Because they are strong and stable, lignocellulosic supramolecular structures in plant cell walls are resistant to decomposition. However, they can be degraded and recycled by soil microbiota. Little is known about the biomass degradation profiles of complex microbiota based on differences in cellulosic supramolecular structures without compositional variations. Here, we characterized and evaluated the cellulosic supramolecular structures and composition of rice straw biomass processed under different milling conditions. We used a range of techniques including solid- and solution-state nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy followed by thermodynamic and microbial degradability characterization using thermogravimetric analysis, solution-state NMR, and denaturing gradient gel electrophoresis. These measured data were further analyzed using an “ECOMICS” web-based toolkit. From the results, we found that physical pretreatment of rice straw alters the lignocellulosic supramolecular structure by cleaving significant molecular lignocellulose bonds. The transformation from crystalline to amorphous cellulose shifted the thermal degradation profiles to lower temperatures. In addition, pretreated rice straw samples developed different microbiota profiles with different metabolic dynamics during the biomass degradation process. This is the first report to comprehensively characterize the structure, composition, and thermal degradation and microbiota profiles using the ECOMICS toolkit. By revealing differences between lignocellulosic supramolecular structures of biomass processed under different milling conditions, our analysis revealed how the characteristic compositions of microbiota profiles develop in addition to their metabolic profiles and dynamics during biomass degradation. Its major components, namely, cellulose, hemicellulose, and lignin, are complex molecules that are abundantly produced in plant cell walls. Cellulose is a linear condensation polymer comprising β (1→4)-linked D-glucose units with a degree of polymerization ranging from 100 to 20,000 Ruminococcus albus is widely recognized for its high cellulolytic activity Thermobifida fusca, a primary degrader of plant biomass in soil, as well as white-; brown-; and soft-rot fungi, hydrolyzes polysaccharides and lignin in lignocellulose to monosaccharides and aromatic rings Lignocellulose has a strong and stable structure and is resistant to biodegradation. However, recent studies have shown that microbial communities mediate plant biomass degradation in animals, including detritivores, ruminants, termites, and omnivores To understand the cellulosic supramolecular structure and composition of biomass, persistent biomass samples must be pretreated using physical and/or chemical techniques such as milling, enzyme hydrolysis, heating, and ionic water extraction Jatropha curcas using FTIR and solution-state NMR In our previous reports, we have performed unique studies using combined solid- and solution-state NMR and other methods in worldwide biomass experiments Moreover, we developed a web-based toolkit, “ECOMICS,” for trans-omics analysis of ecosystems This study focused on the effects of rice straw pretreatment on the cellulosic supramolecular structure and aimed to improve digestibility of lignocellulosic biomass for paddy soil microbiota. Supramolecular structures and composition of biomass were characterized using multiphysicochemical approaches combined with the ECOMICS web tools for multivariate data analysis . Paddy s1) or 10 min (AM2). BM-processed samples were prepared by placing approximately 300–400 mg of FM-processed samples in ZrO2 rotors (interior volume  = 12 ml) containing 50 ZrO2 balls (diameter  = 5 mm) and were then processed using a BM machine at 400 rpm for 1 h (BM1) or 6 h (BM2). Grinding was cyclically performed for 10 min and was then interrupted for the same length of time to prevent an excessive rise in sample temperature. Thus, the number of on/off cycles was set to 6 and 36 for BM1 and BM2 pretreatments, respectively.Samples were prepared by processing five types of rice straw under different milling conditions. Lyophilized, food milling (FM)-processed rice straw samples were processed in a blender. Some FM-processed samples were further ground in an auto-milling (AM) machine  and a planetary BM machine  to produce AM- and BM-processed samples, respectively. AM-processed samples were prepared by grinding 300–400 mg of FM-processed samples in a stainless steel crusher of an AM machine operated at 1550 rpm for 3 min  with a backscattered electron (BSE) detector operating in the variable pressure (VP)-scanning electron microscopy (SEM) mode (column pressure retained at 30 Pa).−1) were obtained using a Nicolet 6700 FTIR  instrument with a KBr disk. The ATR smart iTR accessory with a high-pressure clamp (Thermo Fisher Scientific Inc.) was used. The spectra were obtained using triangular apodization with a resolution of 4 cm−1 and an interval of 1 cm−1. Each background and sample spectrum was obtained from 32 scans. The ONMIC software supplied with the equipment provided baseline and ATR corrections for penetration depth and frequency variations.Attenuated total reflectance (ATR)-FTIR provides information on structural changes in functional groups of rice straw after milling. ATR-FTIR spectra (4500–650 cm1H frequency) with a 54-mm narrow-bore magnet at room temperature (25±1°C). 1D cross-polarization-magic angle spinning (CP-MAS) and 2D 13C-1H heteronuclear correlation (HETCOR) spectra were obtained using a Bruker 4-mm double-tuned MAS probe. For the NMR measurements, approximately 80 mg of sample was placed in a ZrO2 rotor (outer diameter  = 4 mm) with a KelF-made cap. The MAS spinning speed, set to 12,000 Hz, was regulated using a Bruker MAS II pneumatic MAS controller. The contact time was set to 1.0 ms and 5.0 ms, and the recycle delay was 4 s. The MAS frequency was set to 12,000 Hz. The magic angle (54.7°) pulse length for protons was set to 1.8 μs. CP-MAS and cross-polarization-total sideband suppression (CP-TOSS) spectra were also measured using a Bruker DRX-500 spectrometer operating at 500.13 MHz for 1H equipped with the Bruker 4-mm double-tuned MAS probe to check the effect of spinning sidebands (SSB). The MAS spinning speed, set to 6000 Hz (for CP-MAS and CP-TOSS) and 12,000 Hz (for CP-MAS), was regulated using the Bruker MAS II pneumatic MAS controller with careful temperature controls.Solid-state NMR spectra were measured in a B0 field of 18.8 Tesla using a Bruker AV800 spectrometer  solvent per 1 mg of sample. After centrifugation, the supernatants containing the DMSO-d6/Pyridine-d5 (4∶1) solvent were used for NMR experiments. NMR spectra were acquired on a 700.15 MHz (AV700) Bruker Biospin instrument equipped with an inverse (1H coils closest to the sample) gradient 5-mm TBI 1H/13C/15N probe at 45°C. The central DMSO solvent peak was used as an internal reference . 1H-13C HSQC spectra were programed using a Bruker standard pulse sequence “hsqcetgp” (phase-sensitive gradient-edited-2D HSQC using trim pulses in INEPT transfer). NMR spectra were acquired from 14 to −3 ppm in F2 (1H) using 1024 data points for an acquisition time (AQ) of 60 ms, an interscan delay (D1) of 750 ms, and 179–9 ppm in F1 (13C) using 120 increments (F1 AQ  = 5.78 ms) of 340 scans. The obtained spectra were assigned by Bm-char  Sixty milligrams of each milled biomass sample were suspended in 1 ml of methanol. The mixture was heated at 50°C for 5 min in a Thermomixer comfort  and then centrifuged. The supernatants were discarded, and the pellets were resuspended in methanol and centrifuged. The process was repeated twice more, giving a total of three suspensions. The pellets were then suspended in 1 ml of HThermogravimetric (TG) analysis was conducted using an EXSTAR TG/differential thermal analysis (DTA) 6300  instrument. Approximately 9 mg of sample was individually loaded into an aluminum pan and vaporized  in a nitrogen atmosphere with a flow rate of 200 ml/min. Based on the TG data, the thermodegradation kinetic parameters of milling-processed samples were analyzed using Coats and Redfern's integral method 1H- and 1H-13C HSQC NMR measurements of the supernatants, while the microbiota profiles were determined from denaturing gradient gel electrophoresis (DGGE) analysis of the pellets.A soil microbial community from a paddy field was used as seed material. The paddy soil sample was obtained from a private paddy field in Yamagata, Japan, with permission from the owner to conduct this study. To evaluate the biomass degradation profiles of soil microbiota under different milling conditions, 120 g of paddy soil was prepared and mixed with 480 ml of distilled water. Separate 80-ml aliquots of the mixture were poured into six 100-ml vials. Subsequently, 500 mg of biomass samples milled under different conditions (as described above) were added to each vial and incubated at 30°C with shaking at 160 rpm for 34 days. Incubated samples from each day were centrifuged to separate the supernatant from the pellet. The metabolic profiles of soil microbiota were evaluated from 1H inverse TXI probe with triple-axis gradients at 25°C The supernatant was prepared as described above, with slight modifications. All 1D Watergate spectra were acquired on a Bruker Avance DRX-500 NMR spectrometer operating at 500.13 MHz equipped with a 5-mm Metabolites assigned as acetate, butyrate, and propionate were quantified by calibration 2-processed samples were measured using 1H-13C HSQC. NMR spectra were acquired on the 700.15 MHz (AV700) Bruker Biospin instrument equipped with an inverse (1H coils closest to the sample) gradient 5-mm Cryo 1H/13C/15N probe at 25°C. The acetate peak was used as an internal reference . NMR spectra were acquired from 10.192 to −0.788 ppm in F2 (1H) using 2048 data points for an AQ of 133 ms, D1 of 2 s, and 88–48 ppm in F1 (13C) using 142 increments (F1 AQ  = 9.07 ms) of 128 scans. The obtained spectra were assigned using SpinAssign  To assign metabolites produced by soil microbiota, the supernatants from the first and second days of incubation with BM™ DNA Isolation Kit  according to the manufacturer's instructions. The conditions and experimental procedures used for PCR amplification and DGGE performance have been reported elsewhere https://database.riken.jp/ecomics/eclass/) http://rdp.cme.msu.edu/) classifier Microbial DNA extraction was performed using the PowerSoil1H-NMR data were processed as ft2 files using NMRPipe software 1H chemical shifts of −0.5 and 9.5 using FT2DB , a web tool for digitizing 2D data 13C-1H HETCOR spectra were processed using NMRPipe software, and the ft2 files were digitized using FT2DB. Digital 13C-1H HETCOR data were corrected for 13C chemical shift, and data points consistent with the pseudo-13C CP-MAS NMR spectra were summed. The peak separations of 13C CP-MAS NMR spectra were performed using Fityk software (http://fityk.nieto.pl/) The 1H-NMR spectral data point. Thus, the loading plots provide information on band positions or spectral regions responsible for the positions of coordinates or sample clusters in the corresponding score plots. A 2D correlation map of FTIR and 13C-1H HETCOR spectra was calculated as a symmetric matrix using the HetMap web tool for statistical analysis  PCA was performed according to our previous reports 2-processed sample appeared as a granulated powder, indicating structural breakage.To examine the lignocellulosic supramolecular structures of biomass processed under different milling conditions, the structures were observed using SEM (−1 (anomeric vibration at β-glycosidic linkage), 1027 cm−1 (C–O stretching in cellulose and hemicellulose), 1103 cm−1 (vibration of ester linkage), 1120 cm−1 , 1145 cm−1 (deformation vibrations of C–H bonds in benzene rings), 1232 cm−1 (syringyl ring and C–O stretching in lignin and xylan), 1311 cm−1 (C–H in cellulose and C1–O vibration in syringyl derivatives), 1359 cm−1 (C–H deformation in cellulose and hemicellulose), 1413 cm−1 and 1502 cm−1 (aromatic ring vibrations), 1450 cm−1 (asymmetric C–H bonding in CH3 and –CH2–), 1604 cm−1 (aromatic ring vibrations and C = O stretching), 1720 cm−1 , and 2915 cm−1 (C–H stretching) r = 0.7) and number of samples (n = 5) in the HetMap analysis were chosen to obtain significant correlations between the detected characteristic peaks with the lowest percentage of noise. Positive or negative correlations were consistent with the characteristic peaks contributing to the same or opposite directions in the PCA loading plots, respectively. These results were consistent with the previously reported cleavage of the β-O-4 and α-O-4 linkages in lignin observed after BM pretreatment Each milling-processed sample was also measured using ATR-FTIR . The cha13C CP-MAS, CP-TOSS, and 13C-1H HETCOR NMR analyses were performed n–; 63 ppm, CH2OH of carbohydrates (C6 of amorphous cellulose); 66 ppm, CH2OH of carbohydrates ; 72 ppm, CHOH of carbohydrates ; 72–76 ppm, C–OR of lignin; 75 ppm, CHOH of carbohydrates ; 84 ppm, CHOH of carbohydrates (C4 of amorphous cellulose); 89 ppm, CHOH of carbohydrates ; and 105 ppm, OCHO of carbohydrates (C1 of cellulose) 1- and AM2-processed biomass samples, as well as in the sample processed under the FM condition alone. However, a very small C4 peak of crystalline cellulose appeared in the BM1-processed sample but was absent in the BM2-processed sample, indicating physical fracture of the crystalline structure of biomass. This result was consistent with a previous report that peaks of 108 ppm and 60 ppm in 13C NMR spectra broadened after intense milling 3 of lignin was reduced after BM pretreatment. In addition, the PCA score plot showed that milling-processed samples were clustered according to differences between FM/AM and BM pretreatment  and COC vibration, CH2OH of carbohydrates  and asymmetric C–H bonding, CH2OH of carbohydrates  and C–H stretching in cellulose, CHOH of carbohydrates (C4 of amorphous cellulose) and deformation vibrations of C–H bonds on benzene rings, CHOH of carbohydrates (C4 of amorphous cellulose) and aromatic ring vibration, CHOH of carbohydrates (C4 of amorphous cellulose) and asymmetric C–H bonding, CHOH of carbohydrates (C4 of amorphous cellulose) and C–H stretching in cellulose, CHOH of carbohydrates  and deformation vibrations of C–H bonds on benzene rings, OCHO of carbohydrates (C1 of cellulose) and aromatic ring vibration, OCHO of carbohydrates (C1 of cellulose) and asymmetric C–H bonding, and OCHO of carbohydrates (C1 of cellulose) and C–H stretching in cellulose. Negative correlations were found between CH2OH of carbohydrates  and C–O stretching in cellulose and hemicellulose, CHOH of carbohydrates  and vibration of ester linkage, CHOH of carbohydrates  and deformation vibrations of C–H bonds on benzene rings, CHOH of carbohydrates  and aromatic ring vibration, CHOH of carbohydrates (C4 of amorphous cellulose) and C–O stretching in cellulose and hemicellulose, CHOH of carbohydrates  and C–O stretching in cellulose and hemicellulose, and OCHO of carbohydrates (C1 of cellulose) and C–O stretching in cellulose and hemicellulose. These results suggest that several peaks originating from lignocellulose were changed as the biomass structure was altered from crystalline to amorphous state after BM pretreatment.The obtained CP-MAS and HETCOR spectra of milling-processed samples were assigned based on previous reports as follows: 21.5 ppm, CHreatment . Clusterreatment . Homogenreatment . In partreatment  revealed1H- and 1H-13C HSQC spectra of biomass samples extracted using DMSO/pyridine were analyzed , β-O-4-p-hydroxyphenyl/guaiacyl , α-L-fucopyranoside , α-L-arabinofuranoside , and p-coumarate .analyzed  and 5. 1 and PC2 . This se and PC2 . In addi2-processed sample displayed the highest number of peaks in its 1H-13C HSQC spectra  compared with rice straw-incubated samples , and sugar regions (from 3.2 to 4 ppm). Acetate and propionate contributed to PC1 and PC2 in the FM-processed sample and to PC1 in the BM2-processed sample. However, butyrate contributed to PC2 in the BM2-processed sample. These metabolites  were quantified from 1H-NMR spectral data  (Acinetobacter brisouii [DQ832256] (99% identity) and from Bands 6, 8, and 9 were Paludibacter propionicigenes [AB078842] (94% identity), Ignavibacterium album [AB478415] (93% identity), and Azotobacter chroococcum [EF634034] (100% identity), respectively. P. propionicigenes is an anaerobic bacterium associated with the degradation of rice plant residues in Japanese paddy soil I. album was isolated from hot spring water streams. This organism utilizes D-glucose, D-mannose, D-fructose, maltose, and cellobiose 2-processed sample. Fragmentation of the lignocellulosic supramolecular structures of the BM2-processed sample may have enabled these bacteria to utilize the constituent sugars in lignocelluloses to produce the acetate, butyrate, and propionate observed after BM2 pretreatment.The microbiota detected in the DGGE analysis was identified by sequencing and classification, and phylogenetic analysis was performed using RDP classifier and E-class . The bacier.jsp) . The cloThis study investigated the effects of rice straw pretreatment on the cellulosic supramolecular structure. The results will assist in improving digestibility of lignocellulosic biomass for paddy soil microbiota. The effects were successfully evaluated and characterized using the ECOMICS web-based toolkit. This toolkit enables the evaluation of biomass structures and components and integration of heterogeneous matrix data in environmental and metabolic systems. The ECOMICS toolkit revealed that physical pretreatment of rice straw alters the cellulosic supramolecular structure. Thermal degradation profiles were shifted to lower temperatures, and different microbiota profiles with different metabolic dynamics were evolved during the biomass degradation process.2-processed samples revealed differences in the types and quantities of metabolites produced. Further, in this study, the soil microbiota involved in the degradation of structurally different biomass samples were different from those generally associated with degradation. These evaluations and characterizations were successfully achieved using the ECOMICS toolkit.In this study, the cellulosic supramolecular structure of plant biomass was altered using physical milling processes, and as a result, biomass degradation by paddy soil microbiota was affected. The cellulosic supramolecular structures of biomass samples processed under different milling conditions showed distinct conformational differences; for instance, the cellulose structure was altered from crystalline to amorphous state. Extractable lignocellulosic components were degraded to lower molecular mass molecules after 6 h of BM pretreatment. TG/DTG analysis suggested that the activation energies of biomass decomposition were affected by the cellulose conformation. In addition, biomass samples with differences in cellulosic supramolecular structures, although without compositional variations, produced different metabolites on degradation and had characteristic paddy soil microbiota profiles. Degradation profiles of FM- and BMFigure S1Homogeneous correlation analysis of ATR-FTIR using HetMap. The list of 2D heat maps (A) is provided for comparison of differences between the number of samples (from 7 to 1) and thresholds (from 0.5 to 0.9). The 2D heat map at n = 5 and r = 0.7 is displayed (B). 1, COC vibration; 2, C–O stretching in cellulose and hemicellulose; 3, vibration of ester linkage; 4, aromatic skeletal and C–O stretching; 5, deformation vibrations of C–H bonds on benzene rings; 6, syringyl ring and C–O stretching in lignin and xylan; 7, C–H in cellulose and C1–O vibration in syringyl derivatives; 8, C–H deformation in cellulose and hemicellulose; 9, aromatic ring vibration; 10, asymmetric C–H bonding (in CH3 and –CH2–); 11, aromatic ring vibration; 12, stretching of C = O conjugated to aromatic rings; 13, stretching of C = O unconjugated to aromatic rings (oxidized side chains); 14, C–H stretching in cellulose.(TIF)Click here for additional data file.Figure S2Comparisons of CP-MAS and CP-TOSS spectra using the spinning speed set to 6000 Hz and 12,000 Hz. CP-MAS spectra measured using the spinning speed set to 6000 Hz (black) and 12,000 Hz (blue) and CP-TOSS spectra measured using the spinning speed set to 6000 Hz (red) were obtained using the FM-processed sample.(TIF)Click here for additional data file.Figure S3Peak separation of solid-state NMR using Fityk software. (A) FM-, (B) AM1-, (C) AM2-, (D) BM1-, and (E) BM2-processed samples.(TIF)Click here for additional data file.Figure S413C-1H HETCOR and ATR-FTIR spectra.Homogeneous and heterogeneous correlation analysis of  Homogeneous correlation heat map of NMR spectra (A) and heterogeneous correlation heat maps calculated by Pearson (B) and Spearman (C) between 13C-1H HETCOR and ATR-FTIR spectra. 1, CH3 in hemicellulose; 2, aliphatic –(CH2)n–; 3, OCH3 of lignin; 4, CH2OH of carbohydrates (C6 of amorphous cellulose); 5, CH2OH of carbohydrates ; 6 and 7, CHOH of carbohydrates ; 8, CHOH of carbohydrates (C4 of amorphous cellulose); 9, CHOH of carbohydrates ; 10, OCHO of carbohydrates (C1 of cellulose); 11, COC vibration; 12, C–O stretching in cellulose and hemicellulose; 13, vibration of ester linkage; 14, aromatic skeletal and C–O stretching; 15, deformation vibrations of C–H bonds on benzene rings; 16, syringyl ring and C–O stretching in lignin and xylan; 17, C–H in cellulose and C1–O vibration in syringyl derivatives; 18, C–H deformation in cellulose and hemicellulose; 19, aromatic ring vibration; 20, asymmetric C–H bonding (in CH3 and –CH2–); 21, aromatic ring vibration; 22, stretching of C = O conjugated to aromatic rings; 23, stretching of C = O unconjugated to aromatic rings (oxidized side chains); 24, C–H stretching in cellulose.(TIF)Click here for additional data file.Figure S51H-NMR spectra of the control and FM- or BM2-processed incubation samples at Day 8.Comparison of  Red spectra are control and black spectra are FM- (A) and BM2-processed samples (B).(TIF)Click here for additional data file.Figure S61H-NMR spectra.Degradability characterization of metabolic profiles based on  AM1- , AM2- , and BM1-processed  biomass samples were evaluated by the PCA score plots  and loading plots  of metabolic profiles during biomass degradation by soil microbiota.(TIF)Click here for additional data file.Figure S71H-NMR spectra.Time course variations of quantified metabolites based on  Acetate (A: 1.92 ppm), propionate (B: 1.04 ppm), and butyrate (C: 0.88 ppm) were produced during biomass degradation by soil microbiota. Shown are the production quantity at measured times (open symbols) and their summations (closed symbols) in FM- (square symbols) and BM2-processed (circle symbols) samples.(TIF)Click here for additional data file.Figure S8Degradability characterization of microbial community profiles based on DGGE fingerprinting. Microbiota profiles during biomass degradation in FM- (open square) and BM2-processed (open circle) biomass samples were evaluated by the PCA score plots (A) and loading plots (B). Each number (Days 1–19) in the score plots indicates the day of incubation. Detailed informations on the detected bands are shown in (TIF)Click here for additional data file.Figure S9Phylogenetic tree constructed based on partial 16S rRNA gene sequences. The sequences determined in this study and those retrieved from the databases were aligned using CLUSTAL W2. The phylogenetic tree was then constructed using CLUSTAL W2 and Genetyx-tree software by the neighbor-joining method. The 16S rRNA gene fragment was amplified using the Univ954f and Univ1369r primer sets. The clones obtained were expressed as DGGE Bands 1–9. The 16S rRNA gene sequence of Aquifex pyrophilus [M83548] was used as an outgroup to root the tree. Indicated numbers in phylogenetic tree are bootstrap values.(TIF)Click here for additional data file.Figure S10Classification of DGGE band sequences using E-class. Classification of each DGGE band sequence to the phylum (A) and genus level (B) and its summary (C).(TIF)Click here for additional data file.Table S12 samples extracted by D2O solvent detected in the 1H-13C HSQC spectra.The list of annotated metabolites in BM(DOCX)Click here for additional data file.


Fluid replacement has been a usually recommended maneuver in sepsis; however, growing clinical controversies in the management of critically ill patients with severe sepsis have questioned its benefit. Herein, we evaluated the effect of a rapid hyperhydration (HH) therapy in varying stages of sepsis.Escherichia coli 108 CFU/ml intravenous (i.v.) inoculation, LD60, or S9 group, with E. coli 109 CFU/ml inoculation, LD80. Both groups were treated with HH  in the early (E30 minute) and late (L6 hour) phases of sepsis. The mortality was followed up to 30 days (n = 6/group) and the splanchnic microcirculation was monitored by sidestream dark field imaging (SDF) video microscopy at 6-hour and 24-hour periods (n = 3/group/period).Wistar-EPM rats, weighing 200 to 250 g, were submitted to two sepsis models: S8 group, submitted to 2 ml 80), with E30 minute HH, improved the survival rate from 20% to 50%. However, it was less effective as compared with the E6H phase HH, which resulted in an expressive survival rate (from 20% to 70%). These intriguing results suggested that there is an interdependent and time-dependent pathophysiology feature within the host response based on sepsis severity stage and a rapid high-volume reposition. The SDF analysis in control sepsis groups (S8 and S9), without fluid therapy, showed a broadly distributed microcirculation dysfunction in the liver lobules and kidney tubules at 6 hours after sepsis challenge, and such findings were similar between groups, but after 24 hours the survivors showed an improved microcirculation hemodynamic pattern and it was more evident in the S8 group. The survivals of the S8 E30 minute treated group showed less injury at 6 hours and 24 hours as compared with nontreated groups and S8 L6 hour treated animals. In S9 treated groups, both showed a partial repair at 24 hours post sepsis.The HH at the E30 minute phase of S8 improved the survival rate from 40% to 90%, and L6 hour phase HH promoted an 80% survival rate. Besides, the survival rate in S9  and at the later phase of severe sepsis (S9). The underlying pathophysiology evolved in these paradoxical conditions needs to be better elucidated.


The Lymphoid specific tyrosine phosphatase (Lyp) has elicited tremendous research interest due to the high risk of its missense mutation R620W in a wide spectrum of autoimmune diseases. While initially characterized as a gain-of-function mutant, R620W was thought to lead to autoimmune diseases through loss-of-function in T cell signaling by a recent study. Here we investigate the biochemical characters and T cell signaling functions of two uncharacterized Lyp variants S201F and R266W, together with a previously characterized Lyp variant R263Q, which had reduced risk in several autoimmune diseases, including systemic lupus erythematosus (SLE), ulcerative colitis (UC) and rheumatoid arthritis (RA). Our kinetic and functional studies of R263Q polymorphism basically reproduced previous findings that it was a loss-of-function mutant. The other variant S201F reduced Lyp phosphatase activity moderately and decreased Lyp function in T cell slightly, while R266W severely impaired phosphatase activity and was a loss-of-function variant in T cell signaling. A combined kinetic and structure analysis suggests that the R266W variant may decrease its phosphatase activity through perturbing either the Q-loop or the WPD loop of Lyp. As both R266W and R263Q significantly change their phosphatase activity and T cell functions, future work could be considered to evaluate these mutants in a broader spectrum of autoimmune diseases. Protein tyrosine phosphorylations regulated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) are essential signal transduction events mediating the immune response Human Lyp was first identified in 1999, with 90% homology to murine phosphatase PEP in its phosphatase catalytic domain Besides Lyp R620W mutation, human genomics studies have identified several missense polymorphisms which did not display significant correlation to cause immune diseases In our previous study, we purified the catalytic domain of Lyp and solved its crystal structure together with either a specific inhibitor or peptide substrates http://www.ncbi.nlm.nih.gov/snp). Among them, we investigated the phosphatase activity of S201F , R266W  together with R263Q , which ranked top three of population in the sequenced pools of DNA from T1D patients and controls We have looked up SNPs of PTPN22 on the collective NCBI’s Entrez SNP system  wild type and three Lyp variants, 266W, 263Q and 201F in E-coli, and purified these proteins to near homogenity by passing the lysates through NiWith the purified proteins, we checked whether these variants affected their intrinsic phophatase activity toward pNPP, a small aryl phosphate substrate, through steady state kinetic analysis. As a result, the Km of Q263 increased slightly compared with the wild type R263 while the Kcat didn’t change much. The Kcat/Km of Q263 toward pNPP, which reflected the intrinsic phosphatase activity, didn’t show statistics difference compared to the wild type R263. However, Kcat/Km values of the other two Lyp mutants, F201 and W266 decreased significantly to 1/2 and 1/5 compared to the wild type respectively . The chaScheme 1:Lyp substrate specificity partially resides in its catalytic domain, which was revealed by our previous study using an inverse alanine library screening and crystallographic analysis. To determine whether Lyp polymorphisms affect Lyp substrate specificity towards primary peptide sequence, we examined these variants’ activities toward a 9-amino acid phosphor-peptide derived from the Y394 phosphorylation site of Lck, a physiological substrate of Lyp. Polymorphisms of F201, Q263 and W266 had reductions on Kcat/Km values about 20%, 40% and 60% respectively, all with statistics significance. Interestingly, F201 had a better activity towards the peptide substrate compared to pNPP, while Q263 had a decreased activity towards peptides, indicating F201 preferred its physiological substrate over the small artificial substrate while Q263 lost the ability for physiological substrate recognition.The observations that some Lyp variants like S201F displayed relatively different substrate selectivity towards Lck 394 phosphor-peptide and pNPP elicited our interests that these mutants might have additional effects on their physiological substrates. One explanation is that these polymorphisms might affect the protein surface responsible for substrate recognition other than the active sites. As a potent negative regulator of T cell signaling, Lyp could directly dephosphorylate the Lck at 394 position To investigate whether the decrease of these variants’ phosphatase activity directly correlate to their functions, we transfected these mutants of full-length Lyp in human Jurkat T cells and looked at their effects on early T cell signaling. As shown in ES1 value of pH profile for W266-catalyzed pNPP reaction is 4.1±0.1, left shifted 0.17 on the acidic limb compared to that of the wild type Lyp, and the pKES2 value of W266 is determined to be 6.1±0.1, left shifted 0.43 on the basic limb compared to that of the wild type. The observed pKES2 mostly reflects the change of catalysis rate limiting step from E–P hydrolysis to E–P formation, in which the protonation of D195 is the key determinant of these steps Of the three Lyp variants tested, W266 greatly disturbed its activity both in T cell signaling and enzymatically. To provide further evidence that W266 is a loss-of-function mutant in T cell function, we measured the transcriptional activity of activator protein-1 (AP-1) in Jurkat T cells expressing full-length Lyp wild type or W266 variant. AP-1 transcription factor, which is a critical element involved in interleukin-2 production, is a downstream of ERK signaling Our studies revealed the Lyp variant S201F moderately decreased its phosphatase activity and ability in regulating T cell signaling, while R266W and R263Q significantly decreased their activities towards physiological substrates and behaved as loss-of-function mutants in T cells. As shown in The R266 is a structually conserved residue among the protein tyrosine phosphatase superfamily. In the prototype tyrosine phosphatase PTP1B, a conserved R254 at equivalent position forms the bottom of the second “pY” binding pocket and has important structural roles in maintaining its phosphatase activity and substrate recognition. In agreement with our findings, the mutation of PTP1B R254Q has reduced Kcat toward pNPP by 17 folds and increased Km by 23 folds. The R254K mutant decreased its Kcat/Km value by 16 folds toward small substrate pNPP Unlike F201 and W266, which draw few attentions in human diseases, the minor variant Q263 of Lyp has been identified to reduce the risk of several other autoimmune diseases except T1D, including SLE, RA and UC, but increase the susceptibility to infectious disease like PT. Our current results agree with previous finding that R263Q Lyp variant decreased its phosphatase activity and lost its ability in regulation of T cell signaling. Besides R263Q, we have found here that R266W variant of Lyp significantly impaired its phosphatase activity and capacity in regulation of T cell function. Whether R266W also reduces the risk of autoimmune diseases, such as SLE, UC and RA, and how it affects the susceptibility to PT are worth to investigate in future investigations. Such studies, together with inspections on association of more Lyp genetic variants with a broader spectrum of autoimmune diseases and infectious diseases, will help to evaluate Lyp as a drug target for autoimmune diseases.Para-nitrophenyl phosphate (pNPP) was from Bio Basic Inc.(Canada). The 9 amino acid phosphor-peptide derived from the Y394 phosphorylation site of Lck was purchased from ChinaPeptides Co. (China). The Ni-NTA Resin was from Bio Basic Inc. The monoclonal anti-His, anti-Myc and anti-actin antibodies were from Santa Cruz Biotech. Anti-Src/pY416 antibody was purchased from Invitrogen. Polyclonal anti-ERK1/2, anti-phospho-ERK1/2 antibodies were from Cell Signaling. Anti-CD3 (OKT3) was from eBioscience. All other chemicals and reagents were from Sigma.TCATAGACCCTATTCTTGAGCCATGATGTACCTTCAT; R263Q, AGGAAATGCGGACACAGGTTTTCAGTTTGATCC; and R266W, TGGACACAGAGGCGATCCGGGAAATG. All mutations were verified by DNA sequencing from Beijing Genomics Institute.The Lyp mutants R263Q, R266W and S201F were generated by PCR reactions with the QuikChange site-directed mutagenesis kit from Stratagene. The PAGE- purified oligonucleotide primers were from Beijing Genomics Institute (China) and the sequences were as follows: S201F, The catalytic domain of Lyp (residues 1–294) with N- terminal His tag was prepared and used for in vitro study as before E. coli BL21 DE3. In general, 12 liters of Lyp transformed E. coli were cultured, induced by 0.3 mM IPTG at 25°C. After induction, the cultures were pelleted by centrifugation at 4000 rpm. The cell pellets were washed with buffer A  and were resuspended in 120 ml of ice-cold buffer A. The bacterial pellets were sonicated on ice for 5 min, and the lysates were centrifuged at 10,000 rpm at 4°C for 1 hour. The supernatant was then collected and incubated with 4 ml of Ni2+-NTA resin for 2 hour at 4°C. The protein bound Ni2+-NTA beads were pelleted at 1,000 rpm for 6 min and the supernatant was discarded. The beads were washed three times for 5 min each at 4°C. The bound His-Lyp protein was finally eluted with a buffer containing 20 mM Tris pH 8.0, 300 mM NaCl and 200 mM Imidazole. The protein was further purified through CM Sefinose (BBI) with salt gradient elution. The low-salt solution contains 20 mM MES, pH 6.0, 100 mM NaCl, 1 mM EDTA and 2 mM DTT. The high-salt solution contains 20 mM MES, pH 6.0, 1 M NaCl, 1 mM EDTA and 2 mM DTT. After purified by CM Sefinose, the protein was further concentrated and stored at −80°C. Proteins were at least 99% pure by Coomassie staining after SDS-PAGE electrophoresis. Protein concentration was quantified by Bradford Protein Assay Kit.The His-tagged Lyp wild type and mutants were expressed in The His-tagged Src catalytic domain construct was a gift from Dr.lefkowitz lab in Duke University. The Src protein purification and phosphorylation were performed as described previously Initial rate measurements for the Lyp-catalyzed pNPP hydrolysis was determined as described previously The pH dependence was carried out in the following buffers: 100 mM acetate (pH 4.3–5.0), 50 mM succinate (pH 5.0–6.0), 50 mM 3,3-dimethylglutarate buffer (pH 6.0–7.3), 100 mM Tris/HCl buffer (pH 7.5–8.7). All buffers contained 1 mM EDTA and 1 mM DTT, and was adjusted to an ionic strength of 0.15 M with NaCl. The Kcat value for pNPP hydrolysis catalyzed by the wild type Lyp and R266W mutant were determined at 37°C. To fit the Kcat value against pH, ES1 and KES2 are the apparent ionization constants of enzyme-substrate complex in the rate-limiting step, and H is the proton concentration In this equation, KJurkat T cells were purchased from ATCC and grown at 37°C in RPMI 1640 medium supplemented with 10% FBS. Full-length Lyp wild type, S201F, R263Q and R266W mutants were subcloned into the pcDNA4/mycHis plasmid, and T cells were transfected with plasmids by electroporation with Neon (Invitrogen). After transfection, the cells were stimulated with 5 µg/ml anti-CD3 antibody (OKT3) or medium for 5 min. Subsequently, cells were lysed in 50 mM Tris, pH 7.5, 150 mM NaCl, 10 mM NaF, 2 mM EDTA, 10% Glycerol, 1% NP-40, 0.25% Sodium deoxycholate, 1 mM NaVO4, 1 mM PMSF, 0.3 µM aprotinin, 130 µM bestiatin, 1 µM leupeptin and 1 µM pepstatin. Cell lysates were subjected to denaturing SDS/PAGE, and transferred to nitrocellulose membrane. The Western blots were blocked with BSA, and immunoblotted by appropriate primary antibodies followed by incubation with HRP-conjugated secondary antibodies.5 cells were transfected by electroporation with 800 ng of the AP-1-luciferase plasmid, 40 ng of the Renilla-TK plasmid and 1 µg full-length Lyp wild type, R266W mutant or pcDNA4 vector. Forty-eight hours after transfection, Jurkat cells were stimulated with 5 µg/ml OKT3 or untreated for 6 hours. According to Promega’s instruction , dual luciferase activity was measured, and AP-1 transcriptional activity was normalized by Renilla activity.Jurkat T cells overexpressed Lyp and luciferase were used here. 2×10Data analysis was conducted with Image J and Graphpad software. All data were presented as mean ± standard error of the mean, and statistical comparisons were made with ANOVA tests.Figure S1Sequence alignment of Lyp from different species. Residues of position 201, 263, 266 are highlighted in red. The unconservative residues are shown in blue.(TIF)Click here for additional data file.Figure S2Coomassie blue staining of SDS-polyacrylamide gel of purified wild-type Lyp catalytic domain and variants.(TIF)Click here for additional data file.


Recently, a highly dangerous bird flu has infected over 130 patients in China, and the outbreak was attributed to a novel avian-origin H7N9 virus. Here, we performed a systematic analysis of the virus. We clarified the controversial viewpoint on neuraminidase (NA) origin and confirmed it was reassorted from Korean wild birds with higher confidence, whereas common ancestors of pathogenic H7N9 genes existed only one or two years ago. Further analysis of NA sequences suggested that most variations are not drug resistant and current drugs are still effective for the therapy. We also identified a potentially optimal 9-mer epitope, which can be helpful for vaccine development. The interaction of hemagglutinin (HA) and human receptor analog was confirmed by structural modeling, while NA might influence cellular processes through a PDZ-binding motif. A simplified virus infection model was proposed. Taken together, our studies provide a better understanding of the newly reassorted H7N9 viruses. On 29 March 2013, Chinese Center for Disease Control and Prevention (China CDC) isolated and confirmed a new influenza A H7N9) virus that had infected three Chinese patients, with two from Shanghai and one from Anhui Province. It's the first time that H7N9 viruses infect humans13456N9 virus All influenza A viruses encode 8 genes, which can be translated into 11 distinct proteins by different open reading frames (ORFs)611Accurate determination of the origin of highly pathogenic avian influenza (HPAI) H7N9 viruses is the most crucial problem for preventing pandemic367336363In this work, we first performed phylogenetic analyses for each gene in newly sequenced HPAI H7N9 viruses, respectively. The controversial viewpoint of Korea or Czech Republic-origin of NA gene was carefully evaluated and clarified. Also, we estimated the time of the most recent common ancestor (TMRCA) of each HPAI H7N9 gene, with a Bayesian Markov chain Monte Carlo (MCMC) approach113In this work, Neighbor-Joining (NJ) trees for eight genes of newly sequenced H7N9 viruses were constructed . To avoi−3 substitutions per site per year) are considerably greater than S-OIV (2.59 to 3.67 * 10−3 substitutions per site per year)As previously described11A number of genetic mutations or variations have been detected in recent studies33Anti-viral therapy with drugs might be the most effective treatment for H7N9 bird flu before vaccine development34563As an important surface protein, HA is the major molecule for vaccine developed19In Kageyama's results, a 218–230 deletion was detected at the H7N9 NS1 C-terminus1023In this work, we first clarified a controversial viewpoint on the origin of HPAI H7N9 NA3xN9 or H7Nx (x is a number of 1–17 for HA and 1–10 for NA) genes from the phylogenetic trees  on 9 April 2013The nucleotide and protein sequences of 7 newly sequenced HPAI strains of H7N9 viruses were downloaded from the GISAID  database on April 14. The 7 strains were isolated from 4 patients , 2 birds , and 1 environment (A/Environment/Shanghai/S1088/2013 | EPI_ISL_138984). Totally, we obtained 56 nucleotide sequences and 77 protein sequences for eight genes  of H7N9 viruses. We also downloaded 253,527 nucleotide and 320,023 protein sequences of all influenza subtype viruses from the NCBI Influenza Virus Resource  was applied to check and ensure promising distributions of parameters of posterior, prior probability and meanRate achieving moderate. Finally, FigTree v1.4.0  was used to visualize the phylogenetic trees and mark the TMRCA of each gene.To determine the TMRCA of the HPAI H7N9 outbreak, the molecular clock analysis was performed for each gene of HPAI H7N9 samplesWe first analyzed non-synonymous variations between patient and non-pathogenic samples. Because the results of multiple sequence alignments exhibited that the sequences of A/Shanghai/1/2013 are quite different from other patient samples, we separated 4 patient samples into two groups, including the A/Shanghai/1/2013 group and the second group. The sequences of A/Anhui/1/2013 were selected as the representative sequences for the second group. Based on phylogenetic results, the nearest non-pathogenic samples were chosen as benchmark sequences for comparison. Again, because the NA sequences among Korean wild birds are highly divergent, we further aligned 5 NA sequences from Korean wild bird H7N9 viruses . The identical positions in the alignment results were adopted as a benchmark for identifying NA variations in patient samples.http://www.pymol.org/), an open-source molecular visualization tool. With default parameters in PyMOL, the atoms were shown in different colors .Modeller 9.1135The CTL epitope predictor NetCTL 1.2 ServerY.X. designed and supervised experiments. Y.W. performed phylogenetic analysis. T.G., X.L., Y.Y. and J.R. repeated and validated phylogenetic results. H.C. and W.D. performed variation analysis. Z.L. performed structural modeling of HA and NA. Z.P. performed epitope prediction and docking. Z.D. performed PDZ-binding analysis. Y.X. and Z.L. wrote manuscript with contributions of all authors. All authors reviewed the manuscript.Supplementary Figure 1


In April 2009, a novel swine-derived influenza A virus (H1N1pdm) emerged and rapidly spread around the world, including Japan. It has been suggested that the virus can bind to both 2,3- and 2,6-linked sialic acid receptors in infected mammals, in contrast to contemporary seasonal H1N1 viruses, which have a predilection for 2,6-linked sialic acid.To elucidate the existence and transmissibility of α2,3 sialic acid-specific viruses in H1N1pdm, amino acid substitutions within viral hemagglutinin molecules were investigated, especially D187E, D222G, and Q223R, which are related to a shift from human to avian receptor specificity. Samples from individuals infected during the first and second waves of the outbreak in Japan were examined using a high-throughput sequencing approach. In May 2009, three specimens from mild cases showed D222G and/or Q223R substitutions in a minor subpopulation of viruses infecting these individuals. However, the substitutions almost disappeared in the samples from five mild cases in December 2010. The D187E substitution was not widespread in specimens, even in May 2009.These results suggest that α2,3 sialic acid-specific viruses, including G222 and R223, existed in humans as a minor population in the early phase of the pandemic, and that D222 and Q223 became more dominant through human-to-human transmission during the first and second waves of the epidemic. These results are consistent with the low substitution rates identified in seasonal H1N1 viruses in 2008. In April 2009, a novel swine-derived influenza A virus (H1N1pdm) emerged and spread rapidly around the world Since the first appearance of H1N1pdm, one particular amino acid substitution {aspartic acid to glycine at position 222 (D222G)} within the hemagglutinin (HA) molecule has appeared sporadically To identify whether α2,3 sialic acid-specific viruses, which replicate well in swine, were spread during the early phase of the H1N1pdm pandemic and whether α2,3 sialic acid-specific viruses are easily transmitted, the nucleotide sequences of the HA receptor binding site of H1N1pdm in clinical specimens were determined in this study.Several specimens obtained from mild H1N1pdm cases during the first (May 2009) and second (Dec 2010) waves of the epidemic in Japan were analyzed. In addition to the D222G substitution, we focused on two other substitutions {aspartic acid187glutamic acid (D187E) and glutamine223argine (Q223R)}, known to be critical for receptor binding, which cause a shift from α2,6 to α2,3 sialic acid receptor specificity Three nasal swabs (#1–#3) from mild H1N1pdm cases were obtained in May 2009 in Osaka, Japan. Total RNA was extracted from each sample and the receptor binding site (RBS) within HA was successfully amplified using One Step RT-PCR. The PCR products (369 bp) were cloned into pGEM-T easy vector and analyzed by conventional Sanger technology. More than 100 clones in each specimen were sequenced, and the deduced amino acid sequences were analyzed , and S3.Deep sequencing of more than 3,000 clones (reads) per specimen showed high rates of amino acid diversity, with the K119N, N125D, D222G, and Q223R substitutions in specimens #1–#3 . The res5 to 26 hemagglutination titers on the fifth or sixth passage (P5 or P6) (To elucidate how the genomic diversity (and population) of the RBS, including the D222G and/or Q223R substitutions, might change, we inoculated these specimens into embryonated chicken eggs and serially passaged them five or six times in eggs. Hemagglutination was not detected in any samples of the first passage (P1) but was detected in the third or fourth passage and showed 25 or P6) . PCR proD222G and/or Q223R substitutions were detected at even higher rates in the early passaged (P1) viruses  than in P5/P6 in #1, #2, and #3 specimens reduced hemagglutination titers when RBCs were treated with α2,3 sialidase whereas the titers did not change in MDCK-passaged viruses . These rTo elucidate the transmissibility of the G222 and/or R223 viruses in humans, five nasal swabs (#4–#8) obtained from individuals with a mild case of H1N1pdm in December 2010 were examined by deep sequencing analysis. The same region of HA RBS (369 bp) as shown in This study used a high-throughput sequencing approach to analyze more than 2,000 clones from each of eight H1N1pdm samples obtained in 2009–2010. The results showed that α2,3 sialic acid-specific viruses containing D222G and/or Q223R substitutions within the HA molecule were present in the upper respiratory tract as a minor population in patients with mild H1N1pdm infections in the early phase (May 2009) of the pandemic in Japan. However, these substitutions nearly disappeared in the samples from five mild cases in Dec 2010, suggesting that the D222G and/or Q223R mutants showed low rates of human-to-human transmission. Thus, the newly emergent influenza A viruses may have been dual specific but not exclusively α2,6-sialic acid-specific during the early phase of the pandemic and adapted during multiple cycles of human-to-human transmission. These results are consistent with previous reports showing that, during previous pandemics, the receptor binding properties of avian influenza viruses changed after introduction into mammals HA residue 222 plays a critical role in the binding affinity of the galactose moiety of sialosaccharides for the RBS. The D222G mutation would result in a loss of interaction between galactose and K219  because In this study, we performed deep amplicon sequencing using Roche/454 GS-Junior technology. We compared the results ob<1?show=[dh][dh]?>tained by using deep sequencer with those obtained by the conventional Sanger method to evaluate this newly developed technology. Sequencing analysis using next-generation technology is necessary to reveal H1N1pdm genetic diversity in detail; our preliminary sequencing analysis using a conventional cloning approach failed to detect the D222G mutation in specimen #1 . The freThe present study did not confirm whether viruses with the D222G and/or Q223R substitutions actually replicated in humans; however, the D222G and Q223R mutant viruses formed the dominant population when specimens were inoculated into embryonic chicken eggs , indicatSeveral reports have shown that the D222G substitution was associated with severe, and sometimes fatal, cases of H1N1pdm Three and five individual H1N1pdm-positive nasal swabs were provided in May 2009 and December 2010, respectively, in Osaka, Japan. All of the patients examined in 2009 had fever, sore throat, and cough, but were not hospitalized. They were administered oseltamivir after nasal swab-sampling. The patients examined in 2010 had upper respiratory symptoms but were not hospitalized. As a control, five specimens diagnosed with seasonal H1N1 in 2008 in Osaka were investigated. The analyzed samples were unlinked and anonymous in the Osaka Prefectural Institute of Public Health. This study was approved by the ethical review committees of the Osaka Prefectural Institute of Public Health and the Research Institute for Microbial Diseases (RIMD), Osaka University. The Both ethical committees specifically waived the need for consent.g for 10 min. The supernatants were suspended in TRIzol LS reagent  for 60 min. Total RNA was extracted using a PureLink™ RNA Mini Kit (Life Technologies/Invitrogen) according to the manufacturer's instructions. Contaminating DNA was eliminated with DNAase I (Life Technologies/Invitrogen).Each nasal swab was collected in Hanks solution and was centrifuged at 20,000 416TTGAAAGGTTTGAGATATTC435-3′; reverse primer, 5′-784CTAGTGTCCAGTAATAGTTC765-3′.Total RNA was subjected to One-step RT-PCR  to detect the receptor binding site in the HA molecule. The primers used were as follows: forward primer, 5′-Amplified PCR products were cloned into the pGEM-T easy vector . More than one hundred clones per specimen  were sequenced using conventional Sanger technology .Clinical specimens were injected into 9-day-old embryonated chicken eggs for virus isolation Viral samples were serially diluted with PBS and added at a concentration of 0.5% to chicken red blood cells. After incubation at room temperature for 30 min, hemagglutination was observed.PCR products were purified using the QIAquick® PCR purification kit  and were ligated with RL MID adaptors . The PCR products were further purified with Agencourt AMPure XP beads . The quality and quantity of the library was assessed using an Agilent 2100 Bioanalyzer  and TBS380 mini fluorometer , respectively. After amplifying the samples by emulsion PCR, high-throughput pyrosequencing was performed using a GS Junior (Roche Diagnostics GmbH). Data analysis was performed on each sequence read using computational tools as described previously 100 µl of 10% chicken, guinea pig, or horse red blood cells were treated with 10 U of α2,3 sialidase  at 37°C for 18 h and were washed with PBS. After the erythrocyte suspension was diluted to 0.5% (chicken) or 0.75% (guinea pig and horse), the hemagglutination titer was measured as described above.The crystal structure of HA of influenza virus, A/Brevig Mission/1/1918 (H1N1) (Protein Data Bank ID code 2WRG), was used as a template for homology modeling by using a Molecular Operating Environment. SA α2,6-linked structural analogs were used as inputs for docking with the model HA structure.Figure S1Alignment of the amino acid sequences within the receptor binding site of variants other than E187, G222, and R223. Variants other than E187, G222, and R223 (and R223  obtained(TIFF)Click here for additional data file.Figure S2Alignment of the amino acid sequences within the receptor binding site of variants other than E187, G222, and R223. Variants other than E187, G222, and R223 (and R223  obtained(TIFF)Click here for additional data file.Figure S3Alignment of the amino acid sequences within the receptor binding site of variants other than E187, G222, and R223. Variants other than E187, G222, and R223 (and R223  obtained(TIFF)Click here for additional data file.Figure S4Stereo views of the homology-based structural model for binding of sialosaccharides to the RBS of H1N1pdm hemagglutinin. The structural complex of the RBS of H1N1pdm HA with human receptor (orange) was constructed using the co-crystal structure of A/Brevig Mission/1/1918 (PDB ID: 2WRG) as a template. (A) HA of wild type (pink) overlapped with the HA of D222G mutants (green). Position 222 is shown in deep colors. Putative interactions salt-bridge between the sugar and the RBS are shown as black dashed lines. (B) HA of wild type (pink) overlapped with the HA of Q223R mutants (blue). Q223 and R223 are shown in deep pink and yellow, respectively.(TIFF)Click here for additional data file.Figure S5Kinetics of hemagglutination in viruses isolated from egg-passaged samples . Clinical specimens were injected into 9-day-old embryonated chicken eggs and incubated for 72 h. Allantoic fluid was collected (passage 0) and serially passaged in chicken eggs (passage 5–6). The viral growth of each sample was measured by hemagglutination assay using (0.5%) chicken red blood cells.(TIFF)Click here for additional data file.Figure S6Direct sequencing analysis of PCR products (positions 221–227 aa in HA-RBS) amplified from egg-passaged P6 (#1 and #2) and P5 (#3) viruses using a conventional ABI sequencer. The arrows indicate nucleotide substitutions. WT indicates wild type.(TIFF)Click here for additional data file.Table S1Frequency distribution of D222G, D222N, D222V, and D222E variants within the hemagglutinin receptor binding site of viruses obtained from clinical specimens (#1–#3).(TIFF)Click here for additional data file.


Human enterovirus 71 (HEV71) is one of the major pathogen responsible for hand, foot and mouth disease (HFMD). Currently no effective vaccine or antiviral drugs are available. Like poliovirus, EV71 is transmitted mainly by the feco-oral route. To date the majority of the studied EV71 vaccine candidates are administered parenterally. Injectable vaccines induce good systemic immunity but mucosal responses are often unsatisfactory, whereas mucosal vaccines provide both systemic and mucosal immunity. Therefore, oral immunization appears to be an attractive alternative to parenteral immunization.In this report, we studied the efficacy of an orally administered vaccine candidate developed using recombinant baculovirus displaying VP1 (Bac-VP1) in a murine model. Gastrointestinal delivery of Bac-VP1 significantly induced VP1-specific humoral (IgG) and mucosal (IgA) immune responses. Further, we studied the efficacy of the Bac-VP1 associated with bilosomes and observed that the Bac-VP1 associated with bilosomes elicited significantly higher immune responses compared to bilosomes non-associated with Bac-VP1. However, mice immunized subcutaneously with live Bac-VP1 had significantly enhanced VP1 specific serum IgG levels and higher neutralizing antibody titers compared with mice orally immunized with live Bac-VP1 alone or associated with bilosomes.Bilosomes have been shown to possess inherent adjuvant properties when associated with antigen. Therefore Bac-VP1 with bilosomes could be a promising oral vaccine candidate against EV71 infections. Thus, Bac-VP1 loaded bilosomes may provide a needle free, painless approach for immunization against EV71, thereby increasing patient compliance and consequently increasing vaccination coverage. Salmonella-based VP1 vaccines induced both humoral and cellular immune responses against EV71 but mucosal responses were not investigated Human enterovirus 71 (EV71) is a positive-stranded RNA virus belonging to the Enterovirus genus of the Picornaviridae family. EV71 has emerged as the most important neurotropic virus in young children after poliovirus Autographa Californica multiple nucleopolyhedrovirus (AcMNPV), an enveloped double-stranded DNA virus which can drive the expression of foreign genes in mammalian cells without causing cytotoxic effects There is a limitation for oral immunization in humans due to the lack of safe mucosal adjuvants. Most of the currently available adjuvants used in animal models are not acceptable for use in humans 2 inhalation for five minutes.All animal experiments were carried out in accordance with the Guidelines for Animal Experiments of the National Institute of Infectious Diseases (NIID). Experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the Temasek Life Sciences Laboratory, National University of Singapore, Singapore . Mice were housed in individually ventilated cages  provisioned with water and standard food, and they were monitored daily for health and condition. Total limb paralysis or more than 20% body weight loss was used as criterion for early euthanasia. The animals were euthanized by COAutographa californica nuclear polyhedrosis virus (AcNPV), was propagated in Spodoptera frugiperda Sf9 II and Sf9 III cell lines (ATCC) which were grown at 27°C in serum-free medium SF-900 II and SF-900 III (Invitrogen), respectively. EV71 strains were grown in rhabdomyosarcoma (RD) cells in Dulbecco’s modified Eagle medium (DMEM)  at 37°C with 5% CO2. The 50% tissue culture infective dose (TCID50) was determined in Vero cells using the Reed and Muench formula Baculovirus, 5′-GCATTCTGCCTTTGCGGATCTGCAGGGAGATAGGGTGGCAGATG) and VP1-R (5′- CATTTGCGCGTTGCAGTGCTCAAGAGTGGTGATCGCTGTG). In total five primers were designed to construct the novel expression cassette. Primers A (5′- TTTGGCGGCGGCGGCGCATTCTGCCTTTGCGGGAGATAGGGTGGCAGATGTAATTG) and B (5′- CATGATATGGCAAAGGAGGTCCATAGGATCCAAAGAGTGGTGATCGCTGTGCGAC) were used to amplify the entire full length VP1 gene and to introduce additional overlapping regions of the gp64 signal sequence at the N-terminus and H3N2 transmembrane region at the C-terminus of VP1. The resulting amplicons were purified and subjected to a second PCR using primers A and C (TTGGCAGGCCCACATGATGAACCCCAACAAAGCAACACAAAGCAAAAAACATGATATGGCAAAGGAAATCCA) to add the entire sequence of the H3N2 transmembrane domain to the C-terminus of VP1. Primers D (5′-AATCGGTCCGATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCATTCTG) and E (5′- AATAAGCTTTTAATATTGTCTATTACGGTTTTGGCAGGCCCACATGATGAACC) were then used to introduce the entire region of the gp64 signal sequence at the N-terminus and the gp64 CTD (cytoplasmic domain) region at the C-terminus of the above amplicons. Primer D and Primer E were designed to add restriction enzymes sites RsrII and Hind III at both terminuses, which allowed the insertion of the fusion genes into the RsrII/HindIII site of the multiple cloning site of pFASTBacHTA. Finally, the polh promoter was substituted with the ie1 promoter, amplified from white-spot syndrome virus DNA, using AccI and Rsr II restriction sites and the Primers F (5′-CCTACGTATCAATTTTATGTGGCTAATGGAGA) and G(5′-CGCGTCGACCTTGAGTGGAGAGAGAGCTAGTTATAA). The final construct was designated as Bac-VP1.The full-length open reading frame (ORF) of the VP1 gene of EV71 virus (C4 strain) was amplified from genomic RNA of Fuyang EV71-C4 strain (Genbank accession #EU703813) by RT-PCR using primers VP1-F  by site-specific transposition using the Bac-To-Bac system (Invitrogen) as described before To detect the immunofluoresence signals, the Sf-9 II cells were cultured on sterile cover slips (placed in 6-well plates) and infected at an MOI of 0.1. Two days after infection, the cells were fixed in 4% PFA for 1 h at 4°C, rinsed with phosphate-buffered saline , and blocked with 2% bovine serum albumin for 30 min at 37°C. The cells were then incubated with the polyclonal primary antibody raised against bacterially expressed VP1 in guinea pig (1∶300) for 1 h at 37°C, followed by three PBS washes. The cells were then incubated with the secondary FITC-conjugated rabbit anti-guinea pig mAb  for 1 h at 37°C, followed by three PBS washes. The negative control cells were treated the same way. Plasma membrane staining was done according to the manufacturer’s instructions  Protein localization was visualized using a confocal microscope .The virus particles were purified by two rounds of sucrose gradient ultracentrifugation following standard protocols and the viral titers were determined by PFU Bile salt stabilized vesicles (bilosomes) were prepared as previously described Transmission electron microscopy with negative staining was used to visualize the associated antigens present within the bilosomes formulation. Formvar/carbon-coated 200 mesh copper grids were glow discharged and a droplet of vesicle suspension applied. 2% phosphotungstic acid  negative stain was added and the grids were immediately dried. The grid was examined by transmission electron microscopy .7 PFU) associated or non-associated with bilosomes. In addition, a group of mice (n = 10) was subcutaneously immunized with 200 µl of Bac-VP1 and a subsequent booster dose was given on day 21. As positive control, a group of mice (n = 10) was immunized s.c. with 200 µl of inactive EV71 (C4) virus (107 TCID50) with complete Freund’s adjuvant and the subsequent booster dose was administered using incomplete Freund’s adjuvant on day 21. As negative controls, mice (n = 10) were immunized with baculovirus wild type (Bac-wt) virus or PBS. Serum and intestinal lavage fluids were collected as described previously Specific-pathogen-free female BALB/c mice (6 weeks old) were obtained from the Laboratory Animals Centre, National University of Singapore, and maintained at the Animal Holding Unit of the Temasek Life Sciences Laboratory. Prior to oral immunization, all mice were starved for 2 h and fed with sodium bicarbonate buffer (pH 9.6) to protect the antigens from the gastric acid. Ten mice per experimental group (n = 10/group) were immunized intragastrically by oral gavage on days 0, 7, and 21 with 200 µl of live Bac-VP1 or inactive Bac-VP1 . The plates were incubated at 4°C overnight and then incubated with 1% BSA in PBS with 0.05% tween20 (PBS-T) for 1 h at room temperature to prevent non-specific binding, 200 times diluted sera of different time points were added to the plates in duplicates and incubated for 1.5 h at room temperature. After three washes with PBS-T, 1000 times diluted horseradish peroxidase (HRP) conjugated goat anti-mouse immunoglobulins (DAKO) were added into each well. The reaction was developed by 100 µl TMB substrate  and then terminated by 50 µl of 2 M H2SO4. The optical densities at 450 nm were determined using a microwell plate absorbance reader .The VP1-specific serum IgG and mucosal IgA levels were tested separately against purified recombinant VP1 antigen by indirect enzyme-linked immunosorbent assay (ELISA). Briefly, U bottom microtiter plates were coated with purified EV71 capsid protein VP1 in carbonate coating buffer (15 mM Na50) of EV71 virus and incubated at 37°C for 2 h, and the mixtures were added to the monolayer of RD cells in triplicate wells. After incubation for 5 days at 37°C, the neutralizing antibody titers were read as the highest dilution of sera that completely inhibited virus growth.The microneutralization test was performed according to a previously described protocol 7 TCID50 of parental EV71- B4 strain (Genbank accession #AF316321.2). Five days later, the mice were euthanized and their brain tissues were dissected. After homogenization, the tissue lysates were collected, sterile filtered and used for further inoculation (i.p.) in to 1-day-old Balb/c mice. Six mouse brain passages later, the adapted virus caused 100% mortality in 6 day old mice. The protective immunity of vaccinated sera against EV71 infection was assessed in a mouse model of infection. 1-week-old BALB/c mice (n = 6) were intraperitoneally (i.p.) administered with vaccinated sera on day one post challenge with 5 MLD50  of mouse adapted EV71 B4 strain HFM 41 (5865/SIN/00009). Fifty percent mouse lethal dose (MLD50) of the virus required for the experiments was predetermined. Mice from the positive control or negative control group were given an equal amount of vaccinated sera obtained from mice immunized with inactivated EV71 vaccine or Bac-wt or PBS. Survival rates and clinical scores of the mice were monitored daily until 21 days post infection.Mouse-adapted EV71-B4 strain was produced as described previously Brain tissues from the passively immunized mice were homogenized for total RNA extraction by Trizol reagent (Invitrogen). Extracted RNA (1 µg) was used for qRT-PCR using Quantifast SYBR Green RT-PCR kit  according to the manufacturer’s protocol. 5′ UTR EV71 specific primers, i.e UTR-F-5′-TCCTCCGGCCCCTGAATG-3′and UTR-R-5′-GGACACCCAAAGTAGTCGGTTC -3′ were used for amplification. The qRT-PCR thermal cycling conditions were an initial incubation at 50°C for 12 min (reverse transcription), 95°C for 6 min , followed by 40 cycles each of 95°C for 12 s (denaturation), 60°C for 30 s , and 77°C for 15s. Melting curve data were collected from 50–95°C at a ramping of 1°C/5 s, and a finally cooling at 40°C was performed. The reaction was carried out using a Rotor-Gene-Q real time PCR cycler (Qiagen). The relative expression values were normalized to the expression value of the β-tubulin housekeeping gene. Serial ten-fold dilutions of recombinant plasmid DNA containing full-length EV71 genome were included to generate a standard curve for quantitative analysis.The data were expressed as arithmetic mean ± standard deviation (SD). The unpaired two-tailed Student’s t-test was performed to determine the level of significance in the difference between means of two groups. The level of significance was expressed as P<0.05.The recombinant baculovirus (Bac-VP1) was constructed to express the VP1 on the baculoviral envelope by incorporationg a signal peptide and transmembrane domain with a CTD (cytoplasmic domain). Ideally, the gp64 signal sequence directs the translocation of the fusion protein to the insect cell plasma membrane during virus replication and is cleaved after protein anchoring thus exposing the VP1-N-terminus to the outer surface. The H3N2-HA transmembrane domain enables the protein to anchor into the plasma membrane, while the gp64 CTD mediates (1) the recognition of budding baculovirus nucleocapsid with the membrane-bound protein and (2) the incorporation of the anchored protein into the viral envelope. H3N2-HA expression and the position of the gp64 CTD was illustrated in our previous study Sf9-II cells, the cells were infected separately with Bac-VP1 and as a control with wild type baculovirus. Confocal microscopic analysis using plasma membrane stain and VP1 polyclonal anti-guinea pig VP1 antibody (1∶500) followed by respective secondary FITC conjugated antibody revealed that VP1 expressed by the recombinant baculovirus was able to successfully translocate to the plasma membrane of infected insect cells. No signal was detected in Bac-wt infected cells  or PBS served as positive or negative controls respectively. At different time points  anti-sera were collected to determine the VP1-specific serum IgG levels. Mice orally immunized with live Bac-VP1 had significantly (P<0.001) enhanced VP1-specific IgG levels compared with inactive Bac-VP1 . Also, tTo test whether Bac-VP1 can act as an immunogen and elicit a mucosal immune response, mice were immunized orally with live or inactive Bac-VP1 associated with bilosmes or non-associated with bilosmes. Immunizations with Bac-wt or PBS served as negative controls respectively. Mice were vaccinated orally on day 0 and boosted on day 7 and 21. Mucosal IgA antibody titers against VP1 were measured at day 56. The mice immunized with Bac-VP1 significantly (P<0.001) induced higher mucosal IgA levels than the mice immunized with inactive Bac-VP1. Again, the live Bac-VP1 associated with bilosomes showed higher (P<0.001) mucosal responses compared with inactive Bac-VP1 associated with bilosomes. The mucosal responses elicited by mice immunized with bilosomes associated Bac-VP1 were also significantly higher (P<0.01) than those of mice immunized with non-associated Bac-VP1.in vitro microneutralization assay to examine their ability to neutralize live EV71 strains in RD cells. Initially, neutralization assays were performed using the sera from different time points against homologous Fuyang strain (EV71-C4) to analyse their protective potential (5). Interestingly the mice immunized orally with bilosomes associated Bac-VP1 exhibited a maximum neutralization activity of 128 (27). However, the mice immunized orally with bilosome associated or non-associated inactive Bac-VP1 showed low neutralization activity of around 8 (9) compared to sera from the group of mice immunized orally with live Bac-VP1 alone or associated with bilosomes. The complete protection of RD cells using the sera of mice immunized with inactivated EV71 virus was observed to be a maximum of 2048 (211) (1). This increase in neutralization efficiency of bilosomes associated Bac-VP1 compared to non-associated Bac-VP1 might be a direct result of the antigen protection by bilosomes in the gastrointestinal tract. Since neutralization assays were conducted against heterologous EV71 strains, these results also show that our construct elicits cross-protective antibodies.Next, the sera from vaccinated mice were analysed in an otential . Sera frotential   . On the 50 of mouse-adapted EV71-B4 strain in 6 day old mice. Pups treated with sera from mice orally vaccinated with bilosome associated live Bac-VP1 had 100% protection against EV71 infection. However, pups treated with sera from mice orally vaccinated with bilosomes associated inactive Bac-VP1 had only 16.6% protection. Sera from mice orally vaccinated with live Bac-VP1 alone also conveyed 100% protection while mice that received sera from inactive Bac-VP1 vaccinated mice, all succumbed to the challenge (4 copies/ml (The level of protective immunity induced by our vaccine constructs was determined in a suckling mouse model. Passive protective efficacy of the sera (day 56) from vaccinated mice were tested against 5 MLDhallenge . Similarhallenge . There wopies/ml .in vivo in animal models Most human pathogens including EV71 initiate infection via the mucosal surfaces and therefore mucosal immunity is often required to achieve protection against these pathogens. Oral or intranasal administrations are the two main options for mucosal vaccination. Intranasal vaccines can have a detrimental effect on persons with asthma and other chronic pulmonary or cardiovascular disorders 11) which was significantly higher than the neutralization titers of mice vaccinated with oral vaccine candidates. However, mice orally vaccinated with live Bac-VP1 alone or associated with bilosomes additionally induced VP1 specific mucosal IgA response compared to parenteral immunization that induces mainly humoral immune response. Protection against viral infectivity in an animal model has long been considered the best tool to test the efficacy of a vaccine candidate. One limitation of the vaccine efficacy study is the lack of a suitable mouse model of EV71 disease. Due to this, we have performed the passive protection assay in a suckling mouse model of EV71 infection. Sera from mice vaccinated orally with live Bac-VP1 alone or associated with bilosomes could completely protect pups against EV71 infection whereas sera from mice vaccinated orally with inactive Bac-VP1 associated or non-associated with bilosomes did not alleviate clinical symptoms of EV71 infected pups. Therefore, this study revealed that gastrointestinal delivery of VP1 by recombinant baculovirus elicited protective immunity against EV71 in mice. Moreover, the bilosomes associated Bac-VP1 efficiently induced humoral and mucosal immune responses against EV71. Besides its broad range of target cells coupled with its non-replicative nature Interestingly, mice vaccinated parenterally with inactivated EV71 virus induced neutralizing antibodies at a titer of 2048 (2In conclusion, oral vaccination is still considered the easiest way to deliver immunogens and also it is more acceptable to patients and reduces the need for highly trained personnel during mass immunization. Since bilosomes are produced from naturally occurring lipids and possess inherent adjuvant properties when associated with antigens, Bac-VP1 associated with bilosomes could be a viable option for an oral vaccine candidate against EV71 infection. Its advantages, together with its increased immunogenicity and protective efficacy in mouse models, make this recombinant baculovirus vaccine one of the safest vaccines; however it is necessary to investigate further the possible toxicity in animals before its use in clinical trials.


Phosphotyrosine binding (PTB) domains are critically involved in cellular signaling and diseases. PTB domains are categorized into three distinct structural classes namely IRS-like, Shc-like and Dab-like. All PTB domains consist of a core pleckstrin homology (PH) domain with additional structural elements in Shc and Dab groups. The core PH fold of the PTB domain contains a seven stranded β-sheet and a long C-terminal helix.In this work, the PTB domain of Dok1 protein has been characterized, by use of NMR spectroscopy, in solutions containing sub-denaturing and denaturing concentrations of urea. We find that the Dok1 PTB domain displays, at sub-denaturing concentrations of urea, alternate conformational states for residues located in the C-terminal helix and in the β5 strand of the β-sheet region. The β5 strand of PTB domain has been found to be experiencing significant chemical shift perturbations in the presence of urea. Notably, many of these residues in the helix and in the β5 strand are also involved in ligand binding. Structural and dynamical analyses at 7 M urea showed that the PTB domain is unfolded with islands of motionally restricted regions in the polypeptide chain. Further, the C-terminal helix appears to be persisted in the unfolded state of the PTB domain. By contrast, residues encompassing β-sheets, loops, and the short N-terminal helix lack any preferred secondary structures. Moreover, these residues demonstrated an intimate contact with the denaturant.This study implicates existence of alternate conformational states around the ligand binding pocket of the PTB domain either in the native or in the near native conditions. Further, the current study demonstrates that the C-terminal helical region of PTB domain may be considered as a potential site for the initiation of folding. Conversely, other conformational states of protein including partially folded and unfolded states reside at the high energy region of the folding funnel landscape. The folded conformation of a protein, largely populated under physiological solution condition, often undergoes excursion to other low-lying energy states that surrounds the free energy minimumFolding and unfolding studies of protein interaction domains have generated valuable insights toward the protein folding mechanisms 15N-1H HSQC spectrum of the PTB domain is well dispersed, as expected for a folded protein , in comparison to the other  at four different conditions including 0 M urea or native, in the presence of phosphorylated β3 tail and in sub-denaturing concentrations, at 0.5 M and 1 M, of urea , in 7 M urea, encompass all of the major secondary structural elements allowing us to probe the structural and dynamical characterization of the unfolded state of the PTB domain.The 13Cα  shift of αH indicates population of alpha helical conformations while the opposite trend has been observed for the β strand structural propensity 13Cα chemical shift . In a previous study, the secondary shift of 13Cβ has also been seen to be a poor indictor of the secondary structure for the denatured protein Secondary structural propensity of the unfolded states of Dok1 PTB domain has been assessed from secondary chemical shifts and nuclear Overhauser effect (NOE) data. Secondary chemical shifts of 13Cα  and αH , 9 αH/NH  and 6 βH/NH (i to i+2) NOEs were identified for the Dok1 PTB domain in 7 M urea. The C-terminal region of the PTB domain contains several of these medium range and NH/NH NOEs, indicating preference for helical conformations. By contrast, other residues of the PTB domain in the denatured state appear to be sampling the extended region of the φ, ψ conformational space as suggested by the strong sequential αH/NH NOE connectivities. Interestingly, we have observed as many as 26 NOE contacts involving urea and PTB domain  spectra. Analysis of 3D nge NOEs . All togB domain , Fig. S31 (1/T1), R2 (1/T2) and heteronuclear {1H}-15N NOE  and J(H), were determined  and plotted in N) parameter, largely determined by R1, shows similar values across the polypeptide chain, except for the last two residues, Q102 and G103, at the C-terminus (N) for these residues Q102 and G103 are diminished (H), mostly determined by heteronuclear NOE, represent a wider variation across the polypeptide chain whereby higher values are observed for residues, Q102 and G103, at C-terminus along with fewer residues including A12, S39, R70, G74, A91, A95 at the middle and the N-terminus (H) of residues A12, S39, R70, G74, A91, A95 indicate a fast motion without any associated contribution to intermediate time scale motions  J(0) values. There are also a few residues, R70, S73, G74, T77, A83, G85, A91, T94, A95 at the C-terminus with below average J(0) value , J(terminus . J(N) fminished . The valterminus . The hig motions . The J(00) value . The seq0) value .Protein domains essentially act as a molecular interface for their interactions with nucleic acids, proteins and phospholipids. These binding events in turn control different cellular processes from signal transduction to transcription regulation15N-1H HSQC spectra of PTB domain acquired at various concentrations of denaturant revealed detectable binding of the denaturant with the folded protein. Despite a direct binding of urea, the folded conformation of the PTB domain appears to be stable and unfolds only at 7 M urea. The HSQC spectral changes, with low to high concentrations of urea, have indicated an unfolding transition whereby the folded and unfolded states are in slow exchange at the NMR time scale. The PTB domain of Dok1 assumes highly unfolded conformations at 7 M urea without any evidence of long-range interactions. A large number of residues including polar, charged and Gly demonstrated NOE contacts with the denaturant  NOEs and secondary chemical shift parameters indicated that the unfolded PTB domain is in largely extended conformations. The unfolded conformational ensembles of the PTB domain are also dynamically flexible as indicated by low value of hetero-nuclear NOEs. However, non-random conformations of the PTB domain can be seen at the C-terminal region and for a few short segments of the protein. The C-terminus of the PTB domain is sustained by a long helix encompassing residues N86-K100 in its native state. Residues in this segment of the PTB domain demonstrated a consensus pattern of helical conformations in secondary chemical shifts in 7 M urea  values in spectral density mapping were also indicative of fast motion whereas other residues, with higher J(0) and lower J(H), may be experiencing motional restriction at sub-nanosecond timescale. The β-sheet region of the Dok1 PTB domain in 7 M urea did not exhibit any preferred secondary conformations expect for residues at the loops connecting β4, β5 and β6 strands. Most of the residues corresponding to the β-sheet region of the native PTB domain were also found to be motionally flexible, although non-random conformations and motional restriction can be seen for three short regions namely residues A27-V32, L46-L53 and Y56-V61. Above average values of R2/R1 and positive values of heteronuclear NOE have indicated formation of clusters with non-random motions. Further, the above average J(0) and below average J(H) values in spectral density mapping correlate with a relatively slower motion of these clusters in 7 M urea. Such slow motions may also indicate plausible conformational exchange processes contributing to the higher R2 values. Notably, most of the residues in these clusters are non-polar or aromatic and did not exhibit NOEs to urea. This may perhaps explain non-random characteristics of these hydrophobic clusters. A plot of the average area buried upon folding (AABUF) of the domain sequence correlates well with the residues involved in cluster formation . Cells were induced with 1 mM IPTG at an OD600 of 0.6–0.7 and incubated for 6–12 hours at 16°C for protein production. Cells were centrifuged and resuspended in 20 mM Tris-HCl buffer, pH 8.0. Cell solutions were lysed by sonication and the supernatant was applied to Nickel-NTA column for His tag-based affinity purification (Qiagen). The weakly bound proteins were removed from the column by extensive washing with appropriate buffers. Target protein was eluted with buffer containing 500 mM imidazole. Further, purified protein was extensively dialyzed against 50 mM sodium phosphate buffer, containing 100 mM NaCl and 2 mM DTT at pH 6.0. Samples of Dok1 PTB domain were loaded onto a Hiload Superdex 75 16/26 GL preparative column with the AKTA FPLC UPC-900 system . FPLC column was equilibrated with 50 mM sodium phosphate buffer containing 100 mM sodium chloride and 2 mM DTT, pH 6.0. Protein samples were eluted at a flow rate of 0.5 ml/min and detected spectrophotometrically at 276 nm. The amino acid sequence of Dok1 PTB domain was numbered as Q1- G103.His tagged human Dok1 PTB domain  was sub cloned into the pET14b vector and over expressed into 15N-1H HSQC spectra of native and the 7 M urea unfolded state of Dok1 PTB domain were assigned using standard triple resonances NMR methods with a combination of 3-D spectra: HNCA, HN(CO)CA, HNCACB and CBCA(CO)NH αH and side chain protons were assigned by 3D 15N -NOESY-HSQC (mixing time 120 ms) and 3D 15N-TOCSY-HSQC (mixing time 80 ms) experiments in case of urea unfolded sample. NMR spectra were acquired at sample concentrations of 0.6 mM or 1 mM in 50 mM sodium phosphate, 100 mM NaCl, 2 mM DTT and 10% D2O at pH 6. For the unfolded state of the protein, all the conditions were same except 7 M urea had been added from a stock solution of 10 M urea made in same buffer. Temperature for all the experiments was set to 298 K. The proton chemical shift was directly referenced to 1H resonance frequency of 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS) whereas the 13C and 15N dimensions were referenced indirectly. NMR parameters of the unfolded state of Dok1 PTB domain have been deposited to BMRB with accession number 19672.All the NMR experiments were performed on Bruker DRX 600 MHz or 700 MHz spectrometers equipped with cryoprobe. Data acquisition and processing were performed either with Topspin software (BRUKER) or NMRPipe and NMRDraw suite 15N labelled samples. A series of HSQC spectra were recorded at different urea concentrations ranging from 0.25 M to 7 M. The urea induced chemical shift changes in 15N and HN resonances was determined using the following equation which is designated as chemical shift perturbation (CSP), H and WN are weighting factors for 1H and 15N chemical shifts, respectively H and ΔN are the chemical shift difference for amide proton and 15N respectively.Urea titration experiments were recorded at 298 K for Dok1 PTB domain by adding aliquots of denaturant from a 10 M stock made in 50 mM sodium phosphate, 100 mM NaCl and 2 mM DTT at pH 6 to the NMR tube containing Temperature dependence of amide proton chemical shift for the Dok1 PTB domain was performed by recording a series of HSQC spectra at seven different temperatures, 285 K, 288 K, 291 K, 294 K, 297 K, 300 K and 303 K. These experiments were done for native and two different concentrations of denaturant, 0.5 M and 1 M urea, in 50 mM sodium phosphate buffer, 100 mM NaCl, 2 mM DTT, pH 6.0. Temperature dependent experiments were also performed at the native condition in the presence of pTyr747 phosphorylated integrin β3 tail at a protein to peptide ratio of 1∶0.6. For each amide proton, a straight line was fitted to the chemical shift temperature dependence and the residuals were plotted to observe the curved temperature profiles.1 = 1/T1) and transverse (R2 = 1/T2) relaxation rate constants of 15N labeled Dok1 PTB domain were determined by collecting a series of 1H-15N HSQC spectra at different time interval with sensitivity enhancement in a Bruker DRX 600 MHz spectrometer at 298 K temperature 1 measurement were set to 5, 40, 80, 130, 210, 330, 470, 630, 800, 1000 and 3000 ms with repeating the experiments at the time points of 80 ms and 470 ms for error estimation. For T2 measurement the data were acquired with delays of 14.4, 28.8, 43.2, 57.6, 72.0, 86.4, 100.8, 115.2, 129.6, 144.0 and 158.4 ms with the duplicate points at 72.0 ms in this case. T1 and T2 relaxation experiments were repeated twice for all the experimental conditions and a total of 2048 complex data points with 128 complex increments were collected. Relaxation data were also collected for the natively folded PTB domain in 50 mM sodium phosphate buffer, 100 mM NaCl, 2 mM DTT, pH 6.0. The relaxation rate constants were determined by fitting the cross peak intensities to a mono-exponential function as in I = A*exp(R*t), where I is the signal intensity, A is the signaling constant, R is the relaxation constant and t is the time point 1H-15N hetero-nuclear NOE values were determined by using a pair of spectra recorded with and without proton saturation. The NOE experiment with proton saturation was carried out with a 2 s inter-scan relaxation delay followed by 3 s of proton saturation. Similar delays were used for the heteronuclear NOE experiments of the unfolded proteins in urea1H-15N hetero-nuclear NOE experiments spectrum were recorded with 2048 complex data points with 256 complex t1 points. The NOE values were determined as a ratio of peak intensities with and without proton saturation. A reduced spectral density analysis of the 15N relaxation data was carried out for the 7 M urea unfolded state of the Dok1 PTB domain as described earlierThe longitudinal Click here for additional data file.Figure S2Chemical shift changes of selected amide proton resonances with temperature. Sections of 15N-1H HSQC crosspeaks of residues showing curved  and linear  temperature dependence. 15N-1H HSQC spectra were obtained at temperatures of 285 K (red), 288 K (light red), 291 K (green), 294 K (yellow), 297 K (purple), 300 K (cyan) and 303 K (pink) at 0 M, 0.5 M and 1 M urea and in presence of tyrosine phosphorylated β3 tail peptide.(TIF)Click here for additional data file.Figure S3Urea-protein NOEs. Slices of 3D 15N-edited NOESY-HSQC spectra of the PTB domain showing NOEs between urea protons resonating at 5.9 ppm with the amide protons of representative residues of the PTB domain in 7 M urea.(TIF)Click here for additional data file.Figure S4Relaxation parameters of the native PTB domain. Bar diagram showing R1 (panel A), R2 (panel B) and heteronuclear NOE (panel C) of the native PTB domain as a function of residue.(TIF)Click here for additional data file.


Respiratory viral infection is a major cause of asthma exacerbations in both children and adults. Among the respiratory viruses, influenza virus is a particularly important pathogen due to its enormous morbidity and mortality in annual epidemics. The swine-origin influenza A virus, designated as A(H1N1)pdm09, emerged in the spring of 2009 and caused the first influenza pandemic in the 21st century. With the emergence of the novel A(H1N1)pdm09 virus, numerous epidemiologic studies detected asthma as a frequent comorbid condition in patients infected with this virus. Here we review recent reports regarding asthma in patients infected with influenza A(H1N1)pdm09 virus, and we discuss the utility of influenza vaccines and antivirals. Asthma is a chronic airway disease with the symptoms of repetitive cough, wheezing and dyspnea, with reversible airway narrowing accompanied by airway hyper-responsiveness . It is eInfluenza virus causes influenza characterized by a sudden onset of high fever and respiratory symptoms such as cough, sore throat and coryza, as well as systemic symptoms such as headache, muscle ache and fatigue. Influenza epidemics occur yearly during the autumn and winter in temperate regions, whereas the disease patterns in tropical and subtropical regions are less well established . Annual Influenza A viruses can be subtyped according to their two major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Currently, there are 16 subtypes of HA (H1–H16) and nine subtypes of NA (N1–N9), and all have been found in wild aquatic birds, which are the natural reservoir of influenza A viruses. Only two subtypes of these viruses (H1N1 and H3N2) are currently circulating in humans, as seasonal influenza. Influenza A viruses have negative-sense, single-stranded, and eight-segmented RNAs as the genome . It is kIn March and early April 2009, a novel swine-origin influenza A(H1N1) virus, designated as A(H1N1)pdm09, emerged in Mexico and the United States  and rapiFigure 1). The polymerase basic 2 (PB2) and polymerase acidic (PA) gene segments were derived from the avian virus lineage, whereas the polymerase basic 1 (PB1) gene segment was from human A(H3N2) virus. The HA, nucleoprotein (NP), and non-structural protein (NS) gene segments were from classical swine A(H1N1) virus. The NA and matrix (M) gene segments were from the Eurasian avian-like swine A(H1N1) virus.A triple reassortant swine A(H1N2) virus then reassorted with a Eurasian avian-like swine A(H1N1) virus, resulting in A(H1N1)pdm09 virus (A(H1N1)pdm09 virus has none of the known hallmarks of virulent influenza viruses such as highly pathogenic avian A(H5) and A(H7) viruses, except for an amino acid substitution of aspartic acid by glycine at position 222 (D222G) in the HA, which was observed in severe and fatal cases with high frequency. The D222G substitution changes the receptor binding specificity of the virus from α2–6  to α2–3 (avian type) sialylated glycans . This amWe recently reported that A(H1N1)pdm09 viral isolates derived from fatal cases manifested sporadic amino acid changes in the PB2 and PA proteins  more frequently than those derived from mild cases . More reWidespread activity of pandemic A(H1N1) 2009 occurred and reached its peak a couple of months earlier than the usual seasonal influenza in the northern hemisphere, from April 2009 to January 2010 . The AH.Asthma was one of the most common underlying medical conditions among patients hospitalized with A(H1N1)pdm09 viral infection in 2009 worldwide . KloepfeA Canadian group reported that children admitted to a hospital with A(H1N1)pdm09 viral infection tended to have pre-existing asthma to a greater extent compared to the children with seasonal influenza A viral infection (15% vs. 5%), although there was no significant difference in the severity of pre-existing asthma between the groups of children with these infections . An age-In contrast, it is not clear whether A(H1N1)pdm09 viral infection can frequently cause the development of asthma compared to seasonal A(H1N1) or A(H3N3) viral infection. The study by Figure 1).Influenza A viral infection induces the production of interleukin 1 beta (IL-1β), IL-6, IL-8, tumor necrosis factor-alpha (TNF)-α, histamine, protease, interferon (IFN)-α, and IFN-gamma (IFN-γ) from airway epithelial cells and other cells including peripheral blood basophils . These pFigure 1). Two types of influenza vaccines are currently available; inactivated vaccine and live, attenuated vaccine. The live, attenuated nasal-spray influenza vaccine has been approved for use in the United States since 2003. However, it has not been recommended in high-risk groups including asthmatics because its safety is not fully demonstrated. The widespread use of inactivated influenza vaccines contain a trivalent mixture of strains of A(H1N1)pdm09, A(H3N2), and type B viruses likely to circulate during the next influenza season. Many studies indicated that no increase in asthma exacerbations was reported for both vaccinated children and adults pdm09 virus vaccine was conducted in the United States . The resSpecific antiviral drugs against influenza viruses could be used for the treatment and prophylaxis for influenza. Based on their chemical properties and spectra of activity against influenza viruses, the drugs can be classified into two categories: the M2 ion channel inhibitors, i.e., adamantanes (amantadine and rimantadine) and the NA inhibitors, i.e., zanamivir, oseltamivir, peramivir, and laninamivir. Currently, the NA inhibitors are exclusively used for the treatment and prophylaxis of influenza because the circulating strains of A(H1N1)pdm09 and A(H3N2) viruses have a known amino acid substitution of serine by asparagines at position 31 in the M2 protein, which confers resistance to the adamantanes.Few studies have examined the safety of NA inhibitors in patients with asthma. A double-blind, placebo-controlled crossover study indicated that zanamivir inhaled as a dry powder did not significantly affect the pulmonary function and airway responsiveness of subjects (19 to 49 years of age) with mild or moderate asthma . HoweverEpidemiological studies as described above demonstrated that A(H1N1)pdm09 viral infection is closely associated with asthma in both children and adults. Although A(H1N1)pdm09 virus has not shown a high mortality rate similar to that of the highly pathogenic avian influenza virus of the H5N1 subtype, patients with A(H1N1)pdm09 viral infection were more susceptible to asthma exacerbation compared to A(H1N1) or A(H3N2) viral infection. Detailed analyses of virus-host interactions are needed to elucidate the mechanism underlying A(H1N1)pdm09 viral infection-induced asthma.Since March 31, 2013 when the public health authorities of China reported three cases of human infection with an avian influenza A(H7N9) virus, a total of 135 human cases including 44 fatal cases have been reported in China and Taiwan as of August 12, 2013 . The curThe 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.


An H12N8 subtype avian influenza virus (AIV) was isolated from a wild bird in China in 2011. It is the first report of isolation of the H12N8 subtype AIV in Asia. Phylogenetic analysis results suggested it is a reassortant, and all eight gene segments belong to the Eurasian gene pool. Wild aquatic birds are natural reservoirs of all subtypes of avian influenza viruses (AIVs) –3. Long-226 and G228 (H3 numbering) at the receptor binding sites (We isolated an H12N8 subtype AIV from wild aquatic bird feces in November 2011 and named the strain A/wild goose/Dongting/C1037/2011 (H12N8), which is the first report of the detection of an H12N8 subtype AIV in Asia. The eight gene segments were amplified and sequenced by the ABI 3730xl DNA analyzer. The complete viral genome consists of 8 gene segments of negative-sense, single-stranded RNA, and it encodes 11 proteins with the amino acid lengths of 759 for polymerase basic 2 (PB2), 727 for PB1, 80 for PB1-F2, 716 for polymerase (PA), 564 for hemagglutinin (HA), 498 for nucleoprotein (NP), 470 for neuraminidase (NA), 252 for M1, 97 for M2, 225 for nonstructural 1 (NS1), and 121 for NS2 proteins. The amino acid sequence of the HA cleavage site is VPQAQDR↓GLF, representing low pathogenicity in chickens  have been deposited in GenBank under accession no.