Yarrowia lipolytica to improve the specific activity of two fungal endo-mannanases, PaMan5A and PaMan26A, which belong to the glycoside hydrolase (GH) families GH5 and GH26, respectively. Following random mutagenesis and two steps of high-throughput enzymatic screening, we identified several PaMan5A and PaMan26A mutants that displayed improved kinetic constants for the hydrolysis of galactomannan. Examination of the three-dimensional structures of PaMan5A and PaMan26A revealed which of the mutated residues are potentially important for enzyme function. Among them, the PaMan5A-G311S single mutant, which displayed an impressive 8.2-fold increase in catk/KM due to a significant decrease of KM, is located within the core of the enzyme. The PaMan5A-K139R/Y223H double mutant revealed modification of hydrolysis products probably in relation to an amino-acid substitution located nearby one of the positive subsites. The PaMan26A-P140L/D416G double mutant yielded a 30% increase in catk/KM compared to the parental enzyme. It displayed a mutation in the linker region (P140L) that may confer more flexibility to the linker and another mutation (D416G) located at the entrance of the catalytic cleft that may promote the entrance of the substrate into the active site. Taken together, these results show that the directed evolution strategy implemented in this study was very pertinent since a straightforward round of random mutagenesis yielded significantly improved variants, in terms of catalytic efiiciency (kcat/KM).Microbial mannanases are biotechnologically important enzymes since they target the hydrolysis of hemicellulosic polysaccharides of softwood biomass into simple molecules like manno-oligosaccharides and mannose. In this study, we have implemented a strategy of molecular engineering in the yeast  Hemicellulose is a combined designation of a diverse set of abundant non-crystalline carbohydrate polymers, among which mannans are the major component in softwoods Mannans are hydrolyzed by the coordinated action of several types of glycoside hydrolases (GH) among which endo-β-1,4 mannanases (EC 3.2.1.78) are the key enzymes that depolymerize the mannan backbone. They are encountered in families GH5, GH26 and GH113 in the CAZy database Despite the fact that mannanases are largely exploited in biotechnological applications, only a few studies have so far reported on the improvement of mannanase properties using molecular engineering Podospora anserina that belong to the glycoside hydrolase (GH) families GH5 and GH26  in order to improve their activity towards galactomannan. To evaluate the activity of mannanase variants produced in the Yarrowia lipolytica expression host, an in-house high-throughput method based on the reducing sugar assay In this study, a random mutagenesis strategy was used to generate variants of two endo-mannanases from the ascomycete fungus Y. lipolytica was chosen as host to perform molecular engineering of PaMan5A and PaMan26A since a very high reproducibility in protein expression level was demonstrated PaMan5A and PaMan26A were inserted into the Y. lipolytica expression vector in frame with the yeast preprolip2 secretion peptide under the control of the oleic acid-inducible promoter POX2 , which is adequate for excluding false positives variants. The secretion yields of PaMan5A and PaMan26A in deep-well microplates were 6- and 9-fold lower than in shake flask cultures, which is probably due to a better oxygenation of cultures in flasks than in deep-well plates where the ratio volume of culture/volume of container was of 1/2 instead of 1/10. However, levels of mannanase production were in both cases sufficient to deploy a high-throughput screening campaign.The yeast ter POX2 . PositivPaMan5A and PaMan26A, two endo-mannanases from Podospora anserina. The error-prone PCR mutagenesis round was carried out using the wild type genes as templates  and with the conditions appropriate to reach a number of mutations in a range of 2 to 5 mutations per kb. Indeed, a very low number of mutations per kb  would result in too many active variants with unchanged activity, and a high number of mutations per kb would result in too many inactive clones as reported in PaMan5A and PaMan26A, respectively). Y. lipolytica transformation yielded about 5,200 and 5,600 clones for PaMan5A and PaMan26A, respectively. As reported in other studies PaMan5A and PaMan26A, respectively . The Y. lipolytica strain expressing the best PaMan26A variant displayed an increase of activity towards galactomannan of 147% compared to the strain expressing wild-type PaMan26A that corresponded to 12 CV. The Y. lipolytica strains expressing selected PaMan5A variants displayed also increased activity between 8 and 46% compared to the strain expressing wild-type PaMan5A that corresponded to 1.1 to 6 CV. Each of the mannanase-mutant genes was amplified from genomic DNA, subcloned and sequenced. As a result, one single (PaMan5A-G311S), two double  and two triple mutants  were created. Results are summarized in The objective of the directed evolution approach was to improve enzymatic activities of ectively . Since tP. pastoris expression system because (i) it yields higher expression levels than Y. lipolyticaPaMan5A and PaMan26A were already successfully expressed to high yields in P. pastorisY. lipolytica and P. pastoris was roughly 1∶10. All of the mutant enzymes were successfully produced in P. pastoris with yields of approximately 1 g per liter of culture and purified taking advantage of the (His)6-tag. SDS-PAGE analysis of the purified mutants compared to the wild type enzymes revealed similar apparent molecular masses (data not shown). Isoelectrofocusing analysis revealed that all of the mutants displayed similar pIs compared to wild-type enzymes except for the PaMan5A-W36R/I195T/V256A mutant that exhibited an increase of 0.3 unit . Regarding the pH and temperature profiles, no significant difference was observed between variants and parental enzymes (data not shown).For mutant enzymes production, we used the 0.3 unit . This in5) and mannohexaose (M6)  and therefore kcat/KM were considered unchanged and not determined. PaMan5A-K139R/Y223H revealed a decrease of kcat/KM of about 35% and 11% respectively. PaMan5A-G311S displayed an increased kcat/KM of about 37% and 12% towards M5 and M6, respectively.Each mutant was characterized using High Performance Anion Exchange Chromatography – Pulsed Amperometric Detection (HPAEC-PAD) to assess its ability to hydrolyze galactomannan and manno-oligosaccharides: mannopentaose (Mose (M6) . RegardiM apparent values, all of the mutants displayed improved apparent affinity for galactomannan. Although turn-over numbers of almost all of the mutant enzymes were similar to wild-type enzymes, the PaMan5A-W36R/I195T/V256A mutant displayed a 2.5-fold increase of turn-over toward galactomannan resulting in an overall catalytic efficiency improved by 1.8-fold. In terms of catalytic efficiency, the best-performing mutant was the PaMan5A-G311S mutant for which the single amino-acid substitution led to a 8.2-fold increase in kcat/KM due to a drastic improvement of KM whereas the kcat remained unchanged. Other PaMan5A mutants exhibited also to a lower extent increased kcat/KM towards galactomannan, i.e., between 32 and 79% improvement. The PaMan26A-P140L/D416G mutant, which is the unique mutant selected from the 5,600 PaMan26A variants screened, displayed an improved kcat/KM of approximately 30%. All of the mutants were subjected to a paired t-test in comparison with the corresponding native enzyme. Three out of four PaMan5A mutants and the PaMan26A mutant revealed p-values below 0.05 for galactomannan hydrolysis and were therefore considered statistically different from their native counterpart  out of eight are located within the active site cleft and three mutations are located inside the enzyme core . Three mutations were identified in the PaMan5A-W36R/I195T/V256A mutant, among which W36 and V256 that are located close to the catalytic site , indicating a possible hot spot for enzyme improvement to further explore. Concerning the PaMan5A-K139R/Y223H mutant, analysis of the manno-oligosaccharides produced upon hydrolysis of M5 and M6 revealed a potential modification of substrate binding . Therefore the single mutation PaMan5A-G311S does not seem to modify the overall architecture of the enzyme but a slight movement of the β8-strand could move the W315 residue at the surface of the enzyme and explain the decreased KM.The localization of all of the mutated residues of the tic site  and I195 binding . Indeed,antified , suggestantified . Within position . The sin8-strand . The G318-strand , which s8-strand . It is dPaMan26A-P140L/D416G mutant that contained only two mutations, i.e., one in the linker region (P140L) and one at the entrance of the active site (D416G)  out of 12 residues that may confer rigidity to the modular enzyme PaMan26A-P140L/D416G mutant, the P140 residue was substituted by a leucine residue, probably resulting in a decrease of linker rigidity that could partially explain the improved kcat/KM. The D416 amino-acid substitution is located at the edge of the catalytic cleft (One of the most remarkable findings of this study is the activity increase of the  (D416G) . The regic cleft . The favY. lipolytica. All of the selected mutants highlighted improved characteristics when compared to wild-type enzymes, thus validating the approach of mutagenesis and screening that were employed. Examination of the three-dimensional structures of PaMan5A and PaMan26A revealed several hot spots (i) around the active site of both mannanases, (ii) in the core region of PaMan5A and (iii) in the linker region of PaMan26A. The directed evolution strategy implemented in this study was very successful since a straightforward round of random mutagenesis yielded significantly improved variants that would have been difficult to predict using rational site-directed mutagenesis and structure-guided design.Activity improvement of fungal carbohydrate-active enzymes has typically met many obstacles, owing primarily to the lack of efficient expression systems and high-throughput screening methodologies. In this study, molecular engineering of two fungal mannanases was undertaken using random mutagenesis in the yeast Y. lipolytica JMY1212 strain  have been previously described Y. lipolytica JMY1212 strain was cultured in YPD  at 28°C, 130 rpm in baffled flasks or in Petri dishes containing YPD supplemented with agar (15 g/l). The plasmid used for expression in Y. lipolytica was JMP61 that displayed the POX2 promoter for induction by oleic acid Y. lipolytica genome. Subcloning of parental genes was performed using the TOPO TA kit  and TOP10 E. coli competent cells (Invitrogen).The growth media and culture conditions for the PaMan5A and PaMan26A genes  were amplified with primers listed in paman5a and the pPICZαC-paman26a plasmids BamHI and AvrII restriction sites. Y. lipolytica Zeta competent cells were prepared using the lithium acetate method as described in le Dall et alNotI-linearized recombinant plasmid combined with 25 µg of salmon sperm DNA by heat shock at 39°C for 10 minutes and immediately recovered in 1.2 ml of 100 mM lithium acetate. Transformants were plated on YNB agar  and incubated at 28°C for 48 hours.Competent cells were immediately transformed with 500 ng paman5a and PCR1dtmutCBM-F and PCR2r for paman26a. The PCR contained 500 ng of parental gene, 200 µM dNTP, 0.25 µM primers and 2.5 units of Mutazyme. The PCR were made up to 50 µl and incubated at 95°C for 5 min and then at 95°C for 30 sec, 50°C for 30 sec, 72°C for 1 min and 30 sec for 40 cycles followed by 5 min at 72°C. PCR2 was carried out using HiFi polymerase (Invitrogen) to amplify the zeta platform without introducing any mutations. Primers used were PCR1d and GH5EvDRev for paman5a and PCR1d and GH26EvDRev for paman26a. PCR products were gel-extracted using Gel-Extraction kit  and a third overlap PCR (PCR3) was subsequently carried out using gel-extracted products of PCR1 and PCR2 to rebuild the whole zeta fragment containing mutant genes. The primers used for PCR3 were PCR1dL and PCR2rL for the two constructions. The overlap PCR was carried out using i-Star Max II DNA polymerase .Three PCR were carried out for each gene as shown in Y. lipolytica as described above and plated onto YNB in QTrays plates  at a density of about 500 colonies par plate and incubated for 48 hours at 28°C.PCR3 products were subsequently transformed into Y. lipolytica transformants obtained from mutagenesis libraries were subsequently gridded on YNB agar medium containing oleic acid  and Azo-galactomannan  using a QPixII colony picker . The plates were further incubated at 28°C for 48 hours. Mannanase activity was visualized as clear halos around colonies within a blue background.The Ura-positive 540 was measured relative to a mannose standard curve (1 to 20 mM).Mannanase-positive colonies were picked on OmniTrays  containing YPD agar and incubated for 48 hours at 328°C. Further screening was performed as described in PaMan5A and PaMan26A mutant proteins, the selected genes were amplified from genomic DNA using GH5-InFuFOR and GH5-InFuR for PaMan5A and GH26CBM-InFuFOR and GH26-InFuR for PaMan26A, respectively, listed in P. pastoris and protein productions and purification were carried out as described before For heterologous production of Protein concentration was determined by using the Bio-Rad protein assay kit with bovine serum albumin as standard  and UV absorbance at 280 nm. SDS-PAGE was performed in 10% (w/v) polyacrylamide gel (Bio-Rad) using a Pharmacia LMW electrophoresis calibration kit . Native IEF was carried out at 4°C in the Bio-Rad gel system, using pI standards ranging from 4.45 to 8.2. IEF gel was coloured with IEF staining solution .−1 (eight concentrations) and incubated at 40°C for 5 minutes. Reactions were performed in triplicate independent experiments. The reaction was stopped by the addition of 300 µl of 1% DNS reagent and samples were heated at 95°C for 10 minutes. DO540 was measured relative to a mannose standard curve (0 to 20 mM). One unit of endo-mannanase activity was defined as the amount of protein that released 1 µmol of sugar monomers per min. The kinetic parameters were estimated using weighted nonlinear squares regression analysis with the Grafit software .Determination of kinetic parameters on galactomannan was performed using DNS activity assay. Unless otherwise indicated, assay mixtures contained substrate and suitably diluted enzyme in sodium acetate buffer 50 mM, pH 5. Briefly, 1 µg of enzyme was mixed with 190 µl of galactomannan  using a range of substrate concentration from 1 to 20 mg.ml5 and M6, Megazyme) were analysed using HPAEC-PAD. 10 µl of suitably-diluted enzyme were incubated at 40°C for various time lengths with 190 µl of 1 mM substrate in 50 mM acetate buffer pH 5.2. At each time point, 10 µl of reaction mixture were recovered and the reaction was terminated by the addition of 90 µl of 180 mM NaOH. For HPAEC analysis, 10 µl were injected and elution was carried out as described in Monosaccharide and oligosaccharides generated after hydrolysis of manno-oligosaccharides ; 2: PaMan26A wild-type; 3: PaMan26A-P140L/D416G; 4: PaMan5A wild-type; 5: PaMan5A-V256L/G276V/Q316H; 6: PaMan5A-W36R/I195T/V256A; 7: PaMan5A-K139R/Y223H; 8: PaMan5A-G311S.(EPS)Click here for additional data file.


Chlamydomonas reinhardtii, an organism easy to grow and transform and fully amenable to GMP guidelines.The E7 protein of the Human Papillomavirus (HPV) type 16, being involved in malignant cellular transformation, represents a key antigen for developing therapeutic vaccines against HPV-related lesions and cancers. Recombinant production of this vaccine antigen in an active form and in compliance with good manufacturing practices (GMP) plays a crucial role for developing effective vaccines. E7-based therapeutic vaccines produced in plants have been shown to be active in tumor regression and protection in pre-clinical models. However, some drawbacks of in whole-plant vaccine production encouraged us to explore the production of the E7-based therapeutic vaccine in C. reinhardtii chloroplast psbD 5′ UTR and the psbA 3′ UTR, was introduced into the C. reinhardtii chloroplast genome by homologous recombination. The protein was mostly soluble and reached 0.12% of total soluble proteins. Affinity purification was optimized and performed for both tagged forms. Induction of specific anti-E7 IgGs and E7-specific T-cell proliferation were detected in C57BL/6 mice vaccinated with total Chlamydomonas extract and with affinity-purified protein. High levels of tumor protection were achieved after challenge with a tumor cell line expressing the E7 protein.An expression cassette encoding E7GGG, a mutated, attenuated form of the E7 oncoprotein, alone or as a fusion with affinity tags (His6 or FLAG), under the control of the C. reinhardtii chloroplast is a suitable expression system for the production of the E7GGG protein, in a soluble, immunogenic form. The production in contained and sterile conditions highlights the potential of microalgae as alternative platforms for the production of vaccines for human uses.The  Plant-produced proteins represent a significant fraction of pharmaceuticals in advanced preclinical and clinical trial status Chlamydomonas reinhardtii has emerged as a model system, with its three genomes  completely sequenced E. coli, the Chlamydomonas chloroplast allows the formation of disulfide bonds and is able to perform some types of phosphorylation. In addition, it contains low protease levels as well as several molecular chaperones aiding protein folding C. reinhardtii has a single chloroplast, with about 80 genome copies. Consequently, conversion of all copies of the chloroplast genome to the recombinant form (homoplasmy) is facilitated. C. reinhardtii has been used for the expression of recombinant vaccines C. reinhardtii chloroplast demonstrates the high variability in expression levels, indicating that protein yields depend primarily on the intrinsic properties of each protein expressed C. reinhardtii for molecular pharming are still in their infancy: the first biopharmaceutical was expressed in 2003 Microalgae have been proposed as an alternative molecular pharming system. This relatively new platform offers several advantages, including: 1) short time from transformation to scaling up; 2) rapid growth (doubling time of few hours) and ease of cultivation; 3) safety, because microalgae do not harbor human pathogens, many are Generally Regarded As Safe (GRAS) organisms, and grow in axenic conditions facilitating production of biopharmaceuticals in GMP conditions; 4) homogeneity of protein production with the use of controlled bioreactors. In particular, the green unicellular alga E7 oncogene have been explored, with some cases currently in clinical trial High risk HPVs (hr-HPVs) are responsible for 6.1% of total cancer cases worldwide C. reinhardtii and demonstrate its effectiveness in protecting against cancer development in a pre-clinical model.Recombinant E7-based vaccines have been produced in plant systems psbD promoter and 5′UTR and psbA terminator and 3′UTR (expression cassette)  form of the E7 protein, named E7GGG, lacking the pRb interaction ability that causes oncogenic transformation of mammalian cells assette) . The chlassette)  was obtacw15) mutant strain of C. reinhardtii by transformation with glass beads All constructs were introduced in the cell wall-less -mediated transient expression in Nicotiana benthamiana leaves (data not shown).For each construct , 40 indeN. benthamiana by PVX-infection C. reinhardtii. Both forms produced in N. benthamiana were present at lower concentrations with respect to the E7GGG protein produced in Chlamydomonas  of 16 kDa and 18.5 kDa. The two bands were present also after the addition of 10 mM 2-mercaptoethanol and 10 mM dithiothreitol (DTT) and boiling for more than 10′  had no toxic effect on mice when injected sub-cutaneously. Therefore, groups of 8 C57BL/6 mice were immunized 5 times, at 2-week intervals, by sub-cutaneous administration of the following preparations (in the presence of 10 µg/mouse of QuilA adjuvant): (i) soluble algal extract containing the E7GGG protein (1 mg of TSP containing 1 µg of E7GGG/mouse); (ii) purified E7GGG-FLAG protein from Chlamydomonas (2 µg of protein/mouse); (iii) purified E7GGG-His6 from E. coli (2 µg of protein/mouse). As negative controls, mice were vaccinated with either buffer alone, or with C. reinhardtii extract devoid of E7GGG. Both E7GGG-His6 purified from E. coli and E7GGG-FLAG purified from Chlamydomonas induced high titers of specific IgGs after the fourth boost, while the Chlamydomonas E7GGG-containing extract showed a much lower IgG induction  immune response is generally correlated to anti-cancer activity C. reinhardtii, both in crude extracts and as purified protein, compared to the group vaccinated with E. coli E7GGG-His6  response (that represents antigen-specific cytokine mediated inflammation involving Th1 type cytokines)  extract . In the 4 cells from the TC-1 tumor cell line, expressing the E7 antigen, two weeks after the last injection Chlamydomonas crude extract containing E7GGG, as well as purified E7GGG-FLAG from Chlamydomonas and E7GGG-His6 from E. coli elicited tumor protection in 60% of mice (Chlamydomonas E7GGG-FLAG purified protein remained tumor-free for a longer time (100% tumor-free mice after 9 weeks) than the other two groups. Insignificant protection was observed in the two control groups. Taken together, our data indicate that the microalga-produced E7GGG protein is highly immunogenic, both in crude extracts and in purified form, providing effective tumor protection in a preclinical system.Tumor protection was evaluated by challenging vaccinated mice with 5×10 of mice . The groSince biopharmaceuticals have become increasingly important for the treatment of various human diseases, it is very important to improve existing expression systems as well as to develop new ones. In this context, the benefits arising from molecular pharming and, in particular, from the use of microalgae as production platforms , represent a highly attractive perspective.Chlamydomonas chloroplast in a highly soluble form, affording cancer protection in a preclinical animal model. Although the amounts of protein produced are still insufficient for clinical experimentation, the present work shows the possibility of using microalgae for the production of bio-active HPV E7 antigen. Additionally, the obtainment of soluble, purified E7 protein from microalgae offers the possibility to perform detailed biochemical and chemical/physical studies, aimed at verifying its structure and biological activity, which up to now have been performed only on proteins expressed in bacterial systems Important clinical successes have been obtained on patients with HPV16-induced precancerous lesions, treated with vaccines comprising HPV16 E6 and/or E7 proteins Animal experiments performed in this study were conducted according to the Institutional animal use guidelines and the Italian law DL 116/92. All procedures for animal experiments were approved by the Ethical Committee for Animal Experimentation of the Istituti Fisioterapici Ospitalieri (IFO) at the Regina Elena National Cancer Institute. Mice were anesthetized with 40–50 mg of Zoletil  per 1 kg of body weight and all efforts were made to minimize suffering.GLYGYG amino acid sequence instead to the native DLYCYE (amino acids 21–26) motif. The E7GGG protein was fused to two purification tags generating two variants: E7GGG-His6 (carrying the hexa-histidine tag and the thrombin cleavage site fused to the N-terminus); and E7GGG-FLAG (carrying the DYKDDDDKS FLAG tag http://genomes.urv.es/OPTIMIZER) as described E7GGG gene, the E7GGG-His6 sequence was amplified with the forward primer ATGCATCACGGTGATACGCCTACATT-3′5′-TTACAT (the NdeI site is underlined and the start codon is in bold) and the reverse primer GAATTCTAGATTAAGGTTTTTGTGAACAAATAG-3′5′-TT (the EcoRI site is underlined and the stop codon is in bold). To obtain the E7GGG-FLAG gene we created a pCG2 vector harboring the FLAG-tag (indicated as pCG2-FLAG) followed by a stop codon upstream the psbA terminator by annealing the following primers and cloning them in the NdeI and EcoRI sites of pCG2: TATGATTCGAAGATCTgactataaagatgatgacgataaatcaTAAG-3′5′- and TCTAGActgatatttctactactgctatttagtATTTTAAC-3′5′-ACTAAGCT . Subsequently, the E7GGG-His6 sequence was amplified with the same forward primer used to obtain the E7GGG gene, and the reverse primer AGATCTAGGTTTTTGTGAACAAATAGGA-3′5′- ATAA (the BglII site is underlined). The amplicon obtained was then digested with NdeI and BglII and cloned in the pCG2-FLAG vector obtaining the E7GGG-FLAG gene inserted between the psbD promoter/5′UTR and the psbA terminator/3′UTR.We used the mutated form of the HPV16 E7 protein (NP_041326) named E7GGG, firstly realized for genetic vaccination by Smahel and colleagues C. reinhardtii cell wall-less strain cw15 was grown photomixotrophically until mid-log phase  in TAP medium at 25°C under constant illumination of 120 µE m−2 s−1 on a rotary shaker. Cells were harvested by centrifugation and re-suspended in TAP medium to a final concentration of 3×108 cells/ml. Chloroplast transformation was performed as previously described 8 cells/ml) were agitated by vortexing for 20″ at 3,000 rpm in presence of 300 mg of glass beads  and 5 µg of plasmid DNA, and then spread onto TAP/agar plates containing 100 µg/ml spectinomycin. 50 transformants for each construct were propagated for 10 rounds in selective medium to obtain homoplasmic lines. For protein expression and characterization, transformants were grown in the same condition described for the cw15 strain with the addition of spectinomycin 100 µg/ml in the culture media. To verify the correct integration in the chloroplast genome and the obtainment of the homoplasmic state, total DNA was extracted from all transformants with a standard phenol/chloroform extraction protocol. Correct integration and homoplasmy were checked by amplification with the primers indicated in For chloroplast transformation, the 6C. reinhardtii cells were directly re-suspended in a suitable volume of loading buffer  and boiled for 5′ before loading on a 12% SDS-PAGE gel. To analyze the solubility of the E7GGG protein variants cells were re-suspended in 1/20 culture volume of different buffers (described in N. benthamiana extracts were prepared by grinding the tissue to a fine powder in liquid nitrogen. The powder was re-suspended and homogenized with an ultraturrax in 3 volumes (w/v) of buffer containing protease inhibitors . Soluble and insoluble proteins were separated by centrifugation for 20′ at 15,000 g at 4°C, with the resulting supernatant or insoluble pellet used in Western blot analysis. For all other experiments including characterization, purification, mice immunization, soluble proteins extracted using TS buffer  were employed. Protein concentration was estimated using the Bradford assay . For Western blot analysis, proteins were transferred onto a PVDF membrane . After blocking with nonfat milk (5% in PBS), membranes were incubated 2 hours at R.T. with a 1∶3,000 dilution of a polyclonal anti-E7 antibody . Membranes were then incubated for 1 hour at R.T. with a 1∶10,000 dilution of an anti-mouse peroxidase-conjugated secondary antibody  and the bound antibody was detected using the ECL Plus system . Protein quantification was performed by luminometry using a Chemidoc ImageLab system with ImageLab 4.0 software (Bio-Rad).For screening of transformants to identify the best expressor lines, 2×102PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0. E7GGG-FLAG protein elution was performed with 1M Arg-HCl, pH 3.5. Purified proteins were then concentrated and dialyzed against PBS+0.1 mM ZnSO4 using Amicon Ultra-4 (Millipore) dialysis tubes with a 3 kDa cut-off.Affinity purification of tagged, soluble E7GGG variants was performed in native conditions. The purification of the E7GGG-His6 protein was performed using the Ni-NTA affinity resin (Qiagen), while to purify the E7GGG-FLAG protein we used the anti-FLAG M2 Affinity gel (Sigma). In both cases, after optimization of purification conditions in small scale (100 ml of culture), we performed medium scale purifications (2–10 liters of culture) in order to accumulate protein for mice immunization. Briefly, 50 ml of soluble proteins in TS buffer extracted from 2 liters of culture were filtered (0.45 µm) and incubated with 1 ml of affinity resin for 2 hours. Resins were then washed with 200 ml of washing buffer and protein elution was obtained with 4–5 ml of elution buffer. E7GGG-His6 protein elution was performed with 50 mM NaH4) 1) 200 µl of buffer; 2) 500 µl of soluble extract from wt Chlamydomonas; 3) 500 µl of soluble extract from a Chlamydomonas transformant containing about 1 µg of the E7GGG protein); 4) 200 µl with 2 µg of purified E7GGG-FLAG protein; 5) 200 µl with 2 µg of purified E. coli E7GGG-His6 protein. Adjuvant QuilA (10 µg/mouse) was added to all vaccine preparations. One week after the first and the last boost, all animals were subjected to ELISA and spontaneous Delayed-Type Hypersensitivity (DTH) assays, and after the last boost three animals in each group were sacrificed to evaluated cell-mediated immune responses by Enzyme-Linked Immunosorbent Spot (ELISPOT) analysis. All remaining animals were then challenged by sub-cutaneous injection of 5×104 E7-expressing TC-1 tumor cells Four-week-old female C57BL/6 mice  were used. Mice were maintained under specific pathogen-free conditions at the Experimental Animal Department of the Regina Elena National Cancer Institute . Groups of 8 mice were vaccinated subcutaneously on days 0, 14, 28, 42, and 56 with the following preparations  or bicarbonate buffer . Serial dilution of sera in PBS+2% non-fat milk were added to the coated wells, followed by anti-mouse peroxidase-conjugated secondary antibody  diluted 1∶10,000. Colorimetric reaction was induced by adding 100 µl/well v/v H2O2/ABTS  . The ELISA antibody titers were calculated as the log10 of the reciprocal antibody dilution that showed an OD405 value above the cut-off value, which was defined as the average OD405 value of non-immunized sera+3 standard deviations. DTH was performed as previously described 6 cells/well), harvested from three vaccinated mice/group, was added to microtiter wells coated with a rat anti-mouse IFN-γ antibody  along with interleukin 2 . Samples were incubated with or without 1 µg/ml of E7-specific H-2Db CTL epitope  Sera of mice collected one week after the last boosts were analyzed by ELISA assay for the presence of E7-specific antibodies. Microtiter plates were coated with 100 ng/well of E7-His6 protein from Figure S1Sequence of the Chlamydomonas cassette expressing the E7GGG protein variants. The sequence of the E7GGG gene is in red, with the start and stop codons in bold. The E7GGG-His6 gene variant contains the additional sequence highlighted in blue, that comprises the His6-tag and the thrombin site. The E7GGG-FLAG gene variant contains the additional sequence highlighted in green that comprises the FLAG-tag. Restriction sites are underlined.(TIF)Click here for additional data file.Figure S2Verification of transgene integration and confirmation of homoplasmy.A. Integration scheme of the pCG2 plasmid. The integration occurs in psaA intron between nucleotides 158321–160126 (acc. NC_005353.1). The 5′ flanking region (5′ fl.) comprises nucleotides 157103–158321; the 3′ flanking region (3′ fl.) comprises nucleotides 160126–162410. Triangles of different colors joined by horizontal lines indicate primers used to verify correct integration and homoplasmy, and the relative amplicons. Amplicons 1 and 2 indicate correct integration, amplicon 3 indicates lack of homoplasmy, amplicon 4 is a positive control for the presence of chloroplast DNA. B. Sequences of the primers. C. PCR results of four representative transformants (a–d) after 10 rounds of restreaking on selective medium: the presence of amplicons 1 and 2 confirms the correct integration, the absence of amplicon 3 indicates that the lines are homoplasmic for the presence of the transgene.(TIF)Click here for additional data file.Figure S3Growth curves of the best expressors for each protein variant. Cell concentration (cells/ml) was measured at 0, 12, 24 and 48 hours. Control = transformant obtained with the pCG1 vector. Error bars represent standard deviation of three biological replicates.(TIF)Click here for additional data file.Figure S4Comparison of the solubility of the E7GGG protein produced in C. reinhardtii by chloroplast transformation or in N. benthamiana plants by PVX-mediated infection. Immunoblotting of soluble fraction (S) and insoluble pellet (I)  from Chlamydomonas and N. benthamiana extracted using the following buffers: (1) 100 mM HEPES-KOH pH 5.0, 200 mM sucrose; (2) 100 mM HEPES-KOH pH 6.0, 200 mM sucrose; (3) 100 mM Tris-HCl pH 7.0, 200 mM sucrose; (4) 100 mM Tris-HCl pH 8.0, 200 mM sucrose; 5) 100 mM Tris-HCl pH 7.0, 154 mM NaCl; (6) 100 mM Tris-HCl pH 7.0, 200 mM sucrose, 1 mM Triton X-100; (7) PBS .(TIF)Click here for additional data file.Figure S5Optimization of E7GGG-FLAG affinity purification. Western of 10 µl of the following samples: lane 1: E7GGG-FLAG extract before purification; lane 2: flow-through; lanes 3, 4: elution with 0.1 M glycine pH 3.5; lanes 5, 6: elution with 0.1 M glycine pH 2.5; lanes 7, 8: elution with 1 M Arg-HCl pH 3.5; lanes 9, 10: elution with 100 µg/ml FLAG peptide; lane 11: empty resin (negative control). e = eluted fraction r = resin after protein elution.(TIF)Click here for additional data file.Figure S6E7GGG-His6 protein characterization.A. Western blot of 20 µg of TSP extracted from the E7GGG-His6 transformant and treated as follows: lane 1: boiling for 5′ in presence of 10 mM 2-mercaptoethanol; lane 2: boiling for 10′ in presence of 10 mM 2-mercaptoethanol; lane 3: boiling for 5′ in presence of 10 mM 2-mercaptoethanol and 10 mM DTT; lane 4: boiling for 10′ in presence of 10 mM 2-mercaptoethanol and 10 mM DTT; lanes 5, 6: purified E7GGG-His6 protein from E. coli 2 and 5 ng, respectively. B. Western blot after calf intestinal phosphatase (CIP) treatment of 20 µg TSP at 37°C. Lane 1: 40 U CIP 30′; lane 2: 40 U CIP 60′; lane 3: untreated 30′; lane 4: untreated 60′.(TIF)Click here for additional data file.


Pseudomonas aeruginosa is a frequent cause of acute infections. The primary virulence factor that has been linked to clinical disease is the type III secretion system, a molecular syringe that delivers effector proteins directly into host cells. Despite the importance of type III secretion in dictating clinical outcomes and promoting disease in animal models of infections, clinical isolates often do not express the type III secretion system in vitro. Here we screened 81 clinical P. aeruginosa isolates for secretion of type III secretion system substrates by western blot. Non-expressing strains were also subjected to a functional test assaying the ability to intoxicate epithelial cells in vitro, and to survive and cause disease in a murine model of corneal infection. 26 of 81 clinical isolates were found to be type III secretion negative by western blot. 17 of these 26 non-expressing strains were tested for their ability to cause epithelial cell rounding. Of these, three isolates caused epithelial cell rounding in a type III secretion system dependent manner, and one strain was cytotoxic in a T3SS-independent manner. Five T3SS-negative isolates were also tested for their ability to cause disease in a murine model of corneal infection. Of these isolates, two strains caused severe corneal disease in a T3SS-independent manner. Interestingly, one of these strains caused significant disease (inflammation) despite being cleared. Our data therefore show that P. aeruginosa clinical isolates can cause disease in a T3SS-independent manner, demonstrating the existence of novel modifiers of clinical disease. Pseudomonas aeruginosa is a common nosocomial pathogen responsible for acute lung infections, blood stream and catheter-associated infections P. aeruginosa to evade killing by infiltrating neutrophils P. aeruginosa. To date, four effector proteins have been described in P. aeruginosa: exoenzyme S (ExoS), ExoT, ExoU and ExoY. ExoS and ExoT are heterobifunctional enzymes with an amino-terminal Rho-GAP domain and a C-terminal ADP ribosyltransferase domain P. aeruginosa demonstrated that most isolates produce ExoT. Distribution of ExoS and ExoU, on the other hand, is generally mutually exclusive Evasion of killing by neutrophils is a function of the effector proteins injected by in vitro correlates with increased morbidity, higher rates of mortality and relapse in P. aeruginosa ventilator-associated pneumonia and blood stream infections P. aeruginosa are T3SS-negative when assayed in vitroin vivo, but not in culture in vitro. Alternatively, the bacteria could have been passengers that colonized an infected host without being the primary pathogen associated with disease, or, they could be expressing other virulence factors. One report showed that eight clinical isolates from acute P. aeruginosa infections were type III secretion negative isolates, and that three displayed moderate virulence in an animal model of acute lung infection and one other strain retained a limited amount of cytotoxicity against epithelial cells. It was not determined if the observed virulence phenotypes were due to a poorly expressed type III secretion system, or due to other known or novel virulence factors Detectable secretion of type III effectors in vitro. We also examined five T3SS-negative strains and their defined T3SS- mutant derivatives for virulence in an animal model of infection. Taken together, our data indicate that strains that do not express detectable levels of T3SS effector and translocator proteins comprise a complex set of phenotypic groups, including strains that can persist and cause disease in a T3SS-independent manner.Here we characterized 81 clinical isolates from patients with infections of the urinary tract, blood stream, wound infections and acute infections of the respiratory tract. We found that 26 were T3SS negative by western blot. Since all except one of the 26 strains still had type III secretion genes on the chromosome, we generated type III secretion null mutants in 16 strains and determined if they cause cytotoxicity Clinical isolates were de-identified with only the source of the infection passed on to the investigators. The experiments were classified as non-human subject research under 45 CFR46/21 CFR 56 and HIPAA exempt by the University Hospitals Case Medical Center IRB.All animals were housed under specific pathogen-free conditions in microisolator cages and maintained according to institutional guidelines and the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. The infection protocol was approved by the University Hospitals Case Medical Center IACUC [protocol #2012–0105 (E.P.)].P. aeruginosa and E. coli strains were routinely cultured on Luria-Bertani medium (LB). E. coli strains harboring plasmids encoding a gentamicin resistance gene were cultured on LB plates with 15 µg/mL gentamicin, buffered with 10 mM NaPO4 (pH6.9). P. aeruginosa strains with an integrated plasmid conferring gentamicin resistance were cultured on LB plates with 30 µg/mL gentamicin and 5 µg/mL triclosan (Sigma). E. coli strain DH5α was used for plasmid construction and strain SM10 λpirP. aeruginosa by conjugation. P. aeruginosa strains PAO1F and 19660 have been described previously popD and pEXG2-ΔpscD have been described previously pscC1 was constructed by amplifying an internal fragment of pscC (codons 20–417) using primers intC1-5R (5′-AAAAAGAATTCGCGGACTGCTGGCGCTGCTGCCTG-3′) and intC1-3H (5′-AAAAAAAGCTTGGAAGGGTCGGCACGCCAGCCGAAG-3′) and cloning them as an EcoRI/HindIII fragment into plasmid pINT pscUL was constructed by amplifying flanking regions 3′ of genes pscU and pscL using primers pscU-3-1 (5′-AAAAAaagcttGCCCGTGGCAACGAGGAACTGGACAA-3′) and pscU-3-2 (5′-TTCAGCATGCTTGCGGCTCGAGTTACGCCCTGATCGCTATCGGGCAGGGCA-3′) and pscL-3-1 (5′-AACTCGAGCCGCAAGCATGCTGAAGGGGACGCCGGTTGAGGGAACCA-3′) and pscL-3-2 (5′-AAAAAtctagaAGGCGATGCTCGAGGAGGCCTT-3′), respectively. The two flanking regions were joined by splicing-by-overlap extension PCR  or by insertion of a non-replicating plasmid (pscC-). The plasmids were transferred to P. aeruginosa by conjugation using E. coli strain SM10 λpir, where they integrated into the targeted locus by homologous recombination. Cointegrates were selected on plates with 30 µg/ml gentamicin and 5 µg/mL triclosan (to select against the E. coli donor). In the case of the allelic exchange procedure, strains in which the plasmid had exited via a second homologous recombination event were isolated using 5% sucrose plates as a counterselection , since plasmid pEXG2 harbors a sacB gene that confers sensitivity to sucrose in the medium Null mutations of type III secretion genes were constructed by allelic exchange  and ORF1-5-2 (5′-AAAAAGAATTCGCCCAGCCAGTCCATGATCGCCA-3′)], exoU [exoU-3H (5′-AAAAAAAGCTTTCATGTGAACTCCTTATTCCGCCAAG-3′) and exoUD344A-3-1 (5′-AGTAGAGTGGACAGAATTCCAGGcaGGCGGGGTGATGATTAACGTG-3′)], exoT [exoT3-2 (5′-AAAAAAAAGCTTCTAGCCGACCCGTGTGCGGAAAAGA-3′) and exoTE383/385D-3-1 (5′-ATCGATCGAGGGCGATGATCAGGATATCCTCTACGACAAG-3′)], pscU [pscU5X (5′-AAAAAATCTAGAGGAGGAGACGCCATGAGCGCCGAGAAGA-3′) and pscU3H (5′-AAAAAAAAGCTTGATAGCGATCAGGGCGTATCCGTCTGCT-3′)], pscL [pscL5R (5′-AAAAAAGAATTCGGAGGGCGATGAATGCTTCCATTTGTT-3′) and pscL3H (5′-AAAAAAAAGCTTTCAACCGGCGTCCCCTTCCTCCT-3′)] and absence of the T3SS genes [PA1689to3 (5′-ATCGTTCATCCAGACCGCGACCAAGAG-3′) and bglXto5 (5′-AGCAGGACCCGGTCGCCAGCCTCAG-3′)].Presence of PCR was performed with Denville Taq Blue using the manufacturer’s buffer but with the addition of DMSO to 5%.P. aeruginosa strains were grown overnight in a modified LB-medium with 200 mM NaCl, 10 mM MgCl2 and 0.5 mM CaCl2 (LB-MC). On the day of the experiment bacteria were diluted 1∶300 into 3 mL LB-MC from which calcium had been removed by adding EGTA to 5 mM  and grown to mid-logarithmic phase. 1 mL of each culture was removed into a microcentrifuge tube. The bacteria were pelleted by centrifugation and 0.5 mL of the supernatant was transferred into a second microcentrifuge tube. Supernatant proteins were precipitated by adding trichloroacetic acid to a final concentration of 10% and pelleted by centrifugation. Protein pellets were washed 1x with acetone, dried and resuspended in 1x SDS sample buffer normalized for OD600. Samples were separated by SDS-PAGE and either stained for total protein using SYPRO Ruby (Invitrogen) according to the manufacturer’s instructions or transferred to PVDF membrane and probed for the presence of ExoS, ExoT, ExoU, PopB and PopD For secretion assays Antibodies against ExoS, ExoT, PopB and PopD were purified following a published protocol ∧4 A549 cells in RPMI1640 with 10% FBS). P. aeruginosa isolates were grown overnight in LB-MC. The next day, the A549 cells were washed 1x with PBS and 1 mL of fresh RPMI1640 with 10% FBS was added to each well. P. aeruginosa overnight cultures were normalized for OD600, diluted in PBS and used to infect the A549 cells at an MOI of 10. The infection was allowed to proceed for four hours, before the supernatant was removed and the cells were fixed with 3.7% formaldehyde in PBS (20′ at room temperature). Images of individual wells were taken using a Nikon Eclipse TE200 inverted scope. Rounded, intoxicated cells and flat cells were counted. Cytotoxicity was expressed as %rounded A549 cells. The experiment was repeated on three separate days and the results from these three assays were averaged.A549 lung epithelial cells were seeded in 24-well plates  and on the day of the infection back-diluted 1∶200 in tryptic soy broth and grown to an OD600 of 0.2. At this point 1 mL of cells was removed from the culture tube, the bacteria were pelleted by centrifugation, washed twice with PBS-MC  with 5 mM MgCl2 and 0.5 mM CaCl2 added) and resuspended in PBS-MC. The optical density of the washed cells was determined and adjusted to correspond to a concentration of 8×10∧7 colony forming units (CFU)/mL. 5-week-old female C57BL/6 mice were anesthetized by i.p. injection of 0.4 mL 2,2,2-tribromoethanol (1.2%) . The cornea of one eye of the anesthetized mice was scored with three parallel, 1 mm scratches using a 26-gauge needle and 2.5 µL of the bacterial inoculum (2×10∧5 CFU) was spotted onto the scratched cornea. After two hours, two of the mice were sacrificed and their eyes removed. The eyes were homogenized in 1 mL of PBS by sonication in the presence of metal beads and viable bacterial CFU were determined by dilution plating. Images of infected corneas were taken one and two days post infection using a Leica dissection scope. Two days after infection, the remaining mice were euthanized, the infected eye was removed and homogenized by sonication in the presence of beads, as above. Surviving bacterial CFU were determined by dilution plating. C57BL/6 mice were purchased from The Jackson Laboratory .P. aeruginosa express the type III secretion system (T3SS), which is important for virulence, both in animal models of infection and clinical disease. In order to study the virulence of these T3SS negative strains, we screened 81 clinical isolates from the microbiology laboratory in the Pathology department at University Hospitals Case Medial Center for secretion of T3SS-related proteins. We examined production of the main T3SS effectors, ExoS, ExoT and ExoU, as well as the translocator proteins PopB and PopD, which are required for delivery of effectors into targeted host proteins. We excluded isolates from cystic fibrosis (CF) patients, since many of these strains lose expression of the type III secretion system genes during the prolonged colonization of CF patients Previous reports had noted that not all clinical isolates of P. aeruginosa were grown in vitro under T3SS inducing conditions . Culture supernatants were then concentrated, separated by SDS-PAGE, and probed by western blot for the presence of the effectors ExoS, ExoT and ExoU, as well as the translocator proteins PopB and PopD. A representative blot is shown in exoT gene on its chromosome as indicated by PCR, but the protein was not produced. The strain producing only ExoT, on the other hand, was negative for exoS and exoU by PCR. The distribution of effectors did not correlate with any specific site of isolation, aside from the two bronchoalveolar lavage isolates that were ExoU/ExoT producing strains. Overall, our data showed a greater proportion of ExoU-producing strains than had previously been reported Clinical isolates of exoU+/exoT+, 11 exoS+/exoT+, 2 were exoU+/exoS+/exoT+ and 2 only harbored the exoT gene, suggesting that there is no significant skew in effector complement relative to the T3SS effector-positive strains.We found that 26 of the clinical isolates were T3SS negative by western blot. However, all isolates except one harbored copies of at least one of the effector genes on the chromosome as assayed by PCR. A PCR assay for the strains assayed in exoS, exoT and exoU, as well as the apparatus genes pscU and pscL, which are the outermost genes of the gene cluster encoding the secretion apparatus P. aeruginosa. One other strain of P. aeruginosa has been described in the literature that lacks the type III secretion genes, PA7 bglX to amplify across the location of the T3SS apparatus genes. The analysis confirmed that JT87 is missing the T3SS genes since amplification yielded a product that is close to the expected size of 360bp, based on the PA7 genome , arguing that this strain produces a T3SS-independent virulence factor(s).The animal experiments revealed a complexity of phenotypes. Infection with strains JT20 and JT72, which exhibited type III-dependent cytotoxicity in our  cleared . Strain t manner . MoreoveP. aeruginosa virulence in animal models, T3SS negative strains of P. aeruginosa are commonly isolated from the clinic. Here we analyzed 81 clinical P. aeruginosa isolates with respect to their production of type III secreted proteins in vitro. 55 of the strains expressed T3SS effectors, with an approximately equal number of strains producing ExoS and ExoT, and strains producing ExoU and ExoT. 26 of the strains produced no detectable ExoS, ExoT or ExoU, and did not produce the translocator proteins PopB and PopD. These secretion negative isolates displayed a range of virulence-related phenotypes, including type III secretion dependent and independent intoxication of epithelial cells in vitro, in addition to type III secretion independent disease in a corneal model of infection.Despite the prominent role that type III secretion plays in in vitro. Possible causes include a regulatory mutation that prevents expression of the type III secretion genes in vitro, suggesting that this isolate produces a new toxin.Three out of 16 of these T3SS-negative isolates were still able to deliver effectors into host cells, arguing that they encode a functional T3SS that is poorly expressed P. aeruginosa infections ranged from 0% P. aeruginosa isolates from first infected CF patients secreted T3SS proteins. The percentage declined to 29% in chronically infected children and 12% in chronically infected adult CF patients The complement of T3SS negative strains by western blot in this study was relatively high (32%) compared to previous studies, where the frequency of T3SS negative strains isolated from acute P. aeruginosa strains harbored exoS effector genes, whereas 28% harbored exoU. Presence of exoS and exoU was found to be mutually exclusive in almost all instances Analysis of effector distribution in a panel of clinical and environmental isolates found that approximately 72% of P. aeruginosa isolates studied here, or that linger once clearance of the infecting bacteria has begun, resulting in continued pro-inflammatory signaling. JT4, on the other hand, persisted in the cornea and caused disease in a T3SS-independent manner, arguing that this strain either expresses a novel virulence factor or overexpresses a known virulence factor, such as proteases or lipases that can contribute to the virulence of P. aeruginosaP. aeruginosa strain PA7, whose genome sequence was recently reported and found to be devoid of T3SS apparatus and effector genes in vitro, arguing that it encodes a novel virulence factor. Beyond promoting T3SS-independent disease, such new virulence mechanisms may also augment the virulence of type III secretion positive P. aeruginosa isolates. It has already been noted that accessory factors exist that modulate the virulence of T3SS-positive strains Three strains we analyzed, JT4, JT28 and JT87, showed novel phenotypes. Infection with JT28 resulted in significant corneal opacification, despite clearance of the infection. Since opacification is primarily related to infiltration of neutrophils, not bacterial replication P. aeruginosa can not only survive in vivo, but also cause disease. Our discovery of a strain that can cause significant inflammation despite being cleared by the immune system highlights that treatment of some infections, particular in the eye where a significant portion of the disease can be attributed to damage caused by infiltrating neutrophils, could benefit from anti-inflammatory therapy. Moreover our data suggest that new virulence mechanisms remain to be discovered that allow P. aeruginosa to persist and cause disease in a type III secretion independent manner.Taken together, our data demonstrate that type III secretion negative isolates of Figure S1PCR analysis of T3SS gene distribution. A) Presence of exoU, exoS, exoT, pscL and pscU were probed by colony PCR. B) Presence of the genes encoding the T3SS apparatus was probed using primers that bind in the flanking genes, PA1689 and bglX. Wild-type PAO1F and a mutant derivative in which the T3SS apparatus genes had been deleted (ΔpscUL) were used as controls. Presence of pscL and pscU in these strains was probed with the same ORF-specific primer set used in A). A schematic showing the organization of PA1689, bglX and the intervening T3SS-apparatus genes is shown below the gel.(TIF)Click here for additional data file.


H9N2 influenza A viruses have become established worldwide in terrestrial poultry and wild birds, and are occasionally transmitted to mammals including humans and pigs. To comprehensively elucidate the genetic and evolutionary characteristics of H9N2 influenza viruses, we performed a large-scale sequence analysis of 571 viral genomes from the NCBI Influenza Virus Resource Database, representing the spectrum of H9N2 influenza viruses isolated from 1966 to 2009. Our study provides a panoramic framework for better understanding the genesis and evolution of H9N2 influenza viruses, and for describing the history of H9N2 viruses circulating in diverse hosts. Panorama phylogenetic analysis of the eight viral gene segments revealed the complexity and diversity of H9N2 influenza viruses. The 571 H9N2 viral genomes were classified into 74 separate lineages, which had marked host and geographical differences in phylogeny. Panorama genotypical analysis also revealed that H9N2 viruses include at least 98 genotypes, which were further divided according to their HA lineages into seven series (A–G). Phylogenetic analysis of the internal genes showed that H9N2 viruses are closely related to H3, H4, H5, H7, H10, and H14 subtype influenza viruses. Our results indicate that H9N2 viruses have undergone extensive reassortments to generate multiple reassortants and genotypes, suggesting that the continued circulation of multiple genotypical H9N2 viruses throughout the world in diverse hosts has the potential to cause future influenza outbreaks in poultry and epidemics in humans. We propose a nomenclature system for identifying and unifying all lineages and genotypes of H9N2 influenza viruses in order to facilitate international communication on the evolution, ecology and epidemiology of H9N2 influenza viruses. Sixteen HA and nine NA subtype influenza A viruses have been detected in poultry and wild birds around the world During the last two decades, antigenic and genetic analyses of H9N2 isolates showed their gradual and complex evolution In this study, we performed a large-scale genomic sequence analysis of 571 H9N2 influenza viruses isolated from 1966 to 2009. Our analysis identified 74 lineages and 98 genotypes that could be further divided into seven series. Our results suggest that phylogenetic diversity and genotypical complexity exist in H9N2 influenza viruses. We provide a framework for obtaining a panoramic understanding of H9N2 viral evolution and propose a precise nomenclature system for unifying all lineages and genotypes of H9N2 influenza viruses in order to improve our ability to predict the direction of evolution by monitoring changes in the viral genome.A total of 571 complete genomic sequences of H9N2 influenza viruses isolated from 1966 to 2009 was used in this study. These H9N2 viruses, including 552 avian, 14 swine, and 5 human isolates, were obtained from multiple countries in Asia, the Middle East, Europe, Africa, and North America. The numbers of viruses distributed in different hosts and regions are shown in Eight viral gene segments of 571 H9N2 viruses were aligned and analyzed phylogenetically. The panorama view of the resulting phylogenetic trees  illustraFirstly, each of the eight gene segments from H9N2 influenza genomes could be divided into distinct lineages, and a total of 74 lineages were recognized in this study. These distinct lineages are likely the result of long-term ecological and geographical separations of the hosts. Their evolutionary relationships are shown in Secondly, the phylogenetic trees constructed for each gene segment differed in topology and in the way they classified lineages , Table 2Thirdly, these H9N2 virus lineages had marked host and geographical differences in phylogeny . In our In contrast to the BJ/1/94 lineage, the DE/113/95, HK/289/78, Hok/49/98, and HK/Y439/97 lineages were shown here to originate from aquatic birds . The DE/Fourthly, novel reassortant H9N2 viruses can be generated by exchanging gene segments from different lineages. For example, the WI/1/66 lineage clustered Canadian and United States isolates mainly from turkeys, mallard ducks, and shorebirds, and formed an independent North American cluster . NotablyFifthly, some H9N2 virus lineages are represented by single virus or only distributed in single gene segments . For exaFinally, these H9N2 viruses were closely related to H5N1, H5N3, H3N8, H4N3, H4N6, H7N7, H10N4, and H14N5 viruses in their internal gene phylogeny. To be specific, in phylogenetic trees of internal genes, HK/G1/97-like viruses (e.g. HK/1073/99 and HK/1074/99) showed a close evolutionary relationship with the 1997 Hong Kong H5N1 influenza viruses . In phylPhylogenetic analysis of the eight gene segments revealed that H9N2 viruses have undergone multiple reassortments to generate novel genotypes. With the growing accumulation of genetic information from H9N2 viruses, it is necessary to provide a systematic nomenclature for identifying viruses with different sources and gene constellations. The analyzed H9N2 viruses were divided into 98 genotypes based on genomic diversity, and their host and regional distributions are summarized in Panorama genotypic analysis revealed that seven genotypic series of H9N2 viruses differed in their host and regional distributions  and 4. TThe genotype B series containing genotypes B0–B61 was only detected in China and Japan . GenotypThe genotype C series comprising genotypes C0–C3 was only detected in Korea . GenotypThe genotype D series, containing genotypes D1–D12, was widely distributed throughout the world except for the Middle East  and 4. GThe genotype E series representing genotypes E0–E5 was found in China, Canada, and the United States . GenotypPanorama genotypic analysis also revealed that the genotypic complexity of H9N2 viruses resulted from extensive reassortments . For exaWe have analyzed all eight gene segments of 571 publicly-available H9N2 influenza A genomes and provide a panoramic framework for better understanding the genesis and evolution of H9N2 influenza viruses. In this set of 571 H9N2 genomes, we observed multiple novel mutational and reassortant events, such as deletions in NA genes and segment exchanges in internal genes, particularly in the ribonucleoprotein complex genes. After careful analysis of these changes, we have presented the first characterization of the phylogeny and genetic diversity of H9N2 viruses worldwide.Given the importance of classification of lineages for studies on viral epidemiology, evolution, and ecology Our phylogenetic maps provide a framework for describing the history of H9N2 viral circulation in diverse hosts, and also provide new information for further confirming the genetic and evolutionary characteristics of H9N2 viruses. Firstly, panorama phylogenetic analysis reveals that H9N2 viruses are closely related to H3, H4, H5, H7, H10, and H14 subtype influenza viruses in phylogenies of internal genes Our panorama genotypical analysis revealed the complexity and diversity of genotypes among the analyzed H9N2 viruses. These H9N2 viruses included 98 genotypes which were classified into seven series (A–G) representing HK/G1/97, BJ/1/94, KR/96323/96, DE/113/95, HK/289/78, HK/AF157/92, and WI/1/66 lineage viruses, respectively . Of thesOur panorama genotypical analysis also revealed that multiple reassortments of H9N2 viruses were capable of facilitating the development of multifarious genotypes and, ultimately, the emergence of novel genotypes in diverse hosts. For example, genotype B46 reassortant virus was first recognized in chickens in 2004 and became predominant in poultry, pigs, and birds during 2005–2006. These novel genotypical H9N2 reassortants are constantly evolving in diverse hosts, raising the risk of H9N2 influenza viruses to be introduced into humans.In summary, our large-scale sequence analysis of 571 H9N2 viral genomes has revealed the phylogenetic diversity and genotypic complexity of H9N2 influenza viruses worldwide. Results from our study also indicate that multifarious genotypical H9N2 viruses are continuously circulated in diverse hosts throughout the world, raising concerns over their potential role in causing future influenza outbreaks in poultry and epidemics in humans. We have proposed a precise nomenclature system for identifying and unifying all lineages and genotypes of H9N2 viruses which should facilitate international communication on the evolution, ecology and epidemiology of H9N2 influenza viruses.http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html), together with some H3, H4, H5, H6, H7, H10, and H14 subtype reference sequences. GenBank accession numbers for all analyzed sequences are listed in All influenza virus sequences used in this study were obtained from GenBank. Viral sequences that were identical, inaccurate, unclear background, or shorter than 500 bp were removed. The 571 complete H9N2 genomic sequences, collected from multiple countries between 1966 and 2009, were selected from the NCBI Influenza Virus Resource Database (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Genetic distances among the representative sequences were calculated using the model of pairwise distance calcualtion (MEGA version 4.0.2) (http://www.megasoftware.net/). 1000 bootstrap replicates were performed and phylogenetic trees were constructed for each genomic segment by the neighbor-joining method using the MEGA 4.0.2 program.Multiple nucleotide sequence alignments were performed separately on the eight viral gene segments of representative viruses using Clustal W (BioEdit version 7.0.5) , NA (B), PB2 (C), PB1 (D), PA (E), NP (F), M (G) and NS (H) genes of the 571 analyzed H9N2 influenza viruses. Genome sequences of 571 H9N2 viruses from 1966 to 2009 and other subtype reference viruses were selected from the NCBI Influenza Virus Resource Database. The following nucleotide fragments were used in the phylogenetic analysis: HA, 148 to 1230; NA, 20 to 1390; PB2, 1063 to 2246; PB1, 205 to 1428; PA, 796 to 2016; NP, 76 to 996; M, 80 to 947 and NS, 48 to 831. Neighbor-joining trees were constructed using MEGA and bootstrap values are shown for the key nodes. Clades labeled using different colors indicate major H9N2 virus lineages. Human viruses are colored scarlet, swine viruses in deep blue and avian viruses in black. Representative viruses for each lineage are underlined in the same color as clades, and are highlighted in italics together with human and swine viruses.(TIF)Click here for additional data file.Figure S2Phylogenetic characteristics of HA (A), NA (B), PB2 (C), PB1 (D), PA (E), NP (F), M (G) and NS (H) genes of H9N2 influenza viruses. 571 H9N2 viral genomic sequences from 1966 to 2009 were characterized. Neighbor-joining phylogenetic trees were generated with MEGA. Clades labeled using different colors indicate major H9N2 virus lineages.Virus abbreviations are listed in the legend of (TIF)Click here for additional data file.Table S1Gene constellations of different genotypes of the 571 H9N2 influenza A viruses.(DOC)Click here for additional data file.Table S2Accession numbers of nucleic acid sequences for the 571 H9N2 influenza A and other subtype reference viruses used in this study.(DOC)Click here for additional data file.


Antheraea pernyi (A.pernyi), we cloned a novel diapause-associated protein 3 (DAP3) gene from A.pernyi by reverse transcription-polymerase chain reaction (RT-PCR) and studied the biological functions. Sequence analysis revealed that this gene encodes 171 amino acids and has a conserved domain of Copper/Zinc superoxide dismutase (Cu/Zn-SOD). Western blot and qRT-PCR results showed that DAP3 was mainly expressed in the pupal stage, and gradually decreased as diapause development. DAP3 was also expressed in 1st and 5th instar larvae of A.pernyi. In tissues of 5th instar larvae of A.pernyi, DAP3 was mainly expressed in the epidermis, followed by the head, hemolymph and fat body. To identify the SOD activity of DAP3, we constructed a prokaryotic expression vector by inserting the coding region sequence into plasmid pET-28a (+) and obtained the purified rHIS-DAP3 fusion protein by Ni-NTA affinitive column. Importantly, we found the SOD activity of DAP3 fusion protein was approximately 0.6674 U/µg. To further confirm the SOD activity of DAP3 in vivo, we induced the oxidative stress model of pupae by UV irradiation. The results showed that both the mRNA and protein level of DAP3 significantly increased by UV irradiation. Furthermore, the SOD activity of the total lysate of pupae increased significantly at 10 min post UV irradiation and transiently returned to normal level afterwards. These results suggested that DAP3 might be a novel protein with SOD activity getting involved in regulation of diapause in A.pernyi.To better understand the molecular mechanism underlying of diapause in  A.pernyi is an insect that survive the winter season by pupal diapause. However, the detailed molecular mechanisms underlying the diapause of A.pernyi remain unknown. Diapause-associated protein (DAP) is abundant in the fat body or hemolymph in diapause insects; meanwhile, little or no DAP can be found in non-diapause insects Lepanotarsa dacemlineata and then later studied in Diatraea grandiosella, Buesseola fusca, Gastrophysa atrocyanea and Pectinophora gossypidllaDiapause is a specific physiological phenomenon in living organisms, in which growth and activity are temporarily suspended in the developmental stage. Many insects make effective use of diapause in their life cycles to overcome unfavorable seasons 2.−) and forms the first line of defense against ROS. SOD catalyzes the diamutation of O2.−∶2O2.−+2H+→H2O2+O22.−Reactive oxygen species (ROS) generated from metabolic processes attack organic macromolecules, including proteins, nucleic acids and membrane lipids. The oxidative stress not only causes injuries and pathological deterioration, but also leads to many physiological events correlated with cancer, mutagenesis, cell death, degenerative processes and aging Bombyx mori (B.mori) show that the protein is a time-dependent ATPase that may regulate the diapause duration of B.mori eggs B.mori diapause two weeks after eggs had been chilled at 5°C Studies of time interval measuring enzyme-esterase A4 (TIME-EA4) from diapause eggs of the silkworm A.pernyi diapause.In this study, we identified DAP3 (GenBank login number: AFC35302.1) as a novel Cu/Zn-SOD protein, which might play potential roles in regulation of E.coli TG1, BL21 (DE3) is kept in our laboratory. TRIzol Reagent was purchased from Ambion Company (USA). HT Superoxide Dismutase Assay Kit were products of TREVIGEN Company (USA). SYBR Green I and DNase I were purchased from Roche Company (USA).A.pernyi and different tissues of 5th instar larvae of A.pernyi including epidermis, ovary, fat body, hemolymph,midgut,malpighian tubule, trachea, silkgland.After digested by DNaseI for 30 min at 37°C,the RNA was reverse transcribed into cDNA according to the protocol for RevertAid First Strand cDNA Synthesis Kit . We designed two pairs of primer to amplify DAP3 gene  andβ-actin gene , respectively. The reaction system was as follows: 1 µL of cDNA template, 0.5 µM forward/reverse primer, 10 µL of 2×SYBR Green I Master and 7 µL of PCR grade water. The result was detected in the Light Cycler 480 with three repeats for each reaction.We used TRIzol reagent to isolate the total RNA of different developmental stages insects including diapause pupae, non-diapause pupae, pupae in the period of diapause development for different days, moth, eggs, developed eggs and 1st to 5th instar larvae of A.pernyi insects were extracted and the protein concentration was determined by BCA method Total proteins of the A. pernyi by TRIzol Reagent and the RNA integrity was detected with 1% TBE agarose gel electrophoresis. The cDNA fragment was generated using RevertAid First Strand cDNA Synthesis Kit following the manufacturer's potocol. We designed primers for PCR to obtain the open reading frame (ORF) of the DAP3 gene:DAP3-F: 5′-GGGGATCCATGCAACCGACACG-3′ and DAP3-R:5′-GAAGCTTTTACAAAATTCCGATAACACCAC. And the specific PCR procedures is as follows: initial denaturation at 94°C for 5 min; followed by 35 cycles each denaturation at 94°C for 30 s, annealing temperature at 57°C for 30 s, extension at 68°C for 40 s; with final extension at 68°C for additional 7 min.Total RNA was isolated from 50 mg fat body of BamH I and Hind III was inserted into pET-28a(+) vector digested with the same restriction enzymes by Ligation High.The production was transferred into E.coli strain TG1 to screen the postive clone. And the positive recombinant plasmid was confirmed by sequencing.The amplified fragment (492 bp) after digestion with E.coli strain BL21. A single positive colony was incubated in Luria-Bertani (LB) medium with 40 mg/L Kanamycin at 37°C untill the value of OD600 reached 0.5–0.7 and isopropylthio-β-D-galactoside (IPTG) was added to a final concentration of 1 mM for fusion protein expression. After inducing, the pelleted cells were harvested by centrifugation for 20 min at 6,000 rpm and re-suspended in PBS buffer. Subsequently, the cells were homogenized by ultrasonic machine and the fusion protein was purificated by Ni-NTA agarose. The SOD activity of the purified recombinant DAP3 was determined using HT Superoxide Dismutase Assay Kit. In this assay, O2.−, generated from the conversion of xanthine to uric acid and hydrogen peroxide (H2O2) by xanthine oxidase (XOD), convertsthe tetrazolium salt WST-1 to WST-1 formazan, which absorbs light at 450 nm. The SOD reduces O2.−concentrations and thereby lower the rate of WST-1 formazan formation. Absorbance is measured at 450 nm using a standard spectrophotometer.The recombinant plasmid was transformed into 2. After oxidative stress, the pupae were sacrificed for qRT-PCR and Western blot detection. The SOD activity of the total protein in each group was also analyzed.Fresh pupae were divided into four groups: group1was control group with its pupae were not irradiated by UV; group2, 3 and 4 were exposed to UV light for 10, 20 and 30 min, respectively. Each group had 3 pupae and the ultraviolet irradiance is 300 µW/cmThe Western blot results were quantified using ImageJ (NIH) and normalized with β-actin as loading controls. Quantitative data (mean ± SEM) were from at least 3 independent experiments. For significant analysis, oneway ANOVA was performed using Prism 5 (Graph Pad) software (*p<0.05 and **p<0.01 were considered significant difference).A.pernyi DAP3 has a full length of 572 nucleotide acids encoding a putative protein of 171 amino acid residues. The predicted molecular weight is 18.19 kDa and isoelectric point is 6.6. Importantly, an active SOD motif (SOD motif 1 and 2) is found in the protein. As shown in Sequence analysis showed that A.pernyi DAP3 was aligned with Cu/Zn-SOD of Papilio polytes (BAM19071), Aedes aegpyti (XP004535893), Bombyx mori (AB179561), Bombyx mandarina (EF077625), Hyphantria cunea (AB290453), Homo sapiens (NM_000454), Drosophila melanogaster (M24421) and Caenorhabditis elegans (P34697). The homology percent was 69%, 49%, 48%, 48%, 47.3%, 52.3%, 46.6% and 41.3%, respectively.As shown in http://swissmodel.expasy.org/) predicted the 3D structure of A. pernyi DAP3 online. Furthermore, we used software PyMOL to align A.pernyi DAP3 with B.mori TIME-EA4. As shown in SWISS-MODEL  vector which was digested with the same restriction enzymes. The positive clone was confirmed by the double digestion and agarose gel analysis  and transiently returned to normal level at 20 and 30 min  longevity and induces constitutive diapause 2O2 in B.mori revealed that the level of H2O2 was significant higher in the diapause eggs compared to non-diapause eggs, which suggests H2O2 may be involved in the termination of diapause eggs 2O2 could activate the release of diapause hormone (DH) and facilitate the progeny diapause decision by DH without the expression alteration of DH gene 2O2 can be produced by SOD and degraded by catalase enzyme (CAT). Therefore, we speculate that SOD might play indirect roles in regulation of diapause by synthesis of high level of H2O2, just like TIME-EA4 in B.mori diapause egg.Studies in A.pernyi.Conserved domain of Cu/Zn-SOD was identified in DAP3 protein and the homology of DAP3 with Cu/Zn-SOD is highly conserved –2. We al


Heterodera glycines, is an important pest of soybeans. Although resistance is available against this nematode, selection for virulent races can occur, allowing the nematode to overcome the resistance of cultivars. There are abundant field populations, however, little is known about their genetic diversity. In order to elucidate the differences between races, we investigated the transcriptional diversity within race 3 and race 4 inbred lines during their compatible interactions with the soybean host Zhonghuang 13. Six different race-enriched cDNA libraries were constructed with limited nematode samples collected from the three sedentary stages, parasitic J2, J3 and J4 female, respectively. Among 689 putative race-enriched genes isolated from the six libraries with functional annotations, 92 were validated by quantitative RT-PCR (qRT-PCR), including eight putative effector encoding genes. Further race-enriched genes were validated within race 3 and race 4 during development in soybean roots. Gene Ontology (GO) analysis of all the race-enriched genes at J3 and J4 female stages showed that most of them functioned in metabolic processes. Relative transcript level analysis of 13 selected race-enriched genes at four developmental stages showed that the differences in their expression abundance took place at either one or more developmental stages. This is the first investigation into the transcript diversity of H. glycines races throughout their sedentary stages, increasing the understanding of the genetic diversity of H. glycines.The soybean cyst nematode,  Heterodera glycines, causes significant damage to soybean, resulting in annual yield losses between 1.9 and 3.5 million tons in the USA H. glycines have been identified H. glycines have been shown to have high variability Globodera rostochiensis has led to selection for G. pallida in parts of Europe The soybean cyst nematode (SCN), H. glycines populations differing in their virulence have been carried out. RAPD-PCR was developed and employed for distinguishing two H. glycines populations with different virulence characteristics on soybean H. glycines inbred avirulent and virulent lines were generated, and comparative genomic analysis was carried out H. glycines populations rather than differences in proteomes or transcriptomes. Soluble proteins of four SCN races were characterized and a 75 kDa protein was found to be unique to race 3 and race 4 as compared to race 1 and race 2 H. glycines lines Different phytonematode populations have different reproductive capabilities and other traits. For example, clumping behavior differs among root-knot nematode strains In this study, we describe comparative transcriptome analysis between two SCN inbred lines, race 3 and race 4, at three parasitic stages, parasitic J2, J3 and J4 female, respectively. 92 race-enriched genes were identified in the two races at the three developmental stages. GO analysis of the race-enriched genes and gene expression profile analysis of 13 selected genes were also performed. Our results will increase the knowledge of the transcriptional variation between two SCN races.H. glycines inbred lines, race 3 and race 4, were propagated on conventional susceptible soybean genotype Zhonghuang 13 in a greenhouse at 28°C. To isolate different parasitic stages of H. glycines, one-week-old soybean plants were inoculated with preparasitic second stage juveniles (J2s). Infected roots were washed with sterile water to remove the uninfected nematodes from the roots 24 h post inoculation, then the infected soybeans were planted in sterile sandy soil (sand∶soil = 3∶1) Two vitro at 37°C for 12 h. Using the synthesized sense RNAs, double-stranded cDNAs were synthesized and amplified using the BD SMARTer Pico PCR cDNA Synthesis kit according to the manufacturer's recommendations . For the three life stages of H. glycines, parasitic J2s, J3s and J4 females, reciprocal Suppression Subtractive Hybridizations (SSH) were performed in both directions with the PCR-Select cDNA Subtraction Kit  according to the manufacturer's instructions. For each life stage, the forward (race 4 vs race 3) and reverse (race 3 vs race 4) subtractions were designed to select race 4-specific and race 3-specific cDNAs, respectively. In the forward subtraction, 117 ng of cDNA from race 4 was used as the tester and 572 ng of cDNA from race 3 as the driver. In the reverse subtraction, 117 ng of cDNA from race 3 was used as the tester and 572 ng of cDNA from race 4 as the driver. For each subtraction, two tester populations were created separately by ligating suppression adapter 1 or 2R to the blunt-ended RsaI-digested cDNAs. The two tester populations were mixed with a 40-fold excess of drivers (the driver cDNAs had no adaptors) in separate tubes. The cDNA fragments specific to the tester were then amplified by a primary PCR consisting of 30 cycles with PCR primer 1 and a secondary PCR of 16 cycles using nested primers 1 and 2R. The amplified cDNA fragments were purified from the PCR mixture with TIANgel Midi Purfication kit  according to the manufacturer's instructions.The collected nematodes were homogenized in 1 mL TRIzol reagent and subjected to sonication for 3 minutes Escherichia coli cells (DH5α) were used to transform the resulting constructs by electroporation and plated on SOC medium supplemented with 5-bromo-4-chloro-3-indolyl-beta-Dgalactopyranoside  , isopropyl-beta-D-thiogalactopyranoside   and ampicillin (100 µg/mL) . White colonies were picked and sequenced by BGI Tech (China). The sequences were trimmed to eliminate vector sequences, SMART-cDNA primers and SSH-adaptor sequences. The nematode cDNA sequences were then annotated for functional similarities in the GenBank database using the BLAST algorithms (http://www.ncbi.nlm.nih.gov/BLAST).The cDNA fragments generated by the forward and reverse SSH were used to construct subtraction libraries. In each case, 30 ng of purified cDNA fragments was cloned into the pGEM-T Easy vector . 20 primers  and SuperScript III Reverse Transcriptase . qRT-PCR was performed on a Applied Biosystems 7500/7500 Fast Real-Time PCR System  with 20-µL PCR mix containing 2 µL of cDNA, 200 nM of the appropriate oligonucleotide primers, 10 µL of 2× SYBR Premix Ex Taq and 0.4 µL of 50× ROX Reference Dye II . The mix was preheated at 95°C for 15 seconds, followed by 40 cycles of 95°C for 3 seconds, 60°C for 30 seconds. Melting curve analysis was performed from incubation at 65°C followed by 0.5°C incremental ramp up to 95°C. Melting curve lines were used to validate product specificity. The H. glycines glyceraldehyde-3-phosphate dehydrogenase encoding gene  was used as an internal reference −ΔΔCt method Total RNAs were isolated from preparasitic J2s, parasitic J2s, J3s and J4 females using TRIzol regent according to the manufacturer's instructions . mRNAs were amplified using the BD SMART mRNA Amplification Kit  as described above. The mRNAs were then reverse-transcribed using oligo (dT)Gene annotation was carried out using the BLAST2GO program H. glycines race 3 and race 4 at three different sedentary stages , ‘biological regulation’ (11.3%), ‘locomotion’ (9.4%), ‘response to stimulus’ (9.4%) and ‘metabolic process’ (9.4%) . For racH. glycines race 3 and race 4, respectively. Compared to that of race 4, the race 3-enriched genes, such as the cellulase gene (Hg3J2-CT16), putative gland protein G28B03 coding gene (Hg3J2-07F02), and cellulose binding protein gene (Hg4J3-11E01), had 2.3 to 2.9 fold higher expression levels at the parasitic J2 stage, while the 14-3-3 encoding gene (Hg3J4-02E05) showed 131.6 and 45.1 fold higher expression level at J3 and J4 female stages, respectively  and a putative gland protein G28B03 encoding gene (Hg3J2-07F02) . In addiH. glycines race 4 (Hg4J4-CT33) and the one isolated from H. glycines previously (GenBank accession: Q86RQ1) in variable domain II (unpublished data). The biological meaning of the sequence variation for both the CLE genes needs further research. Three genes  showed obvious different gene abundance at preparasitic J2 stage -related protein 2 coding gene (Hg4J4-CT33), putative gland protein G11A06 coding gene (Hg4J4-CT26) and glutathione S-transferase-1 coding gene (Hg4J4-09C10), had diverse expression tendencies between race 3 and race 4, and the differences occurred at J3 and J4 female stages . AccordiH. glycines race 3 and race 4 at the three different sedentary stages that representing the full diversity of parasitic stages present during nematode-host interaction et al have recently established a new method for RNA-seq analysis with strictly limited numbers of nematode gland cells, making it possible to conduct high-throughput analysis of the transcriptional diversities between two SCN races at all the sedentary stages Our data elucidated the transcriptional diversities between Table S1Primers used in qRT-PCR analysis.(DOC)Click here for additional data file.Table S2Validation of race-enriched genes within race 3 and race 4 at three parasitic stages by qRT-PCR.(DOC)Click here for additional data file.


Animal models of allergic asthma indicate that intravascular platelet activation is essentail for the development of allergen induced chronic airway inflammation. P2Y12, the third CysLT receptor, is expressed on platelets and has been an important pathophysiological role in LTE4 mediated pulmonary inflammation. We investigated platelet activation status in asthmatic patients compared to controls.Fifty asthmatic patients and 20 healthy controls were enrolled from Ajou University Hospital, Suwon, Korea Surface expression of P-selectin and P2Y12 on platelets were determined by flowc ytometry. Plasma soluble P-selectin level was measured by ELISA. The asthmatic subjects were classified into two groups depending on high (>mean + 2 SD of controls) and low expression of P2Y12.p<0.001, p=0.001, p<0.001, respectively). No significant correlations were found between clinical parameters and platelet activation markers. Expressions of P2Y12 on platelet did not increase significantly after the treatment with LTE4 or aspirin. Higher expression group of P2Y12 had significantly higher peripheral eosinophil count (P=0.021).The expressions of platelet P-selectin, P2Y12 and soluble p-selectin level were significantly higher in asthmatic patients than in controls, (Platelet activation may play a role in asthma pathogenesis. A possible interaction between platelet and eosinophil via P2Y12 was suggested.


There is a significant clinical overlap between patients with hepatocyte nuclear factor (HNF)-1A and HNF4A maturity-onset diabetes of the young (MODY), two forms of monogenic diabetes. HNF1A and HNF4A are transcription factors that control common and partly overlapping sets of target genes. We have previously shown that elevated serum pancreatic stone protein / regenerating protein A (PSP/reg1A) levels can be detected in subjects with HNF1A-MODY. In this study, we investigated whether PSP/reg is differentially regulated by HNF1A and HNF4A.Quantitative real-time PCR (qPCR) and Western blotting were used to validate gene and protein expression in cellular models of HNF1A- and HNF4A-MODY. Serum PSP/reg1A levels and high-sensitivity C-reactive protein (hsCRP) were measured by ELISA in 31 HNF1A- and 9 HNF4A-MODY subjects. The two groups were matched for age, body mass index, diabetes duration, blood pressure, lipid profile and aspirin and statin use.PSP/reg induction required HNF4A, but not HNF1A. In contrast, crp gene expression was significantly reduced by repression of HNF1A, but not HNF4A function. PSP/reg levels were significantly lower in HNF4A subjects when compared to HNF1A subjects . hsCRP levels were significantly lower in HNF1A-MODY [0.22 (0.17-0.35) mg/L] compared to HNF4A-MODY group , Parallel measurements of serum PSP/reg1A and hsCRP levels were able to discriminate HNF1A- and HNF4A-MODY subjects.Inducible repression of HNF1A and HNF4A function in INS-1 cells suggested that PSP/reg and crp, are differentially regulated by HNF1A and HNF4A, and provides clinical proof-of-concept that serum PSP/reg1A and hsCRP levels may distinguish HNF1A-MODY from HNF4A-MODY subjects.Our study demonstrates that two distinct target genes,  HNF4A are less common than mutations in HNF1A, however over 30 mutations have been identified to date  -, sex-, and age-matched family members . We wereF1A-MODY . There wGlut2, a known HNF1A   . The median and IQR of PSP/CRP ratio was unaffected. Further, we investigated the effects of mutation types. In this study, all mutations in HNF4A were missense mutations while only 4 HNF1A-MODY subjects had missense mutations with the remaining 29 subjects having truncating mutations. We found no difference between the PSP/CRP ratio in HNF1A-MODY subjects and mutation type . When the 2 patients with hsCRP >10 mg/L were excluded, one with a missense mutation and one with a truncation mutation, median PSP/CRP ratio of missense mutations was 0.025 (0.010-0.092) and of truncation mutations ratio is 0.063 (0.037-0.079). Again, we found no significant increase in the PSP/CRP ratio with more severe inactivating truncation mutations in HNF1A-MODY (P = 0.33).HNF1A-MODY subjects showed significantly higher PSP/CRP ratio than HNF4A-MODY subjects [0.06 (0.03-0.08) vs. 0.01 (0.01-0.02), crp mRNA and CRP protein levels in INS-1 cells, while crp gene and protein expression was normal in cells with a suppressed HNF4A function. Conversely, the induction of PSP/reg was inhibited by HNF4A suppression, but was not sensitive to HNF1A suppression. However it should be noted that overexpression of DN-HNF4A may also alter the activity of endogenous HNF1A.The present study provides molecular proof and preliminary clinical proof-of-concept that the combination of serum PSP/reg1A and hsCRP levels may be of clinical use in distinguishing HNF1A-MODY from HNF4A-MODY subjects. Molecular validation of these clinical findings validated that HNF1A suppression negatively regulates HNF1A mutations if there is a history of young-onset diabetes before 25 years old in at least one family member, family history of diabetes (at least two generations), in the absence of pancreatic islet autoantibodies and without the evidence of insulin resistance [HNF4A mutations is recommended when HNF1A genetic analysis does not show a mutation in individuals with clinical features of MODY or in diabetic family members with macrosomia or diazoxide-responsive neonatal hyperinsulinism [Current guidelines for the genetic diagnosis of MODY recommend to test for sistance . Testingsulinism . Neverthsulinism .1c when HbA1c levels are > 9% in type 2 diabetic subjects [1c levels between HNF1A-MODY and HNF4A-MODY subjects even though both groups had relatively low HbA1c levels. HNF1A-MODY subjects had higher HbA1c levels, yet their CRP levels were lower than HNF4A-MODY subjects. We found no association however between hsCRP and HbA1c levels in HNF1A- and HNF4A-MODY subjects. It has been previously shown that there was no association between hsCRP and HbA1c levels in HNF1A-MODY patients [1c >9% may yield a false negative result.Barriers to molecular genetic testing include low availability and high financial cost of genetic testing. The availability of biomarkers which can be used in combination with clinical characteristics will enable clinicians to better identify cases of MODY and/or prioritise DNA testing. Though DNA sequencing is the ultimate proof of a genetic mutation, which cannot currently be substituted by any other test, measurement of PSA/reg1A and hsCRP levels can be performed to help the clinicians to correctly and rapidly identify patients with either HNF1A- or HNF4A-MODY and to aim for genetic counseling. Our proof-of-concept study suggests that parallel measurements of serum PSP/reg1A and hsCRP levels may be able to discriminate HNF1A- and HNF4A-MODY subjects with high confidence. In this study, we have demonstrated that HNF4A-MODY patients showed significantly lower levels of serum PSP/reg1A and significantly higher serum hsCRP levels compared to HNF1A-MODY patients. Our findings support the previous reported value of hsCRP as a diagnostic biomarker for HNF1A-MODY vs. HNF4A-MODY -30. Howesubjects . In our patients . HoweverBecause of the routine availability of hsCRP testing, the clinical significance of hsCRP as a marker in identifying HNF1A-MODY subjects may become significant in the future. However, the combined detection of hsCRP and PSP/reg1A levels may provide an improved mean for better discrimination of HNF4A and HNF1A subjects.In our study, we could not confirm PSP/reg and hsCRP to be normally distributed, which might be due to our small sample sizes in this proof-of-concept study. We applied ROC and LDA to investigate the usefulness of hsCRP and/or PSP/reg as biomarker to distinguish HNF1A- form HNF4A-MODY. Resulting classification rules are not optimal, and need to be confirmed in larger cohorts.HNF1A and HNF4A mutation carriers has significant therapeutic implications as these subjects show sensitivity to low-dose sulphonylureas and subsequent alterations in treatment can improve glycaemic control in the majority of subjects [HNF4A gene is relatively less common than HNF1A mutations and accounts for approximately 5% of MODY cases worldwide [HNF4A mutations are associated with macrosomia, transient and persistent neonatal hypoglycaemia, later age of diabetes onset and the absence of low renal threshold [HNF4A mutation carriers as heterozygous mutations in the HNF4A gene in either parent can impact on pregnancy. The offspring of HNF4A mutation carriers has a 50% chance of inheriting the mutation from either parent and has the risk of macrosomia due to increased insulin secretion in utero [HNF4A mutation subjects is required and close monitoring during pregnancy and immediately at birth is needed to minimize complications of macrosomia and neonatal hypoglycaemia. The potential biomarkers to differentiate HNF4A-MODY from HNF1A-MODY would assist in better identification of HNF4A-MODY subjects and would enable these subjects to receive appropriate treatment and monitoring prior to and during the pregnancy to ensure successful maternal and neonatal outcome.Identification of subjects . HNF4A-Morldwide . Howeverhreshold ,10. It iin utero . Therefocrp and PSP/reg genes by HNF1A and HNF4A on a molecular level. Previous studies have demonstrated that HNF4A- and HNF1A- regulated gene expression are remarkably similar [cis-acting element located upstream of the TATA box of the HNF1A promoter that has a high-affinity-binding site for HNF4A [crp gene, and a loss of crp expression was shown to be directly linked with altered regulation of HNF1A function [crp gene expression. In contrast, we observed a strong decrease in PSP/reg gene expression in response to suppression of HNF4A function, compared to a prominent induction of PSP/reg at the gene and protein levels during suppression of HNF1A function. Our study is therefore also one of the first reports that demonstrate differentially regulated HNF1A and HNF4A target genes.Of note, our study also confirms the clinical observation of a differential regulation of  similar -23, and or HNF4A . Previoufunction ,28,46. IPSP/reg and crp, are differentially regulated by HNF1A and HNF4A. We have provided preliminary clinical proof-of-concept, and have mechanistically validated that the parallel measurements of serum PSP/reg1A and hsCRP levels in subjects with clinically suspected MODY may discriminate HNF1A-MODY from HNF4A-MODY. Our study therefore warrants clinical validation of these preliminary findings in a larger cohort of MODY subjects, as well as type 1 and type 2 diabetes patients.In conclusion, our study demonstrates that two distinct target genes, HNF1A: Hepatocyte nuclear factor-1alpha; HNF4A: Hepatocyte nuclear factor-4alpha; MODY: Maturity-onset diabetes of the young; PSP/reg: Pancreatic stone protein / regenerating protein; hsCRP: High-sensitivity C-reactive protein; DN: Dominant-negative; ELISA: Enzyme-linked immunosorbent assay; Glut-2: Glucose transporter 2; INS-1: Insulinoma cell line-1; ROC: Receiver operating characteristic (ROC); LDA: Linear discriminant analysis.The authors declare that they have no competing interests.MPK carried out recruitment and phenotyping of subjects, participated in the design of the study, statistical analysis, interpretation of data and drafting the manuscript. CB performed experiments in cellular models and participated in interpretation of data and drafting the manuscript. SB participated in recruitment and phenotyping of subjects. SMK performed experiments in cellular models and analysed data. JS performed statistical analysis and participated in manuscript drafting. RG measured PSP/reg and participated in the study design. JHMP supervised experiments in cellular models, participated in the design of the study, interpretation of data and manuscript drafting. MMB conceived of the study, participated in its design and coordination, supervised the study and drafted the manuscript. All authors read and approved the final manuscript.Receiver operating characteristic (ROC) analysis testing all observed protein levels as potential thresholds to distinguish HNF1A- from HNF4A-MODY. A) Analysis of all subjects indicates ratio of PSP/CRP as the best marker based on the highest area under the curve . HNF1A-MODY is predicted with sensitivity of 79% and specificity of 89% when PSP/CRP ratio >0.03 (threshold 3). CRP reaches AUC = 0.79 (blue curve) and predicts HNF1A with sensitivity 73% and specificity 89% when using threshold 2. PSP shows an AUC of 0.76 and high sensitivity of 90% with specificity of 67% when threshold 1 is used to distinguish HNF1A- from HNF4A-MODY. B) Excluding the two subjects with extreme CRP levels, ROC analysis shows similar results but for higher sensitivity. The combination of both proteins shows highest AUC = 0.88 with sensitivity for HNF1A of 84% and specificity to predict HNF4A of 89%. CRP reaches AUC = 0.84 and sensitivity = 77% with specificity = 89% at threshold 2. PSP shows an AUC of 0.75 with high sensitivity of 90% and specificity 67% when PSP > 9.34 ng/ml predicts HNF1A-MODY.Click here for filePerformance of PSP/reg1A and hsCRP as individual or combined classifiers to distinguish HNF1A- from HNF4A-MODY.Click here for fileLinear discriminant analysis (LDA) identifies thresholds to discriminate HNF1A- from HNF4A-MODY. Double lines on vertical axes represent disruption and change of scale. A) Analysis of all subjects  results in the best prediction quality for the combination of both markers. Predicting HNF1A when PSP/CRP ratio > 0.04 has a sensitivity of 64% and a specificity of 89%. Discriminating based on CRP alone reaches 89% to predict HNF4A but only 12% for HNF1A, using a CRP level of 3 mg/l as threshold. A threshold of PSP > 13 ng/ml achieves prediction of HNF1A with sensitivity 45% and specificity 78%. B) LDA of the data set excluding the two subjects with extreme CRP levels, finds similar results for analysis but for CRP as marker. As in A), combination performs best with specificity 89% and sensitivity 65% when PSP/CRP ratio >0.04 is predicted as HNF1A. CRP < 1.25 mg/l correctly predicts 90% of HNF1A subjects, but at the same time only 33% of HNF4A. Using PSP < 12.5 ng/ml as threshold discriminates 45% of HNF1A correctly from 78% of HNF4A subjects.Click here for file