on this expected scenario, wild type females were pollinated with pollen from each of the mAtGCS1 transformants, and the resulting offspring seeds were sown in Kan- and Gen-containing media to assess which construct complements the GCS1 mutation  is hemizygously expressed, were used. As a result, when the +/AtGCS1gcs1 pollen was used, approximately 33.3% of the offspring plants survived in the media , indicating that complementation by the construct was almost completely successful . Furthermore, the results for the +/GHHgcs1, +/GHPgcs1 and +/GPPgcs1 pollen displayed obvious complementation and were comparable with to +/AtGCS1gcs1   . The T-Dmutation . As a poentation . The dif, P>0.5) . At the gcs1 complementation by GAA led to the expectation that the +/GAAgcs1 sperm cells occasionally fail to fuse. To observe the sperm behavior in an ovule, we produced a +/GAAgcs1 line whose sperm cells express HTR10-RFP fusion as a sperm nucleus marker mAtGCS1 constructs which are successful in complementation, such as GHH, GHP and GPP . In this assay, we prepared 3 partially-modified PbGCS1 constructs, designated –HG, –C and –TM/C, besides the normal PbGCS1 construct (PbGCS1 Full). The expected product from each construct is shown in P. berghei parasites by double crossover homologous recombination, by which the endogenous PbGCS1 gene is replaced with each construct. Normal expression of the transgene in these knock-in (KI) parasites was confirmed with RT-PCR assay  in the C-terminus of AtGCS1, and demonstrated that deletion of the C-terminal sequence covering them or an exchange of their positively charged residues, resulted in an obvious reduction of GCS1 function Arabidopsis gamete fusion (unpublished data). As an explanation for our results conflicting with the hypothetical C-terminus contribution, C-terminal domain(s) may be associated with functional localization and/or stability of AtGCS1 molecules on the sperm membrane. Indeed, the functional GHP keeps endogenous 34 amino acids between TM and the GFP insert , the +/GAAgcs1 phenotypes might not be attributed to neofunctionalization by GAA products. We, therefore, conclude that the original gcs1 phenotype, namely no gamete fusion, was converted into delayed gamete fusion by the introduction of GAA. Nevertheless, the fact that some gcs1 sperm cells were complemented by GAA indicates that slightly delayed gamete fusion is not crucial for double fertilization, while extensively delayed fertilization results in a failure of seed development , where only one sperm cell fuses with a female partner cell in an ovule, has been reported in various bserved, . When ovdetected . Since aelopment .GCS1 is highly conserved and putative orthologs have been identified in various eukaryotes, e.g., protists, amoebae and invertebrates, and therefore, its molecular structure and function are important to understanding the common molecular mechanics of fertilization in these organisms. In addition, understanding of malaria parasite fertilization based on GCS1 may be applicable to novel strategies attacking parasites Studies with experimental animals were approved by Animal Care and Use Committee of Gunma University, and followed guidelines of this committee. The transgenic P. berghei was generated under the guidelines of the recombinant DNA experiments committee of Gunma University. The assigned ID for above experiments is 10-007.GAGCTCAGCAGAGCACATCTTATCAATTTCTGAGGC;ATGCS1PROF(SacI) GGTACCAGCAGAGCACATCTTATCAATTTCTGAGGC;GCS1GENF1000(KpnI), GGTACCGAAACCCTAACTTAATCGAATCGTTGAGGC;ATGCS1GENR(KpnI), AAGCTTTCATGGTGAGCAAGGGCGAGGATGAGGC;GFPf2(HindIII), AAGCTTCCTTGTACAGCTCGTCCATGCCGTGAGGC;GFPr2(HindIII), CTTAAGCATGGTGAGCAAGGGCGATGAGG;GFPf(AflII), CTTAAGTTCTTGTACAGCTCGTCCATTACT;GFPr(AflII), GTTAACATGGTGAGCAAGGGCGATGAGGT;GFPf(HpaI), GTTAACCTTGTACAGCTCGTCCATTGACTT;GFPr(HpaI), CACGTGATATGGTGAGCAAGGGCGATAGGT;GFPf(PmlI), CACGTGCTTGTACAGCTCGTCCATTGACTT;GFPr(PmlI), GTCTTGAATCTCGCTGTTCCCAGT;Atgcs1-seqA2, TTAACTCTCACGTAGTCTTTGTTTCC;Atgcs1truer, CGGACACGCTGAACTTGTGG;GFPBDr, GCCCTGAGCAAAGACCCCAA.GFPBDf, GGTACCGATCATGTTGGAGAATAAATGGGGGPbGCSF(KpnI), ATCGATAATTAAAGAAATTATATTTATCTGTCGGPbGCSR(ClaI), GATATCAATTACATGGAATAGTATTTGCAAATTTGGGPbGCS3UTRF(EcoRV), TCTAGAGCCTTTTATAATATGATATTACCCACGGPbGCS3UTRR(XbaI), CATGGATGCATAATATTACATATACTTG-HAP2/GCS1InvR, TATAAATACAATGGAAATCATAGTGTAG-HAP2/GCS1InvF, AGTAGATATAAAAAGGAAAAAGAAATTTAAAAAGAATGGTAGAAG-CInvR, TAAAATTACATGGAATAGTATTTGCAAATTTGTG-CInvF, CCCAATTAACGTTTTAACTGTATTTATATAATAGT-TMR, GACATTGTCCTATTATAGATTTAATGCAF, TGAGTAATTGTTGCAATGGAAACGGGTCAR, CTCTTGCTCAATTGCATGCBF, GTTGTGTTTCCTCCATCCBR, Arabidopsis GCS1 mutant seeds were obtained as previously described. All Arabidopsis plants (Columbia ecotype) were grown in an incubator on a 16-light/8-h dark at 20°C. Plasmodium berghei (ANKA clone 2.34) was intraperitonealy injected into a Balb/c female mouse.The AtGCS1 construct, an AtGCS1 genomic clone containing its own promoter (1.0 kb) and terminator (0.4 kb) was prepared using genomic PCR with the primers ATGCS1PROF(SacI), GCS1GENF1000(KpnI) and ATGCS1GENR(KpnI), and cloned into the pPZP221 binary vector after digestion by the appropriate restriction enzymes. For +/AtGCS1gcs1 production, this construct was simply introduced into +/gcs1 plants. For the production of mAtGCS1 constructs , an GFP cDNA was amplified with the primer pairs GFPf2(HindIII) and GFPr2(HindIII), GFPf(AflII) and GFPr(AflII), GFPf(AflII) and GFPr(HpaI), GFPf(HpaI) and GFPr(HpaI), GFPf(HpaI) and GFPr(PmlI), and GFPf(PmlI) and GFPr(PmlI), respectively. These PCR products were digested and inserted into the AtGCS1 construct as above, using the appropriate restriction enzymes. All the Arabidopsis T1 transformants were generated by Agrobacterium-mediated infiltration, as previously described, and selected in plate media containing 0.01% (w/v) kanamycin and 0.01% gentamycin (w/v). Regarding +/GAAgcs1, the T2 +/gcs1 plants homozygously expressing GAA (+/+/GAAgcs1) were also recovered. For confirmation of the suspected transcription, total RNA samples were extracted from the flowers of each of the transformants, reverse-transcribed, and amplified with primer pairs Atgcs1-seqA2 and GFPBDr, and GFPBDf and Atgcs1truer (primer sets A and B in For production of the gcs1 transformants described above. The resulting offspring seeds were sown in the same media as in the transformant selection. Surviving and dead seedlings were counted in the plate media.Emasculated wild-type pistils were pollinated with pollen from the +/gcs1 plants were crossed with the transgenic Arabidopsis expressing HTR10-RFP (sperm nucleus marker), and the F1 +/gcs1 plants hemizygously expressing the marker were obtained. The F1 plants were transformed with the GAA construct and the T1 +/GAAgcs1 plants homozygously expressing HTR10-RFP were selected in the media as above. Their hand-pollinated pistils were disassembled at 24 HAP and the developing ovules were isolated and observed under fluorescence microscopy. The Arabidopsis lines, expressing H2B-RFP and ΔFWA-GFP driven by the pEC1 and pFWA promoters respectively, were crossed, and the F2 plants homozygously expressing both markers were recovered. The obtained plants were pollinated with +/+/GAAgcs1 pollen and the fertilized ovules were observed at 30 HAP.The +/PbGCS1 Full construct, two PbGCS1 gene fragments covering a region from its own promoter (0.6 kb) to terminator (1.5 kb), and only the terminator (0.6 kb), were prepared using genomic PCR with two primer sets, PbGCSF(KpnI)/PbGCSR(ClaI) and PbGCS3UTRF(EcoRV)/PbGCS3UTRR(XbaI), respectively. Each PCR fragment was cloned into each side of the selectable marker gene (TgDHFR-ts) in pBS-DHFRPbGCS1 Full construct with the three primer sets, –HAP2/GCS1InvF/–HAP2/GCS1InvR, –CInvF/–CInvR, and –CInvF/–TMR, and the PbGCS1 Full construct as a template. The PCR products were treated with DpnI to digest the PbGCS1 Full template plasmid, and the 5′ end was phosphorylated and then self-ligated. The resultant constructs were sequenced and cut by KpnI and XbaI. The linearized constructs were then introduced into the parasite genome by double crossover homologous recombination so that the constructs come under the control of the endogenous PbGCS1 promoter. The parasite transfection, pyrimethamine selection, and dilution cloning were repeated tree times for each construct, as described previously For production of the Thin blood films prepared from mice infected with transgenic parasites were stained with Giemsa, and then parasitemia and the female gametocyte ratio were calculated. The infected blood was mixed with fertilization medium and cultured for 16 h. The thin blood films were prepared and the ookinate ratio was calculated. The efficiency of fertilization was expressed as the ratio of females which were fertilized with males and transformed into ookinetes. The assay was repeated three times for each clone. Thus, the assay was repeated 9 times for each transgenic parasite.Figure S1Precise observation of unfertilized GAAgcs1 sperm cells. (A–C) Detailed observation of an unfertilized +/GAAgcs1 sperm pair. In the +/GAAgcs1 line, in which the sperm nuclei are labeled with HTR10-RFP, an unfertilized sperm pair is occasionally visible in an ovule. In a visualized embryo sac under differential interference contrast (DIC) microscopy (A), such sperm pairs were detected in the vicinity of female gametes as GFP and RFP signal pairs . (A–C) are an identical field group. (D–F) are magnification of the area indicated by the arrow in (A–C), respectively, and they are merged in (G). The arrowheads indicate the sperm pairs. CN, central cell nucleus; EN, egg cell nucleus; DSY, degenerated synergid cell. Scale bars, 25 µm (A); 5 µm (D).(TIF)Click here for additional data file.


This article was republished on March 19, 2014, because of errors in Figures 3-5. The publisher apologizes for these errors.File S1Originally published, uncorrected article.(PDF)Click here for additional data file.File S2Republished corrected article.(PDF)Click here for additional data file.


A dramatic increase in the frequency of the H275Y mutation in the neuraminidase (NA), conferring resistance to oseltamivir, has been detected in human seasonal influenza A/H1N1 viruses since the influenza season of 2007–2008. The resistant viruses emerged in the ratio of 14.3% and quickly reached 100% in Taiwan from September to December 2008. To explore the mechanisms responsible for emergence and spread of the resistant viruses, we analyzed the complete genome sequences of 25 viruses collected during 2005–2009 in Taiwan, which were chosen from various clade viruses, 1, 2A, 2B-1, 2B-2, 2C-1 and 2C-2 by the classification of hemagglutinin (HA) sequences. Our data revealed that the dominant variant, clade 2B-1, in the 2007–2008 influenza emerged through an intra-subtype 4+4 reassortment between clade 1 and 2 viruses. The dominant variant acquired additional substitutions, including A206T in HA, H275Y and D354G in NA, L30R and H41P in PB1-F2, and V411I and P453S in basic polymerase 2 (PB2) proteins and subsequently caused the 2008–2009 influenza epidemic in Taiwan, accompanying the widespread oseltamivir-resistant viruses. We also characterized another 3+5 reassortant virus which became double resistant to oseltamivir and amantadine. Comparison of oseltamivir-resistant influenza A/H1N1 viruses belonging to various clades in our study highlighted that both reassortment and mutations were associated with emergence and spread of these viruses and the specific mutation, H275Y, conferring to antiviral resistance, was acquired in a hitch-hiking mechanism during the viral evolutionary processes. Influenza viruses cause annual epidemics in many countries and occasional worldwide pandemics. Influenza vaccine and antiviral drugs are two useful measures for preventing influenza and reducing the impact of epidemics. Currently, there have been two classes of antiviral drugs available for preventing and treating influenza illness: M2 ion channel blockers, adamantanes (amantadine and rimantadine), and neuraminidase inhibitors . Since the 2005–2006 influenza season, most human influenza A/H3N2 viruses circulating worldwide were found to carry S31N in the M2 protein, conferring to adamantane-resistance in vitro and in vivo, compared to the corresponding wild-type There are three key factors affecting the evolutionary processes of antiviral resistant influenza viruses from their original appearance to worldwide spread. The first factor is the frequency of drug resistant mutants arising de novo during treatment, the second is the ability to overcome the obstacle of viral fitness caused by resistant mutations In this study, we determined the frequency of the H275Y mutation in the NA to survey the oseltmivir-resistant seasonal influenza A/H1N1 viruses in Taiwan and found that a sudden and dramatic increase in the frequency of the H275Y mutation has been detected since September, 2008. Therefore, we combined the data of influenza epidemic surveillance as well as oseltamivir-resistance testing and complete genetic analysis of the circulating viruses during 2005–2009 to investigate the mechanisms responsible for emergence and spread of these resistant viruses in Taiwan.From January 2005 to August 2008, a total of 47 seasonal influenza A/H1N1 viruses in Taiwan were tested for the presence of H275Y substitution in the NA and only two (4.2%) isolates, A/Taiwan/0045/2006 and A/Taiwan/3293/2008, were shown to be positive for this substitution. However, the frequency of viruses harboring H275Y started to increase from September 2008  and quickly reached 100% (37/37) in December . BecauseFrom 2005 to 2009, three influenza epidemics in 2005–2006, 2007–2008 and 2008–2009 in Taiwan were caused predominantly by seasonal influenza A/H1N1 viruses . Among tTo investigate the relationship between variant evolution and emergence of the oseltamivir-resistant viruses, we determined the prevalence of the H275Y substitution in various clade viruses in Taiwan. Of the HA clade 1 and 2A viruses, none was found to carry H275Y substitution  for oseltamivir against the 25 representative viruses were determined  and also the resistant virus, A/Taiwan/3293/2008, showed all 275Y type , indicating quasi-species genotypes were not detected in these two viruses.The 50% inhibitory concentrations (ICtermined . Among thttp://www.cdc.gov/flu/weekly/pastreports.htm). The global frequency of resistant viruses increased from 44% (April 2008–September 2008) to 96% (October 2008–March 2009) In this study, we provided a comprehensive analysis of the influenza A/H1N1 viruses collected in Taiwan during 2005–2009. The combined data of influenza epidemic surveillance, oseltamivir-resistance testing and complete genetic analysis of the circulating viruses during 2005–2009 revealed the evidence that the annual epidemiological replacement of various influenza A/H1N1 variants associated with emergence and spread of the oseltamivir-resistant influenza A/H1N1 viruses. In the early 2007–2008 influenza season, oseltamivir-resistant influenza A/H1N1 viruses were detected first in Europe , mainly in Norway . The frequency of oseltamivir-resistant viruses in Europe increased to 24.3% (727/2992) at the end of 2007–2008 influenza season. These viruses exhibited a NA H275Y substitution (N1 numbering), conferring resistance to oseltamivir and emerged without antiviral pressure Genome segment reassortment contributes to the increase of influenza diversity and is associated with severe epidemics and disaster pandemics. Influenza A viruses caused four human influenza pandemics in 1918 (H1N1 subtype), 1957 (H2N2 subtype), 1968 (H3N2 subtype) and 2009 (H1N1 subtype), and those viruses all emerged through complex inter-subtype reassortment events Worldwide spread of antiviral-resistant influenza viruses has been observed for subtypes H3N2 and H1N1. Genomic reassortment and additional substitutions also have been considered as important in the evolutionary processes of emergence and spread of adamantane-resistant influenza A/H3N2 viruses in 2005–2006 and a hitch-hiking mechanism was used to explain the occurrence of the S31N substitution in M2 protein of this particular variant Some substitutions R194G, V234M and R222Q of NA proteins were involved in buffering deficiencies on folding or stability of neuraminidase caused by the H275Y substitution and restore viral fitness This study was conducted through the national influenza surveillance network in Taiwan, which is coordinated by the Centers for Disease Control (CDC), Department of Health, Taiwan. This surveillance points cover approximately 75% of basic administrative units of Taiwan  The partial HA sequences of the influenza viruses from 2005–2009 were determined by conventional RT-PCR and sequencer (ABI 3730). Based on phylogenetic analyses and amino acid substitutions of analyzed sequences, we classified these seasonal influenza A/H1N1 isolates into four distinct HA clades: 1, 2A, 2B and 2C, which were designated by a previous study CAC) for tyrosine (encoded by TAC) at amino acid position 275 in the NA protein were identified as oseltamivir resistant. To focus in more detail on the genetic diversity and phylogenetic histories of the oseltamivir-resistant seasonal influenza A/H1N1 viruses during 2005–2009 in Taiwan, 25 representative isolates belonging to various HA clades, 1, 2A, 2B and 2C, which were classified based on the HA sequences and designated according to a previous study http://www.mbio.ncsu.edu/BioEdit/bioedit.html). A phylogenetic tree was constructed by the neighbor-joining method and 1,000 bootstrap replications were performed to evaluate the robustness. The vaccine strains A/New Caledonia/20/1999 and A/Brisbane/59/2007 also were included in the eight phylogenies as reference sequences.Virus isolates identified as influenza A/H1N1viruses were tested for oseltamivir resistance by analysis of NA sequences through a conventional PCR. A 907 base-pair DNA fragment spanning nucleotide position 823–825  of NA gene was amplified and the nucleotide sequences also were determined. Isolates with a C to T substitution at nucleotide position 823 that could result in the substitution of histidine (encoded by 50) analysis of oseltamivir for influenza A/H1N1 viruses was determined using the NA-Star Influenza Neuraminidase Inhibitor resistance Detection Kit (Applied Biosystems) according to the manufacturer's recommendations. Briefly, 25 µl of half-log dilutions of neuraminidase inhibitor (NI) from 0.03 to 1000 nM were mixed with 25 µl of virus dilution with equal amount viruses of 16 hemagglutination (HA) unit in each well of a white 96-well microplate and incubated 10–20 min at 37°C. Two wells of the mixture destined to be negative controls contained only assay buffers, instead of NI, and culture medium, instead of viruses, also were included. Then, 10 µl of diluted substrate was added to each well and incubated for 10–30 min at room temperature, followed by the addition of 60 µl of accelerator, the chemiluminescent signal being measured immediately. The software GraphPad Prism version 4.00 was used to determine the IC50 values.The 50% inhibitory concentration Click here for additional data file.Figure S2Phylogenetic relationships of the NP and NS segments of influenza A/H1N1 viruses in Taiwan. The phylogenetic analyses were constructed using the neighbor-joining method with 1000 bootstrap replications. Branch values of more than 75 are indicated. All of the phylogenies were rooted with the A/New Caledonia/20/1999. Different clades were shown by different colors.(TIF)Click here for additional data file.Table S1Amino acid substitutions in various proteins, differing between clades 2B-1 and 2B-2 in the 92 global isolates.(DOC)Click here for additional data file.