Within these structures oligomeric intermediates are considered to be particularly pathogenic. To test this hypothesis we have used a conformational targeting approach where particular conformational states, such as oligomers or fibrils, are recognized Drosophila melanogaster. The effect of KW1 is observed to occur selectively with flies expressing Aβ(1–40) and not with those expressing Aβ(1–42) or the arctic variant of Aβ(1–42) This finding is consistent with the binding preference of KW1 for Aβ(1–40) oligomers that has been established in vitro. Strikingly, and in contrast to the previously demonstrated in vitro ability of this antibody fragment to block oligomeric toxicity in long-term potentiation measurements, KW1 promotes toxicity in the flies rather than preventing it. This result shows the crucial importance of the environment in determining the influence of antibody binding on the nature and consequences of the protein misfolding and aggregation.We show that oligomer targeting with the KW1 antibody fragment, but not fibril targeting with the B10 antibody fragment, affects toxicity in Aβ-expressing While our data support to the pathological relevance of oligomers, they highlight the issues to be addressed when developing inhibitory strategies that aim to neutralize these states by means of antagonistic binding agents. At the end of these cycles we applied a final extension step of 5 min at 72°C. The polymerase chain reaction products were separated on an agarose gel and photographically imaged. Primers used to amplify B10 and KW1 cDNA were designed based on the coding sequences of KW1 and B10 genes. Primers used to amplify Aβ cDNA were designed based on the coding sequences of Aβ transgene. Amplified rp49 cDNA served as a loading control. See Additional fileMagnetic protein A coupled beads (Invitrogen) were pretreated by blocking 10 μl of the resuspended beads in 100 μl 2% BSA in PBS + 0.025% TritonX-100. After incubation for 15 min, the beads were washed twice in 100 μl PBS + 0.025% TritonX-100 for 5 min. 3 μg of the dissolved antibody (6E10) were added to 100 μl PBS +0.025% TritonX-100 and incubated with the beads for 15 min to allow coupling of the antibodies to the beads. After this pre-treatment, 10 to 20 fly heads were homogenized with a plastic pistil in 20 μl PBST containing the cOmplete protease inhibitor mix. The suspension was sonicated for 1 min, centrifuged for 7 sec and the supernatant was transferred into a fresh tube. This solution was then diluted with 80 μl PBS + 0.025% TritonX-100, mixed with the pre- treated beads and incubated for 20 min. The beads were washed thrice with 100 μl PBS + 0.025% TritonX-100 (5 min) and transferred into a fresh tube. Specifically bound proteins were eluted from the beads with 20 μl 50 mM glycine (pH 2.8) and incubation for 10 min. Afterwards the beads were boiled in 20 μl 1× NuPAGE® LDS Sample Buffer (Invitrogen) and applied onto the gel to check for unspecifically bound proteins. All steps were performed at room temperature, unless indicated otherwise. The resulting fractions were analysed using WB. Anti-myc antibody was used to detect the expressed myc-tagged B10 antibody fragment, while 6E10 was used against Aβ.To test for possible interactions between KW1 and Aβ peptide, a modified protocol had to be used, because fly KW1 (unlike fly B10) was found to directly bind to protein A beads. Therefore, the bead pre-treatment consisted only of the blocking of 10 μl resuspended beads , incubation for 15 min and two washing steps as described above. After this pre-treatment, 10 to 20 fly heads were prepared as described above and mixed with the pre-treated beads. The further steps were carried out as described above. Anti-myc antibody was used to detect the expressed myc-tagged KW1 antibody fragment via WB, while 6E10 was used against Aβ. All steps were carried out at room temperature.Thioflavin T (ThT) and 8-naphthalene-1-sulfonate (ANS) spectra were recorded at room temperature using the LS 55 fluorescence spectrometer (Perkin Elmer). All spectra represent averages of 5 scans. ThT fluorescence was excited at 450 nm and the emission spectrum was recorded between 460 and 700 nm. All ThT spectra were recorded with samples containing 15 μM ThT and 20 μM Aβ. ANS emission spectra were measured from 380 to 700 nm, whilst exciting at 374 nm. In this measurement samples contained 200 mM ANS and 20 μM Aβ peptide.1–20 μg from each Aβ peptide species were blotted onto nitrocellulose membrane  using pore sizes of 0.1 μm or 0.45 μm. The membrane was blocked for 1 h with 2% bovine serum albumin (BSA) solution in TBST, which is Tris- buffered saline , containing 0.01% Tween 20. Equal loading was confirmed with Ponceau red staining of unblocked control membranes. Membranes were washed thrice in TBST for 5 min, before they were further incubated for 1 h with 4 μg/ml B10AP or KW1AP in TBST. B10AP or KW1AP binding was visualized with NBT/BCIP reagent (Pierce). All steps were carried out at room temperature. Densitometric quantifications of the scanned blots were carried out with TotalLab 100 software.CR absorption was measured at room temperature using the Lambda 900 spectrometer (Perkin Elmer). The samples contained 15 μM CR with or without 25 μM Aβ peptide. Absorbance spectra were recorded from 400 to 700 nm with 3 scans per spectrum. CR absorption spectra of Aβ fibrils or buffer control always contained 50 mM sodium borate buffer, pH 9.0, while Aβ oligomers or controls were measured in 10% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP).−1 cm−1. From the DMSO stock aliquots were diluted with buffer and other reagents to yield the final sample. These samples contained always a volume of 100 μl, 25 μM Aβ(1–40), 20 μM ThT, 50 mM HEPES buffer (pH 7.4), 50 mM NaCl, a protease inhibitor cocktail  (1×) and, where appropriate, 5 μM KW1. The DMSO concentration was always less than 5%.Aggregation kinetic measurments are based on time-resolved ThT fluorescence measurements, carried out online in a 96-well plate and by using a FLUOstar OPTIMA (BMG Labtech) plate reader (37°C). ThT fluorescence was recorded by using excitation and emission wavelengths of 482 nm and 490 nm, respectively. Each measurement cycle consisted of 30 min incubation followed by orbital shaking at 100 rpm for 10 seconds immediately before the measurement. Samples were prepared by initially disaggregating Aβ(1–40) peptide as described. The disTEM specimens of Aβ oligomers and fibrils were prepared by placing 5 μl of each sample solution onto a Formvar/carbon copper grid  followed by 1 min of incubation. The grid was washed by dipping it subsequently into 3 droplets of water (~50 μl) and the specimen were counterstained with 3 droplets (~50 μl) of 2% (wt/vol) uranyl acetate. Samples were examined using a Zeiss 900 electron microscope that was operated at an acceleration voltage of 80 kV. Samples were imaged at a magnification of 30,000 × .3, 1.2 mM KH2PO4, 4.9 mM KCl, 2.5 mM CaCl2, 2 mM MgSO4, 10 mM glucose).For measuring the influence of various Aβ species on LTP isolated hippocampal slices (400 μm thickness) were prepared from 4-months old C57/B16 mice as described previously. The sliWe prepared three different Aβ-containing samples by incubation of 100 μM Aβ with or without 20 μM KW1 in 50 mM HEPES buffer (pH 7.4) and 50 mM NaCl for 5 days at 37°C without shaking. The sample incubated without KW1 was divided into two parts. One was applied to the slice as it was, to the other one we added KW1 15 min before addition to the slices. Sample solutions containing 1 μM Aβ were applied to the slices in the pre-chamber for 2 hours. The respective control solution contained no Aβ. The slices were then transferred into a submerge-type recording chamber and were allowed to recover for at least 30 min before starting the electrophysiological experiments. The recording chamber was constantly perfused with ACFS at a rate of 2.5 ml/min at 32 ± 1°C.Synaptic responses were elicited by stimulation of the Schaffer collateral commission fibers in the stratum radiatum (CA1 region) using lacquer-coated stainless steel stimulating electrodes. Glass electrodes  were placed in the apical dendritic layer to record fEPSPs. The initial slope of the fEPSP was used as a measure of this potential. The stimulus strength of the test pulses was adjusted to 30% of the EPSP maximum. During baseline recording, single stimuli were applied every minute (0.0166 Hz) and were averaged every 5 min. Once a stable baseline had been established, long-term potentiation was induced by applying 100 pulses at an interval of 10 ms and a width of the single pulses of 0.2 ms (strong tetanus) three times at 10 min intervals.KW1 and B10 crystal structures were displayed as ribbon diagrams by using the program PyMOL (DeLano Scientific). The structures have the following protein database identification numbers: 3LN9 (B10) and 3TPK (KW1),31.E. coli and purified according to established procedures[B10AP and KW1AP were recombinantly expressed in ocedures,30.2. Cells were seeded into 96-well plates at a density of 50 000 cells/well in 100 μl cell culture medium and grown at 37°C. After 24 h the medium was removed and fresh medium was added together with the Aβ samples or controls to be analyzed.SH-SY5Y cells were grown in Dulbecco’s Modified Eagle Medium  supplemented with 10% heat-inactivated fetal bovine serum and 2% Pen/Strep (PAA Laboratories) at 37°C with 10% COThe Aβ samples were obtained by initially dissolving disaggregated Aβ at high concentration in 100% DMSO. The peptide was then quantified and diluted to a final concentration of 100 μM into 50 mM HEPES buffer pH 7.4, containing 50 mM NaCl. If applicable, the solution also contained 20 μM B10 or KW1, and it was incubated for different periods of time at 37°C. After incubation the Aβ-containing analytes were added to the cells to reach a final concentration of 1 μM Aβ peptide. The cells were then incubated for 24 h at 37°C with the analytes before measuring the cellular effects with one of two assays.Cell metabolic activity was assessed with a FLUOstar Omega 96-well plate reader (BMG LABTECH) by using the Cell Proliferation Kit I  or LDH-Cytotoxicity Assay Kit II (BioVision) according to the manufacturer’s protocol. Statistical analyses were carried out using the paired t-test implemented with SigmaPlot11 (Systat software).Drosophila melanogaster flies within the central nervous system using the elavc115-Gal4 driver[Drosophila expressing B10 or KW1 alone under the control of the elavc155Gal4 system confirmed that both proteins were stably expressed in KW1 and B10 flies. The two proteins relatively inert properties, as they do not discernibly affect the fly phenotype when expressed in isolation, as determined by locomotive behaviour , Aβ1-42) or the early onset AD-associated E22G mutant Aβ(1–42)arc to investigate KW1’s effects on Aβ neurotoxicity. The resulting fly lines are termed KW1; Aβ40, KW1;Aβ42 and KW1; Aβ42arc. Whilst expression of Aβ(1-40) or KW1 alone do not cause significant neurotoxicity in  or the ein vitro data that KW1 preferentially interacts with Aβ(1-40) oligomers [Concerning B10, there was also no evidence that insufficient concentrations or a complete lack of interactions might explain the absence of effects of B10 expression on the flies. The ratio B10 to Aβ(1-42)-arc present in the fly brain (1:3) . Co-IP fin vitro work is that KW1 is produced in the brain of the fly concomitantly with Aβ(1-40) peptide and is present during fibril or oligomer formation  we find a significantly perturbed fibrillation pathway -transgenic animals was highly surprising, given that it had previously been shown to antagonize Aβ(1-40) oligomer induced neurotoxicity Figure A,B, as m Figure C. Indeed Figure C. KW1 re Figure D,E, simi Figure D,E.The non-fibrillar aggregates formed by KW1 co-incubation with disaggregated Aβ(1–40) expose more surface hydrophobicity, as measured by increased binding of the dye 8-anilino-1-naphthalenesulfonic acid (ANS), than the more fibrillar aggregates formed in the absence of KW1 Figure F. They ain vivo can be rationalised by the ability of KW1 to promote the formation of hydrophobic non-fibrillar Aβ(1–40) aggregates. We applied different Aβ(1-40) preparations to the slice for 2 hours prior to electrophysiology recordings. We found that a 2 hour pre-incubation of the slices with 5-day old non-fibrillar Aβ(1–40) aggregates formed in the presence of KW1, prior to their addition to the culture, significantly inhibits LTP compared to untreated cultures /KW1 preparations to human neuroblastoma (SH-SY5Y) cells and measurement of their effects on neuronal metabolic activity with the 3--2,5-diphenyltetrazolium bromide (MTT) assay. Co-incubation of Aβ(1-40) with KW1 throughout the aggregation reaction generated aggregates that modulate the activity SH-SY5Y cells and induce a marked ~12% reduction of the MTT value Figure A, whereain vivo  assay, which measures membrane disruption and thus reports more directly on cellular toxicity than MTT, shows no significant effects of Aβ on SH-SY5Y cells Figure C, we fin Figure E. Therefin vivo with an artificial Affibody binding protein  is able to interfere with Aβ toxicity in vivo[in vivo. Whilst the fibril-binding B10 antibody fragment is unable to perturb the neurotoxicity associated with any of the forms of Aβ studied here, the oligomer-specific KW1 antibody fragment shows a potent and highly specific effect and alters toxicity in the fly model. This observation is consistent with the view that oligomeric intermediates are more toxic than mature fibrils.In this study we show that conformational targeting is a suitable approach to probe the process of Aβ aggregation and its neurotoxic consequences in an intact living organism, the fruit fly. Previous research already established that targeting of Aβ monomers y in vivo and so wUnexpectedly, KW1 promotes rather than neutralizes toxicity. This finding is in sharp contrast to many previous studies, which report oligomer-binding conformational ligands to antagonize the detrimental effects of oligomers Figure A,B, presin vitro by allowing Aβ(1–40) peptide to aggregate in the presence of KW1, we obtain a result that is now fully consistent with the fly data and demonstrates an increased Aβ activity in both LTP  peptide for prolonged periods of time. If we remodel this situation  Figure H and MTT Figure A. The me Figure C. This e Figure E. Indeed Figure E and ANS Figure F. Moreovin vitro observation that KW1 preferentially interacts with Aβ(1–40)-derived oligomers compared with Aβ(1–42) or Aβ(1–42)-arc-derived intermediates -expressing flies and mirrors the a priori, and the same protein, KW1, can either prevent or enhance the neurotoxicity of Aβ(1-40) depending on the time point in the aggregation process at which it is added. We believe that this effect could be in particular a problem of approaches that solely aim to target oligomers in order to antagonize their activity by binding. In the case of proteins with additional activities, such as full-blown antibodies with functional Fc-parts, however, these effects could be overruled as they may alert the immune system to induce the specific destruction of the bound ligand. This explains why oligomer-specific antibodies may be functional in vivo[These data have significance for the design of Aβ-modifying therapeutic strategies for the treatment of Alzheimer’s disease. First, if Aβ-dependent AD pathology does not only depend on a single Aβ state, strategies to bind and to neutralize Aβ peptide more generally or to simultaneously prevent formation of multiple peptide assembly states may be preferable for achieving efficient therapeutic design rather than targeting a single, though highly toxic, molecular state. Second, the biological activity of state-specific binders might be highly context-dependent, as may not only block the binding of oligomers to their cellular receptors, but also modulate the peptide self-assembly reaction. Therefore, the biological consequences of a binder are difficult to predict l in vivo,55.in vivo. By combining these studies with in vitro analysis of the biophysical properties of the antibody fragments and their interactions with Aβ, it is possible to provide significant insights into the molecular basis of the pathogenesis of conformational diseases and perhaps to generate successful strategies, or to detect potential pitfalls, when devising therapeutic solutions to these devastating diseases.Understanding the kinetics and underlying mechanisms of Aβ aggregation in the brains or patients suffering from AD, and the balance of these process with those that facilitate degradation and clearance of aggregates, is crucial for maximising the efficacy of therapeutic strategies based on the modulation of Aβ aggregation. Conformational targeting with appropriately designed antibody fragments is not only a way to determine the types of aggregates that are present in the intact nervous system of a living animal, but also to analyse the functional effects arising from specific perturbations of the mechanisms of Aβ peptide aggregation All authors declare that there are no competing interests.JW, RR, MW, MW and LML carried out research, KGR, CMD, UH, DCC, LML and MF designed research, JW, MW, LML. and MF wrote the paper. All authors read and approved the final manuscript.Figure S2. B10AP and KW1AP do not cross-react with Drosophila proteins. Figure S3. Drosophila Schneider S2 cells are able to express functional KW1 and B10 antibody fragments. Figure S4. Phenotypic effects of KW1 and B10 expression on Aβ-transgenic flies. Figure S5. B10 interacts with Aβ peptide in tissue samples. Figure S6. Concentrations of Aβ, B10 and KW1 in fly head homogenates.  Amino acid sequences of the expressed polypeptide chains. Click here for file


V. cundinamarcensis latex, (P1G10), display healing activity in animal models following various types of lesions. P1G10 or the purified isoforms act as mitogens on fibroblast and epithelial cells by stimulating angiogenesis and wound healing in gastric and cutaneous ulcers models. Based on evidence that plant proteinases act as antitumorals, we verified this effect on a murine melanoma model. The antitumoral effect analyzed mice survival and tumor development after subcutaneous administration of P1G10 into C57BL/6J mice bearing B16F1 low metastatic melanoma. Possible factors involved in the antitumoral action were assessed, i.e., cytotoxicity, cell adhesion and apoptosis in vitro, haemoglobin (Hb), vascular endothelial growth factor (VEGF), tumor growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α) content and N-acetyl-glucosaminidase (NAG) activity. We observed that P1G10 inhibited angiogenesis measured by the decline of Hb and VEGF within the tumor, and TGF-β displayed a non-significant increase and TNF-α showed a minor non-significant reduction. On the other hand, there was an increase in NAG activity. In treated B16F1 cells, apoptosis was induced along with decreased cell binding to extracellular matrix components (ECM) and anchorage, without impairing viability.The proteolytic enzymes from  However, it became apparent that tumor-killing drugs also target normal cells. Moreover, because of the inherent genetic instability of neoplastic cells, exposure to chemotherapy eventually results in selection of drug-resistant clones [i.e., angiogenicity, loss of adhesion and/or migration and tumor antigenicity.During the last decades, a direct strategy has dominated the field of cancer therapy; the selected target was the tumor cell itself and any cytotoxic drug that destroyed tumor cells t clones . To avoivβ3, αvβ5, α4β1, and α5β1), are expressed in tumor cells and its microenvironment, modulating tumor initiation, progression, and angiogenesis [The significance of angiogenesis and metastasis for tumor development are well established and malignant cells in the melanoma microenvironment produce large numbers of angiogenic stimulators that enhance endothelial cell proliferation and migration of vascular endothelial growth factor (VEGF), and fibroblast growth factor FGF-2) leading to vessel growth  leading . In addiCarica candamarcensis (V. cundinamarcensis), a member of Caricaceae family and common to many areas of South America. This fraction contains 14 proteinase isoforms, involved in plant protection against predators [V. cundinamarcensis display five to seven-fold higher activity compared to proteolytic enzymes from C. papaya, a probable consequence of an adaptive response to its habitat. The fraction induces skin epithelization after wound healing caused by dermoabrasions [P1G10 is a proteolytic fraction from redators . Interesbrasions , burns [brasions , and gasbrasions . P1G10 abrasions . In mostbrasions  and no mbrasions . In suppbrasions ,13 demonSeveral studies link the use of proteolytic enzymes to oncotherapy, ,15,16. MThis evidence prompted us to evaluate the possible antitumoral effect of P1G10 using a B16F1 melanoma syngeneic model in C57BL/6J mice and likely mediators involved in its action.In preliminary studies we characterized the chromatographic, electrophoretic profile and amidase activity of the P1G10 fraction. Herein, we analyze by mass spectroscopy the size of the antitumoral proteolytic fraction. One major peak of 25,481.6 Da and two minor peaks of 23,775.2 and 22,140.2 Da are shown in . The smap < 0.05), and 5 mg/kg  P1G10, relative to the control group (ANOVA Bonferroni post-test), as shown in The experimental design to analyze the antitumoral activity involved three animal conditions: the control only injected with vehicle or, P1G10 at 1 or 5 mg/kg. The antitumor activity was evaluated on day 15th following treatment with P1G10, which initiated on day four, after B16F1 cell inoculation. The tumoral mass showed significant reduction in groups treated with 1 mg/kg , reduced the number of lung metastatic nodules in mice [Prior studies demonstrated that plant proteinases suppressed tumor growth, invasion and lung metastasis of murine melanoma . Bromela in mice . As seen in mice ,23,24. W in mice ,25. On t in mice .catk/mK between 57,407 and 375 M−1·s−1 while the various bromelain fractions show catk/mK between 0.00975 and 0.00038 M−1·s−1; in other words the catalytic efficiency of P1G10 is at least 4 × 104 higher than bromelain [Using PFLNA as substrate the enzymes composing P1G10 display romelain . In summp < 0.01, Student’s t test). However, similar VEGF levels were found in serum samples from tumor treated animals relative to untreated tumor bearing controls , following P1G10 treatment (The effect of P1G10 (5 mg/kg) on cell and/or angiogenic markers  was evaluated in tumor tissue, but, VEGF was also measured in serum samples from tumor bearing animals. controls C. In addreatment F.99mTc. B16F1 melanoma-bearing mice were divided into three-interval groups; 5th, 10th and 16th days, treated with 5 mg/kg P1G10 daily, and with the last treatment, each group received a similar dose containing 99mTc-P1G10, as well. The radioactive uptake in each tumor was corrected by variations of the total radioactivity received by each animal. The distribution of 99mTc-P1G10 was expressed as a partition coefficient (Kp) between tumor and blood. The Kp values were corrected by the mass variation among tumors. p < 0.05) and 16th days  relative to day five, ANOVA, Bonferroni’s post-test. To account for possible changes in blood and organ distribution of P1G10 influenced by tumor, separate control experiments were conducted to assess the distribution of 99mTc-P1G10 in normal and tumor bearing animals. The results of these experiments confirmed that there were no changes in blood and organ distribution between normal and tumor bearing animals (results not shown).The distribution of P1G10 in tumoral tissue during treatment was evaluated in the presence of P1G10 previously labelled with w/v) induced angiogenic parameters in dorsal sponge-discs implanted in a non-tumor rodent model [in vivo distribution experiments with 99mTc labelled P1G10. 99mTc-P1G10 within the tumor between 5 and 15 days, suggesting an antiangiogenic effect. Regardless of this effect, the biodistribution of P1G10 in non-tumoral tissues was similar in non-tumor and tumor bearing animals (data not shown).In previous studies, it was demonstrated that 0.1% P1G10  non-significant increase compared to the tumor control without P1G10. The TGF-β role is unclear; some evidence assigns a tumor growth suppressive function in certain malignancies, while others link the increase in TGF-β expression following gene transfection to angiogenesis in prostate carcinoma ,33. It sThe minor non-significant decrease of tumoral TNF-α D is an uin vitro and in vivo [Among cells infiltrating tumor stroma, macrophages are of particular importance ,39 by se in vivo , while l in vivo .50) by P1G10 was similar in normal  and tumoral cell lines . These values ranged between 12.7 and 28.7 µg/mL and the lowest IC50 corresponded to tumoral cells.As shown in p < 0.01) or 24 h exposure , compared to the control  after 4 h . Loss of adhesion at 25 µg/mL P1G10 became significant at 2 h (32%) and later at 24 and 48 h, it became similar to the higher dose (92%) . There was no difference in viability of P1G10 treated cells in suspension compared to the untreated group, suggesting that cell competence was not affected (data not shown).The effect of P1G10 on cell adhesion was initially studied on polystyrene plates. p < 0.01 and p < 0.001, respectively, ANOVA, Bonferroni’s post-test). The effect was stronger for laminin and collagen I. Cell adhesion to collagen I became evident at 1 µg/mL of P1G10, while binding to collagen IV was significant at ≥10 µg/mL .The ability of B16F1 cells to adhere onto different extracellular matrix components (ECM) after exposure to P1G10 was studied using Millicoat Screening kit . Cells were exposed to 100 µg/mL P1G10 and then seeded onto plates coated with collagen I, collagen IV, fibronectin, laminin or vitronectin. The attached cells were stained with crystal violet and quantified by spectrophotometry at 540 nm. 4β1 integrins to collagen I, which is the main component of ECM mediating melanoma cell attachment [In many stages during tumor progression the interactions between cell–surface–adhesion molecules and ECM, and/or cell–cell interaction suffer changes ,45. In Btachment ,47,48. Ttachment ,50,51. Ntachment . It is lC. papaya papain at 10–20 mg/mL induced apoptosis in Jurkat cultured cells [The relationship between loss of cell adhesion and apoptosis observed with P1G10 in ed cells .Male C57BL/6J mice (18–25 g), obtained from the Centro de Bioterismo (CEBIO/ICB/UFMG), were housed at 22 ± 2 °C under a 12/12 h light/dark cycle with free access to food and water were used. The animals were acclimatized to the laboratory environment for at least 24 h before assays. All animal handling and experimental procedures were conducted with prior approval by the Ethics Committee in Animal Experimentation at the Federal University of Minas Gerais (Process n.103/2007).l-cysteine, 5 mM DTT and 10 mM EDTA pH 5.0 in 1 M sodium acetate solution. The suspension was centrifuged at 4000× g 10 min at room temperature and the filtered supernatant  was chromatographed through Sephadex G-10 (25 mm × 400 mm) previously equilibrated with 1 M sodium acetate pH 5.0 at room temperature. The first protein fraction (P1G10) containing the bulk of proteolytic activity was concentrated by ultrafiltration  and stored at −20 °C until use. The recovered sample was standardized by its proteolytic activity, SDS-PAGE electrophoresis and HPLC.The isolation of P1G10 was described previously . BrieflyMatrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) was used to characterize the molecular mass of proteolytic components present in P1G10. Samples (25 µg) were mixed with α-cyano-4-hydroxy-cinnamic acid (matrix) dissolved in 0.1 g/100 mL trifluoroacetic acid and processed in a UltrafleXtrem MALDI-TOF mass spectrometer , using Protein Calib Standart 2  as standard.2 atmosphere.The B16F1 cell line, low-metastatic melanoma, was a gift from the Instituto Ludwig de Pesquisa sobre o Câncer ; CHO (Chinese ovary hamster) and BHK 21 (Baby hamster kidney) cells were gifts from the Pan-American FMD Center, Rio de Janeiro, Brazil. CIPs  and Ehrlich cell lines were courtesy of Helena Nader, Biochemistry and Immunology, Federal University of São Paulo, Brazil and Geovane Cassali, Pathology, Federal University of Minas Gerais, respectively. The 4T1 cell line was obtained from the American Tissue and Cell Collection, USA. B16F1, 4T1, Ehrlich, CHO and BHK21 cells were grown in RPMI medium  and CIPs were grown in F12-HAM (GIBCO). The media were supplemented with 10% heat-inactivated fetal bovine serum (FBS) (GIBCO), 1% penicillin/streptomycin (GIBCO) and cultured at 37 °C in a humidified 5% CO5 cells/100 µL) were subcutaneously (s.c.) inoculated into the right flank of C57BL/6J anesthetized mice, as recommended by the NCI Division of Cancer Treatment and Diagnosis (DCTD), USA for evaluation of new antineoplastic agents. The cervical s.c. injection of P1G10 started four days after tumor cell inoculation.A murine melanoma model was established according to Zuberek and co-workers . Brieflys.c. daily treated with P1G10 (1 or 5 mg/kg) or saline (control). After 15-days treatment, mice were sacrificed in a CO2 chamber and the tumoral mass measured.Animals bearing B16F1 melanoma were s.c., daily, or 5-FU 20 mg/kg, i.p. on days one, five and 10. After treatment, the animals were kept under a dark/light cycle with food and water ad libitum. The deaths were scored daily during the fifteen days after treatment was completed.The animal survival was evaluated as described by Baez and coworkers . Animals99mTc-labeled P1G10, according to Lemos and coworkers [99mTc-P1G10 (5.4 × 106 cpm/mg protein) 5 mg/kg. The mice were sacrificed at different intervals . The radioactive distribution in tumor, blood and specific organs was measured in a γ-counter .The evaluation of P1G10 levels in the tumor was done with oworkers . The assv/v) Triton X-100 was added to suspend the sediment, mixed briefly and centrifuged for 10 min at 3000 rpm, 4 °C. One-hundred µL of the supernatant was added to 100 µL of p-Nitrophenyl-N-acetyl-β-d-glucosaminide dissolved in 0.1 M citrate/sodium phosphate buffer, pH 4.5 in a 96-well plate and incubated for 10 min at 37 °C, and 100 µL 0.2 M glycine, pH 10.6 buffer was added to stop the reaction. The enzyme activity was determined spectrophotometrically at 405 nm using an ELISA reader and expressed as OD/mg tumor [These experiments were carried out according to Plunkett and Hailey . To 100 g, 4 °C and the supernatant recovered for cytokine assay. Cytokines  were determined by immunoassay using anti-mice DuoSet®, R&D Immunoassay Kit , as described by Teixeira and coworkers [Sample supernatant (100 µL) obtained as described above, or serum (150 µL) were mixed with 500 µL PBS pH 7.4 plus 0.05% Tween-20 and centrifuged for 30 min at 12,000× oworkers .3 cells/well), were incubated with different concentrations (0.01–100 µg/mL) of sterile P1G10 for 72 h. At the end of this period, 10 µL of 5 mg/mL MTT  was added onto each well and incubated at 37 °C with gentle stirring for 4 h. The cells were then lysed with dimethyl sulfoxide (DMSO) and the absorbance determined at 540 nm using an ELISA reader.The cytotoxic effect of P1G10 cells was evaluated by the MTT -dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide) assay . Briefly2 humidified atmosphere. To evaluate the role of caspases on DNA fragmentation, 40 μM ZVAD-FMK were added to the culture 40 min before P1G10. A total of 2 × 105 cells detached from plates were suspended in 0.3 mL hypotonic fluorochrome solution containing 50 µg/mL propidium iodide and 0.1% Triton X-100 in 0.1% sodium citrate. After 2–4 h incubation at 4 °C in the dark nuclei fluorescence was measured in a FACS flow cytometer .DNA fragmentation as indicator of apoptosis was assessed by cell cycle analysis of total DNA content, as described by Nicoletti and coworkers , with sl3 cells/100 µL/well in uncoated plates and incubated at 37 °C in 5% CO2 humidified atmosphere. After 24 h, cells were exposed to P1G10  or control solution (RPMI 1640 plus 10% FBS) for up to 48 h at 37 °C in 5% CO2 humidified atmosphere. Cells that remained attached were rinsed twice with PBS and 100 µL of 10 µg/mL resazurin dissolved in RPMI 1640 supplemented with 10% FBS, was added.Adhesion to polystyrene plates. B16F1 cells were seeded at a density of 2 × 10® . A suspension of B16F1 cells at 5 × 105 cells/mL was mechanically dissociated and exposed to P1G10 (1 to 100 μg/mL) or RPMI 1640 plus 10% FBS (control) for 2 h. Then, 100 µL of cell suspension were seeded onto ECM coated plates and incubated at 37 °C in 5% CO2 humidified atmosphere for 1 h. The adherent cells were stained with crystal violet as recommended by the manufacturer and quantified at A540 with an ELISA reader. The results were expressed as percentage adhesion of treated cells relative to untreated control cells (assigned 100% adhesion) after blank subtraction.Adhesion to plates coated with extracellular matrix components (ECM), CytoMatrix™ Cell Adhesion Strips, ChemiconThese findings show that the antitumoral effect on murine B16F1 melanoma by P1G10 results in tumor mass reduction and survival increase similar to that observed with 5-FU. We hypothesize that these findings are linked to reduced angiogenesis resulting from decreased VEGF. It is unclear whether these effects arise as consequence of the loss of adhesion and or apoptosis observed. Finding a group of proteinases that induce epithelization of normal tissue and at the same time inhibit tumoral growth tissue is unique, and may be used to probe pathways relevant to cell regeneration and tumorigenesis.


Enterovirus 71 (EV71) is major cause of hand, foot and mouth disease. Large epidemics of EV71 infection have been recently reported in the Asian-Pacific region. Currently, no vaccine is available to prevent EV71 infection.E. coli resulted in the formation of chimeric virus-like particles (VLPs). Mice immunized with the chimeric VLPs elicited anti-VP4N20 antibody response. In vitro microneutralization experiments showed that anti-chimeric VLPs sera were able to neutralize not only EV71 of genotype C4 but also EV71 of genotype A. Neonatal mice model confirmed the neutralizing ability of anti-chimeric VLPs sera. Eiptope mapping led to the identification of a “core sequence” responsible for antibody recognition within the peptide.The peptide (VP4N20) consisting of the first 20 amino acids at the N-terminal of VP4 of EV71 genotype C4 were fused to hepatitis B core (HBcAg) protein. Expression of fusion proteins in in vitro. The “core sequence” of EV71-VP4 is highly conserved across EV71 genotypes. The chimeric VLPs have a great potential to be a novel vaccine candidate with a broad cross-protection against different EV71 genotypes.Immunization of chimeric VLPs is able to elicit antibodies displaying a broad neutralizing activity against different genotypes of EV71  Picornaviridae family. The virion is around 30 nm in diameter containing a single-stranded positive-sense RNA genome of approximately 7500 nucleotides [Human enterovirus 71 is a non-enveloped RNA virus of the leotides . VP4 is leotides . CrystalEV71 and coxsackievirus A16 (CA16) have been identified as the two major etiological agents of hand, foot and mouth disease (HFMD) ,9. LargeThe evidence that maternal mice vaccinated with the EV71 virus-like particles (VLPs) can confer protection to neonatal mice against lethal challenge reveals an essential role of neutralizing antibody in the protection against infection . To deteE. coli. The resulting fusion proteins were able to spontaneously assemble into chimeric VLPs, which elicited virus-neutralizing antibody response. We further identified a highly conserved linear neutralizing epitope in the N-terminus of EV71 VP4 by epitope mapping experiments. Our results suggest that chimeric HBcAg particles carrying a neutralizing epitope of EV71 VP4 could be a promising vaccine candidate against EV71 infection.In the present study, the peptide consisting of N-terminal residues 1–20 of EV71 VP4 of genotype C4 was fused to hepatitis B core antigen (HBcAg) and expressed in E. coli, respectively.The gene sequence and amino acid sequence of peptide VP4N20 as well as its insertion position in HBcAg are shown in Figure in vivo and in vitro, we then investigated whether the recombinant proteins can form particles. Electron microscopy analysis showed that both HBc-N149 and HBc-N149-VP4N20 proteins were able to efficiently form particles with the size around 25–30 nm  or HBcAg VLPs (HBc-N149) and received booster injections 3 weeks later. Mice immunized with PBS were used as negative controls. The immunized animals were bled at week 0, 2, 5, 8 for serological analysis. The results showed that anti-VP4N20 antibody became detectable in chimeric VLPs-immunized mice at 2 weeks after inoculation. The titers were enhanced by booster injection and reached a maximum at week 5. No anti-VP4N20 antibody response was detected in the HBcAg VLPs -immunized group and the PBS group. Our results indicated that chimeric particles were able to induce anti-VP4N20 immune responses  and a variant of the prototype strain of EV71, BrCr-TR (genotype A) were used for in vitro neutralization assay. As shown in Figure 2 was obtained at week 5 post-immunization  were inoculated intraperitoneally (i.p.) with the virus-sera mixtures that had been incubated overnight at 37°C. After 7 days, control mice receiving EV71 with either PBS or anti- HBcAg VLPs sera started to show symptoms, such as reduced mobility, limb weakness, limb paralysis, and death  was fused to HBcAg protein. HBcAg particles have been extensively exploited as a carrier to improve the immunogenicity of foreign protein segments presented on their surface. As expected, the fusion proteins were able to assemble into chimeric VLPs in bacteria as efficient as unmodified HBcAg. Immunization of the chimeric VLPs was able to elicit VP4N20 specific antibody in mice. in vitro[Neutralizing antibodies play an important role in the immune defense against picornavirus infection. In the case of poliovirus, antibodies raised against VP4 and the N termini of VP1 of poliovirus serotype I were capable of neutralizing the poliovirus virions ,37. Simiin vitro. VP4 plain vitro, indicatin vitro,41. The in vitro. The expin vitro,42. FurtIn summary, this study identified an immunodominant epitope located at the N-terminal of EV71 VP4 protein. The fusion proteins of HBcAg and N-terminal of EV71 VP4-derived peptide were able to spontaneously assemble into chimeric VLPs. Mice immunization with these chimeric VLPs elicited neutralizing antibodies against EV71 of different genotypes. The “core sequence” responsible for immune stimulation was found to be highly conserved across different EV71 genotypes.XhoI and NdeI and subsequently inserted into the vector pET22b(+) . DNA fragment encoding HBc-N149 was amplified by using the primers P1u, P2d (5′-TGGGCAGCAATCTGGAAGATCCGGCGAGCCGCGAACTG-3′), P2u (5′- ACCAGTTCGCGGCTCGCCGGATCTTCCAGATTGCTGCCCA-3′) and P1d by using HBc-N149-VP4N20-encoding gene as a template and further inserted into the vector pET22b (+). The accuracy of the constructs was confirmed by sequencing. E. coli strain BL21 (DE3)  were used for protein expression.The peptide (VP4N20) that corresponds to first 20 residues at the N-terminal of VP4 of EV71 (Bj08) was inserted to HBcAg (HBc-N149) loop region between amino acids 78 and 79. The fusion protein was named as HBc-N149-VP4N20. To construct the plasmid expressing the fusion protein, DNA fragment encoding HBc-N149-VP4N20 was synthesized and amplified using primers P1u (5'- CCGCTCGAGCACCACGGTGGTT-3') and P1d (5'- GGAATTCCATATGGATATTGATCCGTATAAAG-3'). The PCR products were double-digested by 600 of 0.4-0.6. Protein expression was then induced by 0.1 mM of isopropyl-β-d-thiogalactopyranoside (IPTG). After shaking at 37°C for 5 h, the bacteria were collected by centrifugation at 12,000 rpm for 10 min at 4°C, and the pellets were resuspended in 100 ml of balance buffer . For protein purification, the bacterial cells were lysed by ultrasonication, followed by centrifugation at 13,000 rpm for 15 min at 4°C to remove bacterial debris. The clear supernatant was applied to a Ni Sepharose column  according to the manufacturer’s recommendations. The columns were washed with washing buffer  and bound proteins were eluted with elution buffer . The peak fractions were collected and analyzed by SDS-PAGE. The purity of the samples was determined by densitometric scanning. The proteins were dialyzed to PBS buffer (pH7.4) for 8 hours at RT and examined by electron microscopy and used for immunology studies.Overnight cultures of BL21 (DE3) cells harboring the recombinant plasmids were diluted 1:400 in 1 L of LB broth containing 100 μg/ml ampicillin, and grown until reaching an ODElectrophoresis was performed in 12% SDS polyacrylamide gels and the recombinant proteins were detected by Western blotting using a monoclonal antibody (mAb) against the polyhistidine (His) tag in the C-terminal region of the fusion protein. Briefly, the transferred PVDF membrane was blocked with 2% (w/v) BSA in TBS for 1 h at 37°C, and washed thrice with TBS – 0.05% (v/v) Tween 20, then the membrane was incubated with a 1:5,000 dilution of anti-His tag  in a 0.2% BSA-TBS – 0.05% Tween 20 solution for 1 h at 37°C, and washed thrice with TBS – 0.05% Tween 20. Protein bands were probed with 1:2,000 dilution of HRP-conjugated goat anti-mouse IgG  and washed thrice as described above. Chemiluminescence was applied as instructed by the manufacturer .The formation of HBcAg VLPs and chimeric VLPs (HBc-N149-VP4N20) was analyzed by negative staining electron microscopy according a previously described method . BrieflyPathogen-free female BALB/c mice were purchased from Beijing HFK Bioscience Co. . All animals were housed at pathogen-free conditions. Animal experiments were performed in accordance with current guidelines for the Care and Use of Laboratory Animals of Experimental Animal Center of Military Medical Sciences and approved by the center. For mice experiments, five female BALB/c mice (6–8 weeks) per group were vaccinated intramuscularly (i.m.) with recombinant proteins HBc-N149 (5 μg/mouse) or HBc-N149-VP4N20 (5 μg/mouse) at week 0. The second injection was performed at week 3. QuickAntibody™ from KBQ Biotechnology Co.  was used as an adjuvant. Control group was immunized with PBS plus adjuvant. The immunized animals were bled at week 0, 2, 5, 8 for antibody detection.2CO3–NaHCO3, pH 9.6) overnight at 4°C. After washing with PBS-0.05% (v/v) Tween 20 thrice, the plates were blocked with 2% (w/v) BSA in PBS for 2 h at 37°C. Sera were tested at 2-fold serial dilutions starting at 1:100. The plates were incubated at 37°C for 1 h and washed thrice with PBS-0.05% Tween 20. HRP-conjugated goat anti-mouse IgG  was then added into each well in a 1:2000 dilution, and incubated at 37°C for 1 h. The plates were washed thrice and developed with TMB solution  in a dark room for 15 min, and the enzyme reaction was stopped by adding 2 M H2SO4. The absorbance at 450 nm was measured using a microplate reader .Direct ELISA was used for detection of antibodies in the sera of immunized animals. The peptide VP4N20 was synthesized by Scilight-Peptide  and conjugated with Bull Serum Albumin (BSA-VP4N20). The peptides were purified using high-pressure liquid chromatography. ELISA plates (96-well) were coated with 250 ng/well of BSA-VP4N20 in coating buffer (50 mM NaEnzyme-linked immunosorbent assay (ELISA) protocols were used for all the epitope mapping experiments. Peptides truncated at the carboxyl end or the amino terminus were purchased from Scilight-Peptide . The peptides were conjugated with Bull Serum Albumin (BSA). The purity was higher than 90%.50, and incubated overnight at 37°C. Next, 100 μl of cell suspension containing 10,000 RD cells was added to wells containing the virus/antiserum mixtures and incubated at 37°C. After 7 days, the cells were observed to evaluate the appearance of cytopathic effects. Neutralization titer was defined as the highest serum dilution that could completely protect cells from developing cytopathic effects.EV71 BJ08 (genogroup C4) and BrCr-TR (genogroup A), were propagated in RD cells. Virus titers were determined using RD cells by the microtitration method and expressed as the 50% tissue culture infective dose (TCID50) according to the Reed–Muench method. Two-fold serial dilutions of sera were prepared using Minimum Essential Medium  containing 2% FBS. The EV71 stock was diluted to a working concentration of 100 TCID50/50 μl. The neutralization assay was conducted using 96-well plates. In each well, 50 μl of diluted serum sample was mixed with 50 μl of EV71 at 100 TCIDin vivo infection experiments were performed. Briefly, 50 μl of sera or PBS were incubated with 10 LD50 of EV71 BrCr-TR (50 μl in sterile RD cell supernatant) at 37°C for 2 hour. Groups of one-day-old BALB/c suckling mice (n = 10 per group) were inoculated intraperitoneally (i.p.) with the virus-sera mixture or virus-PBS mixture. All mice were monitored daily for clinical symptoms and death for up to 16 days after inoculation.To evaluate protective efficacy of the immunized sera against EV71 infection, The authors declare that they have no competing interests.The authors’ contributions to this research work are reflected in the order shown. MZ contributed to the majority of experimental works and the writing of the manuscript. YB and WL carried out protein expression and purification. YH and XX participated in virus preparation and their characterization. SC participated in in vivo neutralization assay. WS and XS directed the research, designed and coordinated the project, analyzed the data, and wrote the manuscript. PC and YZ conceived the study and participated in its design. All authors read and approved the final manuscript.


These mutations produce conformational changes that reveal novel antibody binding epitopes. A monoclonal antibody, clone C4F6 - raised against the ALS variant G93A of SOD1, has been identified as specifically recognizing a conformation shared by many ALS mutants of SOD1. Attempts to determine whether non-mutant SOD1 adopts a C4F6-reactive conformation in spinal tissues of sporadic ALS (sALS) patients has produced inconsistent results. To define the epitope recognized by C4F6, we tested its binding to a panel of recombinant ALS-SOD1 proteins expressed in cultured cells, producing data to suggest that the C4F6 epitope minimally contains amino acids 90–93, which are normally folded into a tight hairpin loop. Multiple van der Waals interactions between the 90–93 loop and a loop formed by amino acids 37–42, particularly a leucine at position 38, form a stable structure termed the β-plug. Based on published modeling predictions, we suggest that the binding of C4F6 to multiple ALS mutants of SOD1 occurs when the local structure within the β-plug, including the loop at 90–93, is destabilized. In using the antibody to stain tissues from transgenic mice or humans, the specificity of the antibody for ALS mutant SOD1 was influenced by antigen retrieval protocols. Using conditions that showed the best discrimination between normal and misfolded mutant SOD1 in cell and mouse models, we could find no obvious difference in C4F6 reactivity to spinal motor neurons between sALS and controls tissues.Greater than 160 missense mutations in copper-zinc superoxide dismutase-1 (SOD1) can cause amyotrophic lateral sclerosis (ALS)The online version of this article (doi: 10.1186/2051-5960-2-55) contains supplementary material, which is available to authorized users. One of the first dominantly-inherited fALS-associated genes to be identified was the SOD1 gene; mutations in SOD1 account for 10-20% of all fALS cases. There are now more than 160 missense mutations within this gene that have been described in ALS patients {http://alsod.iop.kcl.ac.uk/}. On the basis of studies in various animal models, it is thought that the mutations in SOD1 cause a gain of toxic properties to produce the progressive paralytic symptoms observed in fALS patients. Importantly, the symptoms and CNS pathology observed in patients harboring SOD1 mutations are very similar to those observed in non-inherited forms of disease, suggesting that there could be related mechanisms of pathogenesis.Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of upper and lower motor neurons. Approximately 90% of the cases are sporadic (sALS) in origin whereas 10% are familial (fALS) and caused by mutations identified in more than 10 different genes {The toxic properties of mutated SOD1 are thought to arise from mutation-induced conformational changes leading to SOD1 misfolding and aggregation. Wild-type SOD1 (WT) can acquire some of the same properties as mutant SOD1 when oxidized and stripped of metal cofactors; these preparations have also been shown to be toxic when administered to cells . Indeed,The anti-hSOD1 antibody, C4F6, which has been widely used in studies to identify misfolded SOD1, was reported to show strong immunoreactivity to denatured G93A, significant reactivity (but much lower) to other hSOD1 mutants, and very low reactivity to denatured WT hSOD1 . WT hSODThe only information currently available on the nature of the epitope is that it is located in exon 4 of the hSOD1 protein , which cAll the strains of transgenic mice used in this study have been previously described: WT hSOD1 over-expressing mice used here were the B6SJL-Tg(SOD1)2Gur/J hybrid line , G93A hSTissues from sALS cases were collected with patient consent and handled under protocols approved by the Johns Hopkins Institutional Review Board. All samples were coded and de-identified. Spinal cord tissues from a total of 25 sALS cases and 5 non-disease controls were made available for analysis  analysis. Animals were anesthetized with isoflurane and perfused transcardially with 20 ml of phosphate-buffered saline followed by 20 ml of 4% paraformaldehyde. Spinal cords were immediately removed and placed in 4% paraformaldehyde for 24–48 hours at 4°C prior to paraffin processing.2O2 in PBS following multiple PBS washes for the sections that underwent antigen retrieval. Following blocking of non-specific staining with 10% normal goat serum in PBS containing 0.5% Tween-20 (PBST), sections were incubated overnight at 4°C with either the C4F6 antibody  at a 1:500 dilution or the SEDI antibody (kind gift from Janice Robertson) at a 1:250 dilution in PBST with 3% normal goat serum. The sections were then incubated with a biotinylated secondary anti-mouse antibody  diluted 1:500 in PBST with 3% normal goat serum followed by incubation with the ABC-horseradish peroxidase staining kit . Sections were developed using 0.05% w/v 3,3’-diaminobenzidine  in PBS containing 0.0015% H2O2 and counterstained with hematoxylin. Images were taken using an Olympus BX60 microscope.To characterize the pathologic features of human sALS tissues, 10 μm sections were immunostained with ubiquitin (Rabbit anti-ubiquitin (1:500) from DAKO, CA), which revealed skein-like inclusions, cystatin-C (Rabbit anti-cystatin C (1:100) from Millipore/Upstate Biotech, Catalog #ABC20., Billerica, MA), which revealed Bunina-like inclusions, or by H&E staining, which revealed Lewy- body-like inclusions and Bunina body inclusions. All staining of human tissues used a steam/citrate buffer antigen retrieval protocol. Immunohistochemistry on mouse tissues was performed on 5–7 μm sections. Sections were deparaffinized and either left in water, incubated in 95% formic acid for 20 minutes followed by overnight incubation in 6 M guanidine hydrochloride at room temperature, or steamed in 10 mM citrate buffer with 0.05% Tween 20, pH 6.0 for 30 minutes. Sections were blocked of endogenous peroxidases by immersion in 0.3% HTissue sections were stained with periodic acid Schiff by first oxidizing them in 0.5% periodic acid  solution in water for 5 minutes. Following a rinse in distilled water sections were placed in Schiff’s reagent  for 15 minutes. Sections were washed in warm tap water for 5 minutes and then counterstained in hematoxylin for 1 minute and washed again in water for 5 minutes. Slides were then dehydrated and coverslipped.WT and mutant hSOD1 untagged proteins were expressed from plasmids based on the mammalian pEF-BOS expression vector, and have been previously described , 22–26. Transfection of cells for ICC was performed on glass coverslips that were previously coated with 0.5 mg/ml poly-L-lysine in 1× phosphate buffered saline solution (PBS). A total of 0.8 μg of vector DNA was transfected per well, using Lipofectamine 2000 . For biochemical analyses, a total of 4 μg of vector DNA was used to transfect cells in 60 mm poly-L-lysine coated dishes . Each transfection experiment was repeated a minimum of 3 times.After rinsing the cells 3 times in 1× PBS, transfected cells were fixed with 4% paraformaldehyde in 1× PBS for 15 minutes. Cells were then permeabilized using ice-cold 100% methanol for 5 minutes followed by incubation in 20% normal goat serum in 1× PBS. Immunostaining was then performed with hSOD1 (1:1000) and C4F6 (1:1000) antibodies in 1× PBS with 10% normal goat serum and incubation overnight at 4°C. Cells were then incubated for 1 hour with secondary antibodies  Alexafluor goat anti-rabbit 568 for hSOD and Alexafluor goat anti-mouse 568 for C4F6 diluted 1:2000 in 1× PBS with 10% normal goat serum. Cells were treated with 4’,6-diamidino-2-phenylindole, dihydrochloride, stock 14.3 mM (DAPI)  diluted 1:2000 in 1× PBS for 10 minutes to visualize nuclei. Fluorescence was visualized on an epifluorescence Olympus BS60 microscope.Recombinant hSOD1 proteins were expressed and purified as previously described .Recombinant hSOD1 proteins (1.5 μg), as indicated in figure legends, were spotted onto a nitrocellulose membranes. The proteins were allowed to dry on the membrane for 30 minutes at 25°C and then the membrane was submerged in PBS for 5 minutes. The membranes were then incubated in 4% paraformaldehyde for 30 minutes followed by 5 washes in PBS for 5 minutes each. The membranes were then treated with varying conditions: 1) submerged in 6 M guanidine hydrochloride for 30 minutes or 2) submerged in 10 mM citrate buffer with 0.05% Tween 20, pH 6.0 and steamed for 30 minutes. After each treatment membranes were again washed in PBS 5 times for 5 minutes each. Odyssey blocking buffer  was then used to block the membranes for 1 hour. Immunostaining was performed by incubating the membranes in a solution of C4F6 (1:1000) and hSOD (1:2500) antibodies diluted together in Odyssey blocking buffer with 0.1% Tween 20 at 4°C overnight. Membranes were rinsed in PBST 5 times for 5 minutes each and then incubated in a solution containing both the IRDye 680RD goat anti-mouse and IRDye 800CW goat anti-rabbit antibodies  diluted in Odyssey blocking buffer containing 0.1% Tween 20 and 0.01% sodium dodecyl sulfate for 45 minutes at room temperature. Membranes were then rinsed 4 times in PBS-T for 5 minutes each, 1 time in PBS for 5 minutes, and then imaged using the Odyssey Infrared Imaging Systems . Densitometric analysis was performed using Odyssey software version 3.0.Following transient transfection for 48 hours with SOD1 constructs as described above, cells were harvested in PBS with 1:100 v/v protease inhibitor cocktail . The cells were then lysed by sonicating the samples two times for 15 seconds each before low speed centrifugation (~800 × g) for 10 minutes. Protein concentrations of the supernatant were then determined by bicinchoninic acid assay as described by the manufacturer . Various protein concentrations, as indicated in figure legends, were boiled for 5 minutes in Laemmli sample buffer with β-mercaptoethanol and electrophoresed in 18% Tris-Glycine gels . Following transfer, membranes were blocked in Odyssey blocking buffer (Odyssey) and subsequent processing and imaging using the Odyssey Infrared Imaging Systems (LI-COR) was performed as described for the recombinant dot-blots.The conformations (rotamers) adopted by the side chains of residues E40, D90, K91, D92, and G93 for WT SOD1 were visualized by superimposing 7 structures of 32 subunits that are available in the protein data bank (PDB)  as explained in figure legends.From previous characterization of the C4F6 antibody, it had been established that it does not recognize mouse SOD1 . In the To further demonstrate the importance of this amino acid, we analyzed immunoblots of cell lysates containing the WT, G93A, and D90A proteins separated by denaturing SDS-PAGE using the hSOD antibody for assessing protein loading and the C4F6 antibody to determine relative affinity Figure . Three dth and 6th beta sheets of hSOD1 and are in contact with the loop between the 3rd and 4th beta-sheets  show that residues 90–93 are generally found in a similar conformation as observed in WT SOD1. Thus, in comparing the WT and G93A variants in the region that contains a critical component of the epitope there is not an obvious conformational signature that we see as providing specificity for C4F6 binding. The C4F6 antibody was raised against recombinant apo G93A protein, the structure of which was included in the structures we analyzed and which completely aligns with that of metallated G93A protein. Thus, the conformational element caused by the G93A mutation that enables C4F6 binding does not appear to be due to a stable change in local structure around the 90–93 loop.In studies of WT and mutant SOD1 expressed in cultured cells, our studies reported here, and previously , indicatIn regard to the conformational specificity of C4F6, our data indicate that the condition in which we have the greatest confidence that non-native conformation of the mutant SOD1 is driving reactivity is when used for immunocytochemistry in cell models. In our studies of cells over-expressing human SOD1 encoding ALS mutations other than G93A , the antTo determine the utility of the antibody in fixed tissues and the effect of common antigen retrieval procedures on binding, we examined C4F6 reactivity to spinal cords from mice overexpressing WT and mutant human SOD1. Treatments that we assumed would denature SOD1, treatment in formic acid and 6 M GdnHCl or heat treatment in citrate buffer, resulted in a significant increase in C4F6 staining in spinal cords from all SOD1 variants tested. Similarly, we observed increased C4F6 reactivity to purified SOD1 spotted onto nitrocellulose membrane after heat/citrate treatment. At least for the case in which heat/citrate treatment were used, we have confidence that some of the increased reactivity of the antibody to tissue is due to direct effects on the protein rather than facilitating better access of the antibody to its epitope. Because of the much greater affinity of the C4F6 antibody for the G93A protein, we cannot be sure whether the immunoreactivity detected in these mice is indicative of conformational change. Notably, in contrast to non-transgenic mice and mice expressing high levels of WT hSOD1, untreated spinal cords of paralyzed mice expressing the G37R and L126Z mutants showed at least some C4F6 immunoreactivity throughout the grey matter; either in the form of diffuse neuropil reactivity in the G37R mice or as discrete puncta in the L126Z mice. In all three mutant mouse models, antigen-retrieval treatments significantly increased the intensity of reactivity and, in the L126Z mice, elaborated fibrillar inclusions. However, these treatments also increased reactivity in the WT hSOD1 mice and thus we attribute C4F6 reactivity in this case to relaxation of the WT SOD1 structure to loosen the tight bend in the backbone around amino acids 90–93. Thus, although antigen retrieval methods do appear to enhance C4F6 binding to accumulations of SOD1 in pathologic structures, indicating the utility of the antibody, users should note that antigen retrieval methods that could denature SOD1 may produce C4F6 reactivity that is not necessarily indicative of the conformational state that existed in living tissue.The C4F6 monoclonal antibody is one of several that have been used that were generated by immunizing mice with human G93A SOD1 , 40. To As described in the Introduction, there have been two prior studies of whether spinal motor neurons from cases of sALS show specific immunoreactivity to C4F6 antibody , 15. OneAdditional file 1: Table S1: Human Tissue Characteristics. (DOCX 20 KB)Additional file 2: Table S2: Details for SOD1 structural analysis. (DOCX 15 KB)Scale bar 100 μm. (PDF 207 KB)Additional file 3: Figure S1: Antigen-retrieval impacts SEDI immunoreactivity to hSOD1 in transgenic mouse spinal cord tissues. Tissue sections from mice overexpressing WT, G93A, and G37R, or non-transgenic mice for controls, were stained with SEDI following either no antigen retrieval or formic acid and 6M guanidine hydrochloride (FA & GdnHCl). All tissues from mice expressing mutant hSOD1 (G93A and G37R) were harvested from paralyzed mice. The WT hSOD1 tissue was from mice ~18 months of age. (a), WT (b), and G93A (c) hSOD1 overexpressing mice were stained with C4F6 as controls. (d) C4F6 staining of human tissue from non-disease controls revealed very little immunoreactivity.  In human tissue from sALS cases (2 cases shown), occasional spheroid deposits appeared to be C4F6 immunopositive. For both the control and sALS cases the images shown are from tissues not treated for antigen retrieval. The spheroid deposits were identical in appearance and frequency to structures identified as corpora amylacea (highlighted with arrows) by the Periodic Acid-Schiff stain. Scale bar 200 μm. (PDF 40 MB)Additional file 4: Figure S3: C4F6 immunoreactivity in tissue from sALS cases without antigen retrieval. Tissues from nontransgenic mice (c) but stained cells transfected with G85R SOD1 (d). Following transient transfection with WT , G93A , G85R , or D90A  cells immunostained with the A5C3 antibody only showed reactivity to G93A (j). Nuclei were stained with DAPI (blue). Scale bar 50 μm. (PDF 5 MB)Additional file 5: Figure S2: C4F6 and A5C3 differ in their specificity for SOD1. C4F6 revealed no immunoreactivity to WT SOD1


Avian influenza viruses have caused human infection and posed the pandemic potential. Live poultry markets are considered as a source of human infection with avian influenza viruses. Avian influenza routine surveillance of live poultry markets is taken annually in China. We isolated the 2 H11N9 influenza virus from the surveillance program. To better understand the risk caused by these new viruses, we characterize the genetic and pathogenicity of the two viruses.Viral isolation was conducted with specific pathogen-free (SPF) embryonated chicken eggs. Whole genome was sequenced, and phylogenetic analysis was conducted.Two H11N9 viruses were identified, with all 8 segments belonging to the Eurasian lineage. The HA, NA, M, NS and PA genes were similar to virus isolates from ducks, and the NP, PB2 and PB1 gene segments were most similar to those viruses from wild birds, indicating that the H11N9 viruses might represent reassortant viruses from poultry and wild birds. The HA receptor binding preference was avian-like, and the cleavage site sequence of HA showed low pathogenic. The NA gene showed 94.6 % identity with the novel H7N9 virus that emerged in 2013. There was no drug resistance mutation in the M2 protein. The Asn30Asp and Thr215Ala substitutions in the M1 protein implied a potentially increased pathogenicity in mice. Both viruses were low-pathogenic strains, as assessed by the standards of intravenous pathogenicity index (IVPI) tests.Two reassortant H11N9 avian influenza viruses were detected. These viruses showed low pathogenicity to chickens in the IVPI test. Public health concern caused by the reassortant H11N9 viruses should be emphasized during the future surveillance.The online version of this article (doi:10.1186/s40249-016-0149-2) contains supplementary material, which is available to authorized users. Please see Additional file Wild waterfowl are considered to be the natural reservoir of influenza A virus. There are currently 16 HA subtypes (H1-H16) and nine NA subtypes (N1-N9) circulating in birds . The 16 Since 1900, H1, H2 and H3-subtype influenza A viruses have infected humans and have circulated within the human population. The first human infection with a highly pathogenic avian influenza H5N1 virus occurred in Hong Kong in 1997 . SubsequEpidemiological investigation has shown that live poultry markets (LPMs) are important sources of human infection with avian influenza viruses . Avian iSince 2009, we have undertaken a nationwide surveillance program to monitor avian influenza viruses in poultry-related environmental samples. In this study, we isolated two reassortant H11N9 viruses from an LPM in Jiangxi province during routine surveillance in 2009. Genetic and molecular evolutionary analyses indicate that they are low-pathogenic avian influenza viruses. To our knowledge, this is the first report to identify a reassortant avian H11N9 virus in environmental samples from an LPM.6 U/L penicillin G, 200 mg/L streptomycin, 2 × 106 U/L polymyxin B sulfate, 250 mg/L gentamicin, 60 mg/L ofloxacin-HCl, 0.2 g/L sulfamethoxazole and 5 × 105 U/L nystatin .Our nationwide routine avian influenza virus surveillance of environmental samples has been conducted in China since January 2009. The samples, including bird feces, drinking or contaminated water, and environmental swabs, were collected monthly from LPMs, slaughterhouses and farms. From January 2009 to December 2013, 33 049 samples were collected from 142 cities in 31 provinces. The samples were placed into 15 mL tubes containing viral transport medium and sent to the laboratory for further testing. The viral transport medium consisted of 4 mL of minimum essential medium containing 0.5 % BSA, 10 % glycerol, 2 × 10An RNeasy Kit  was used to extract viral RNA from the samples. Influenza A virus RNA was detected by real-time PCR  targeting a matrix gene on a Stratagene Mx3005P thermocycler. The real-time RT-PCR amplification protocol included steps of 45 °C for 10 min and 95 °C for 10 min and then 40 cycles of 95 °C for 15 s and 60 °C for 45 s.Virus-positive samples were shipped to the Chinese National Influenza Center for viral isolation. The samples were inoculated into the allantoic cavities of 9-day-old embryonated chicken eggs that were then incubated at 37 °C for 48 h before being chilled at 4 °C overnight. The allantoic fluid was harvested, and virus identification was performed with a hemagglutination assay with 0.5 % turkey red blood cells.Eight gene segments of each virus were amplified with a One-Step RT-PCR kit  according to the manufacturer’s protocol. Complete genome amplification was performed using specific primers . PCR proSequences were assembled and edited with Lasergene 8.1 (DNASTAR). Neighbor-joining (NJ) trees were constructed using MEGA 5.0. The bootstrap value was tested with 1000 replications for each gene segment. Homology analyses of nucleic acids and amino acids were performed on the NCBI website with BLAST. All of the reference sequences used in the phylogenetic comparison were obtained from GenBank.Fresh infective allantoic fluid with a HA titer > 16 was diluted 1:10 in sterile isotonic saline. A sample (0.1 ml) of the diluted virus was injected intravenously into each of 10 6-week-old SPF chickens. All of the chickens were examined daily for 10 days and scored based on the condition of each chicken: 0 , 1 (sick), 2 (severely sick) and 3 (dead), as described in the OIE/WHO guidelines .The proportion of influenza M gene-positive environmental samples was 7.9 %, and the positive viral isolation rate was 1.5 %. The H2, H3, H4, H5, H6, H7, H9 and H11 subtypes were detected between year 2009 and 2013. Among all of the viral isolates, 2 H11N9 avian influenza viruses were separately isolated from two LPMs in Wuning city, Jiangxi province. Both viruses were isolated in 2009 and were named A/environment/Jiangxi/26/2009(H11N9) (JX/26/2009) and A/environment/Jiangxi/28/2009(H11N9) (JX/28/2009). JX/26/2009 was collected from Xinning LPM on July 8, 2009, and JX/28/2009 was isolated from Guai LPM on July 8, 2009. The complete genome sequences of the influenza viruses have been deposited in GenBank under accession numbers KC881287 to KC881302.JX/26/2009 and JX/28/2009 were highly homologous, with greater than 99 % identity in the nucleotide sequences of each segment. The lengths of the PB2, PB1, PA, HA, NA, NP, M and NS genes were 2341, 2341, 2233, 1754, 1453, 1565, 1027 and 890 bp, respectively. The HA gene showed the highest identity (99 %) with the Eurasian A/duck/Chiba/11/2007(H11N9) virus, whereas the NA gene sequence showed 99 % identity with the Eurasian A/duck/Vietnam/G30/2008(H11N9) virus. The NA genes of the viruses were 94.6 % identical to A/Anhui/1/2013(H7N9), the novel A(H7N9) virus identified in March 2013 in China. The nucleotide sequences of the internal genes showed high similarity with other avian influenza viruses from wild birds or ducks. The viruses in GenBank that had the highest amino-acid sequence homology to our newly identified viruses are shown in Table The results of a phylogenetic analysis showed that the eight gene segments of the 2 H11N9 viruses isolated in this study all belonged to the Eurasian lineage, which was distinct from the branch of North American lineages in the tree , A/Mallard/Korea/GH171/2007(H7N9), A/Duck/Jiangxi/25186/2009(H7N6), A/Wild duck/Korea/CSM20-5/2009(H4N6), A/Duck/Jiangxi/26326/2010(H7N7) and A/Duck/Nanjing/1102/2010(H4N8), respectively.The HA cleavage sites of the 2 H11N9 viruses were AIASR/G, which is characteristic of low-pathogenic avian influenza viruses. The Q226 residue of HA indicated that the receptor binding sites still recognize the α-2, 3 receptor (in H3 numbering). The mammalian-adaptation mutations of PB2 (627 K and 701 N) were not found in either of the viruses, indicating that the viruses originated from an avian source.The N30D and T215A substitutions in the M1 protein, which have been associated with increased H5N1 pathogenicity in mice , were foThe IVPI scores were calculated according to the OIE/WHO guidelines . The IVPH11N9 has been reported in wild waterfowl in Brazil, Belgium, the Republic of Kazakhstan, and Japan –19. ThisFrom 2009 to 2013, two H11N9 avian influenza viruses were isolated during routine surveillance. The samples were collected from chicken cages in LPMs. Phylogenetic analyses indicated that all the gene segments belonged to the Eurasian lineage. The HA, NA and NP genes originated from H11N9 viruses, whereas the internal gene segments clustered with H7N7, H7N6 and H4N6. The Eurasian-lineage HA gene clusters with recently reported H11N9 viruses that have been found in wild birds or ducks (Fig. Among influenza A viruses, H5 and H7 are considered to be notable avian influenza viruses, but not all H5 and H7 viruses display high pathogenic. Low-pathogenic avian influenza viruses can be precursors of highly pathogenic viruses , 25. TheReassortment among different influenza viruses is considered to be the main mechanism for the generation of new viruses, which may bear enhanced mammalian transmission and pandemic potential. It has been reported that H11N9 viruses can be transmitted directly to hunters from ducks . New H11Two H11N9 influenza viruses were isolated from the live poultry markets in Jiangxi during the routine surveillance. All the segments of the viruses were belonging to the Eurasian lineage of influenza virus genes. The segments of the viruses are originated from both poultry and wild birds. The character of the genes origination highlights the important of Jiangxi province as the possible hot place of mixture the influenza viruses from both migratory wild birds and poultry. The NA genes of the two viruses are different from the novel H7N9 virus of 2013. The Both viruses were low-pathogenic strains from the IVPI tests and the molecular genes markers. The reassortant virus may possess new characteristics that may be of concern to public health, and continuous surveillance should be emphasized.IVPI, intravenous pathogenicity index; LPM, live poultry market; r RT-PCR, real-time reverse transcription-polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; SPF, specific pathogen free


Rps26 is an essential protein of the eukaryotic small ribosomal subunit. Previous experiments demonstrated an interaction between the eukaryote-specific Y62–K70 segment of Rps26 and the 5′ untranslated region of mRNA. The data suggested a specific role of the Y62–K70 motif during translation initiation. Here, we report that single-site substitutions within the Y62–K70 peptide did not affect the growth of engineered yeast strains, arguing against its having a critical role during translation initiation via specific interactions with the 5′ untranslated region of mRNA molecules. Only the simultaneous replacement of five conserved residues within the Y62–K70 fragment or the replacement of the yeast protein with the human homolog resulted in growth defects and caused significant changes in polysome profiles. The results expand our knowledge of ribosomal protein function and suggest a role of Rps26 during ribosome assembly in yeast. RPS26a and RPS26b. Previous studies demonstrated that Rps26 interacts with the 5′ untranslated region of mRNA via the eukaryote-specific 62-YXXPKXYXK-70 (Y62–K70) motif. Those observations suggested that this peptide within Rps26 might play an important and specific role during translation initiation. By using alanine-scanning mutagenesis and engineered strains of the yeast Saccharomyces cerevisiae, we found that single amino acid substitutions within the Y62–K70 motif of Rps26 did not affect the in vivo function of the protein. In contrast, complete deletion of the Y62–K70 segment was lethal. The simultaneous replacement of five conserved residues within the Y62–K70 segment by alanines resulted in growth defects under stress conditions and produced distinct changes in polysome profiles that were indicative of the accumulation of free 60S subunits. Human Rps26 (Rps26-Hs), which displays significant homology with yeast Rps26, supported the growth of an S. cerevisiae Δrps26a Δrps26b strain. However, the Δrps26a Δrps26b double deletion strain expressing Rps26-Hs displayed substantial growth defects and an altered ratio of 40S/60S ribosomal subunits. The combined data strongly suggest that the eukaryote-specific motif within Rps26 does not play a specific role in translation initiation. Rather, the data indicate that Rps26 as a whole is necessary for proper assembly of the 40S subunit and the 80S ribosome in yeast.The eukaryotic ribosome consists of a small (40S) and a large (60S) subunit. Rps26 is one of the essential ribosomal proteins of the 40S subunit and is encoded by two almost identical genes, IMPORTANCE Rps26 is an essential protein of the eukaryotic small ribosomal subunit. Previous experiments demonstrated an interaction between the eukaryote-specific Y62–K70 segment of Rps26 and the 5′ untranslated region of mRNA. The data suggested a specific role of the Y62–K70 motif during translation initiation. Here, we report that single-site substitutions within the Y62–K70 peptide did not affect the growth of engineered yeast strains, arguing against its having a critical role during translation initiation via specific interactions with the 5′ untranslated region of mRNA molecules. Only the simultaneous replacement of five conserved residues within the Y62–K70 fragment or the replacement of the yeast protein with the human homolog resulted in growth defects and caused significant changes in polysome profiles. The results expand our knowledge of ribosomal protein function and suggest a role of Rps26 during ribosome assembly in yeast. Saccharomyces cerevisiae, the ribosome consists of two subunits , which contain 79 ribosomal proteins. Fifty-nine ribosomal proteins are encoded by duplicated genes. Twenty-one duplicated genes are translated into identical polypeptides, while the remainder are translated into very similar proteins  and RPS26b (located on chromosome 5), which are 92% identical. On the protein level, Rps26a differs from Rps26b by only two residues (E106D and D113A) in the C-terminal domain of the protein. Both proteins consist of 119 amino acids and possess a molecular mass of approximately 14 kDa (http://www.yeastgenome.org/). The Δrps26a Δrps26b double deletion is lethal, indicating that Rps26 is essential for the life of yeast . Be. Bein vil P site  (22). Bal P site , 24. In in vivo function of Rps26. Only simultaneous replacement of the highly conserved residues Y62, P65, K66, Y68, and K70 with alanine resulted in moderate growth defects and changes in polysome profiles that were indicative of the accumulation of free 60S subunits. The combined findings of this study point toward an important role for Rps26 in ribosome assembly and subunit joining; however, the findings are inconsistent with the idea of a specific role of the Y62–K70 segment during translation initiation.In order to gain further insight into the mechanism of Rps26 function and, specifically, into the role of the Y62–K70 segment, we employed yeast as a model organism. Based on published data , we expeIt was previously reported that Rps26 contacts the 5′ untranslated region of mRNA , 21 via rps26a Δrps26b deletions was rescued by the expression of RPS26a from a URA3-based plasmid  complemented the growth of the Δrps26a Δrps26b strain on yeast extract-peptone-dextrose (YPD) medium at 30°C or 40°C in the presence of the translational inhibitor paromomycin, high concentrations of dithiothreitol (DTT), or NaCl. Only at low temperature or if the strains were grown at alkaline pH did the Rps26a5A strain display a moderate growth defect  in strain SC288 (see TEF1 promoter (Tef1Promo) in strain SC306. Mutated versions of RPS26a  Wild-type (WT) RPS26a or versions of RPS26a with the alanine substitutions for conserved amino acid residues (shown in boldface) in the 62-YXXPKXYXK-70 motif were expressed under the control of the TEF1 promoter. YPD plates were cultivated at 30°C or 15°C for 2 days or 5 days, respectively. Download Figure S1, PDF file, 0.2 MB.Drop test analysis of growth phenotypes of yeast strains expressing Rps26 variants with single alanine substitutions within the 62-YALPKTYNK-70 peptide. (A) Wild-type Copyright © 2016 Belyy et al.2016Belyy et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00109-15.3Figure S2 -myc-tagged Rps26. Wild-type (Rps26) or c-myc-tagged Rps26a (Rps26a::c-myc) was expressed under the control of the TEF1 promoter in S. cerevisiae (rps26a::LEU2 rps26b::TRP1) . Download Figure S2, PDF file, 0.1 MB.Drop test analysis of yeast strains expressing wild-type or C-terminally cCopyright © 2016 Belyy et al.2016Belyy et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the del9) on the functionality of Rps26. The results of the experiment revealed that Rps26del9 failed to rescue the lethality of the Δrps26a Δrps26b mutation , which shares 63% amino acid sequence identity with yeast Rps26  and Rpl24 (large subunit) by Western blotting, the amount of 18S rRNA  and 28S rRNA (large subunit) by agarose gel electrophoresis, and the area below the 40S and 60S peak curves by polysome profiling  Schematic representation of PCRs with the primers used to check marker gene insertions into RPS26a/b. Annealing areas and direction of amplifications are shown by colored arrows. (B) Agarose gels showing results of PCRs with the wild-type yeast (WT) and S. cerevisiae (RPS26a/rps26a::LEU2 RPS26b/rps26b::TRP1) (strain SC222). Lanes (M) contain molecular mass markers; actual sizes are shown on the left and right sides of the gels. The products of reactions with primer pairs #734/#519, #734/#724, #733/#746, and #733/#521 are shown. The appearance of 1.1-kb and 1.25-kb bands with the marker-specific primer pair #521/#519, as well as the synthesis of 2.37-kb and 2.8-kb bands in addition to the 1.7-kb band for LEU2 and TRP1, respectively, demonstrates correct insertion of the LEU2/TRP1 markers into the RPS26 loci. Download Figure S4, PDF file, 0.1 MB.PCR analysis of the Copyright © 2016 Belyy et al.2016Belyy et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00109-15.6Figure S5 S. cerevisiae (RPS26a/rps26a::LEU2 RPS26b/rps26b::TRP1)  Tetrad analysis of b strain . (B) MarCopyright © 2016 Belyy et al.2016Belyy et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 5A, or Rps26-Hs were noticeable but not statistically significant .The recently solved crystal structure of the 40S ribosomal subunit revealed a dumbbell-like fold of the yeast protein, in which peripheral, α-helical regions are joined by a handlelike structure consisting of two symmetrical, antiparallel β-sheets  19). Th. Th19). runcated , and RpsThe molecular mechanism by which Rps26 affects the assembly of 80S ribosomes is currently not understood. One possible role of Rps26 in protein synthesis seems to be linked to its interaction with initiation factor eIF3 , which i5A or Rps26-Hs. This was likely due to specific degradation of small subunits containing mutated or heterologous versions of Rps26. Pathways directed to degradation of defective ribosomes and ribosomal subunits, like ribophagy . Genomic DNA from S. cerevisiae strain D273-10B (TRP1 and LEU2 marker genes. S. cerevisiae strain MH272-3fα (ura3 leu2 his3 trp1 ade2) and the diploid strain MH272-3fα/a (ura3/ura3 leu2/leu2 his3/his3 trp1/trp1 ade2/ade2) (Tables S1 to Cloning was performed in D273-10B  was usede2/ade2)  are the e2/ade2) , pYCplace2/ade2) , pYEplace2/ade2) , YEpTef5e2/ade2) , and pES10.1128/mSphere.00109-15.8Table S2 Table S2, DOCX file, 0.1 MB.Plasmids used in the current study. Download Copyright © 2016 Belyy et al.2016Belyy et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00109-15.9Table S3 Table S3, DOCX file, 0.1 MB.Primers used for gene cloning/mutagenesis in the current study. Download Copyright © 2016 Belyy et al.2016Belyy et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00109-15.10Table S4 S. cerevisiae deletion mutants. Download Table S4, DOCX file, 0.1 MB.Primers used in PCR analysis of Copyright © 2016 Belyy et al.2016Belyy et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli strains were grown in LB medium supplemented with the appropriate antibiotic. Yeast strains were grown on rich medium  or on minimal medium containing 0.67% yeast nitrogen base without amino acids  with 2% glucose (SDex) or 2% galactose . SDex and SGal media were supplemented with the appropriate additives .LEU2 and TRP1 marker genes for RPS26 gene disruptions were amplified with their own promoters and terminators from the S. cerevisiae D273-10B genomic DNA. Mutations rps26a::LEU2 (Δrps26a) and rps26b::TRP1 (Δrps26b) were constructed by replacing nucleotides 19 to 324 of RPS26a or RPS26b with the functional LEU2 or TRP1 gene cassette, respectively. Yeast transformations were performed by the lithium acetate method   isolate (SC254) was selected for subsequent experiments. YCplac33-RPS26a was replaced by pRS313-based plasmids encoding different Rps26 variants via the 5-fluoroorotic acid (5-FOA) method  or with the TEF1 promoter (pRS313-based plasmid p896 and YCplac33-based plasmid p887).For expression in yeast, RPS26a was amplified with primers #721 and #725 (a full list of primers used for PCR is shown in 5A) and the Rps26del9 variant lacking 9 amino acid residues (62-YALPKTYNK-70) were generated by the PCR splicing method . Constitutively expressed RPS26a and del9rps26 with a COOH-terminal c-myc tag were constructed in pESC-His, containing the TEF1 promoter instead of the original GAL1/10 promoter, by exchanging the stop codon for a cysteine codon. Subsequently, the fragments were transferred, together with the TEF1 promoter, into the low-copy-number vector pRS313 using EcoRI/ClaI restriction endonuclease sites (plasmids p1687 and p1692).The human gene coding for Rps26-Hs (pET-15-rps26e) was a generous gift from G. Karpova  . The cod595) were spotted onto YPD, SDex, or SGal plates containing the required supplements. The plates were incubated for the times and temperatures indicated in the legends to the figures in which the results of the experiments are shown.Mutant strains were analyzed on agar plates by the drop test. To that end, 5-fold serial dilutions of overnight agar cultures adjusted to the same optical density at 595 nm , and transferred into 2-ml tubes. Extracts were prepared by vortexing yeast cell suspensions with glass beads in the presence of protease inhibitor cocktail  and 1 mM phenylmethylsulfonyl fluoride (PMSF) using a FastPrep-24 device . Extracts were clarified by centrifugation at 8,000 rpm for 10 min, followed by centrifugation at 13,500 rpm for 15 min at 4°C (Yeast strains were grown overnight in liquid medium to an ODn at 4°C .A260 units in 60 µl) were loaded on top of 90 µl of a 25% sucrose cushion in the lysis buffer and were subjected to ultracentrifugation in a CS 150NX micro-ultracentrifuge  equipped with an S100AT3 rotor at 95,000 rpm for 35 min  were loaded on top of an 11-ml linear 15-to-55% sucrose gradient in the lysis buffer and subjected to ultracentrifugation for 2.5 h at 39,000 rpm . Fractions were collected from top to bottom with a density gradient fractionator monitored at A254  and were subsequently analyzed by Western blotting using specific antisera as indicated in the legends to the figures in which the results of the experiments are shown.For polysome profile analysis, clarified supernatants  were mixed with 80% N,N-dimethylformamide and heated at 65°C for 15 min  antibody , yeast anti-Rpl24 antiserum, anti-Sse1 antiserum, and anti-Rps9 antiserum (Yeast extracts were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate buffer  followedntiserum .


Brassica oleracea var. capitata) to elevated Cu2+ levels resulted in leaf chlorosis and lesser biomass yield at ≥2 µ M. Root nitrate content was not statistically affected by Cu2+ levels, although it was substantially decreased at ≥5 µ M Cu2+ in the shoot. The decrease in nitrate contents can be related to lower nitrate uptake rates because of growth inhibition by Cu-toxicity. Shoot sulfate content increased strongly at ≥2 µ M Cu2+ indicating an increase in demand for sulfur under Cu stress. Furthermore, at ≥2 µM concentration, concentration of water-soluble non-protein thiol increased markedly in the roots and to a smaller level in the shoot. When exposed to elevated concentrations of Cu2+ the improved sulfate and water-soluble non-protein thiols need further studies for the evaluation of their direct relation with the synthesis of metal-chelating compounds .Arable soils are frequently subjected to contamination with copper as the consequence of imbalanced fertilization with manure and organic fertilizers and/or extensive use of copper-containing fungicides. In the present study, the exposure of stone-head cabbage ( At the protein level, Cu serves as a co-factor for various enzymes such as Cu/Zn-superoxide dismutase (Cu/ZnSOD), cytochrome c oxidase, ascorbate oxidase, amino oxidase, laccase, plastocyanin (PC), and polyphenol oxidase , zinc (Zn) and molybdenum (Mo) are essential for the growth and development of plants, but they rapidly get toxic at higher levels . Cu cont oxidase . Howeverradicals .Plants exposed to elevated levels of Cu show unspecific toxicity symptoms. Elevated Cu levels in soils primarily result in stunted root growth and leaf chlorosis . Copper viz. phytochelatins), which are most likely glutathione-derived compounds.  seeds were germinated to sand in a green house. The seedlings collected at ten days after germination were transferred on an aerated 25% modified Hoagland nutrient solution in a 11 liter container  in a greenhouse for 10 days. The nutrient solution consists of 1.25 mM Ca(NO3)2.4H2O, 1.25 mM KNO3, 0.25 mM KH2PO4, 0/0.5 mM MgSO4.7H2O, 11.6 µM H3BO3, 2.4 µM MnCl2.4H2O, 0.24 µM ZnSO4.7H2O, 0.08 µM CuSO4.5H2O, 0.13 µM Na2MoO4.2H2O and 22.5 µM Fe3+-EDTA with supplemental concentrations of 0, 2, 5 and 10 µM CuCl2 and pH 5.9–6.0. The nutrient solution was continuously aerated with Aqua-Supreme—Air Pump—Model AP-4.The photoperiod was 14 h. 30 and 25 °C (±5 °C) temperatures were set for day and night respectively, whereas the relative humidity was maintained at 60–70%.Stone-head cabbage  followed by centrifugation at 800 Frozen root and shoot material was homogenized in de-mineralized water (10 mL per g fresh weight) and one layer of Miracloth filter was used to filter the homogenate. The supernatant was incubated in a water bath at 100 °C for 10 min. The remainder was centrifuged for 15 min (0 °C) at 30,000 g. The anions were separated by HPLC and   one layer of Miracloth which was then centrifuged at 30,000 g for 15 min (0 °C). Extraction medium containing 80 mM sulfosalicylic acid, 1 mM EDTA, and 0.15% (w/v) ascorbic acid with an Ultra Turrax at 0 °C (10 mL per g fresh weight) was used for the homogenization of fresh plant matter. The resultant homogenous material was passed through2+ (≥2 µM) in nutrient solution caused chlorosis of both the shoot and young emerging leaves, that ultimately reduced both root and shoot biomass production  and carotenoid contents of stone-head cabbage were significantly decreased upon exposure at ≥2 µM Cu2+ (a/b and chlorophyll/carotenoid ratios when exposed to increased Cu2+ concentrations (10 µM Cu2+). Ten µM Cu2+ exposure resulted in the start of rapid development of shoot chlorosis and significantly faster reduction in chlorophyll a contents of chlorophyll b and carotenoids, ultimately leading to a significant reduction in chlorophyll a/b and chlorophyll/carotenoid ratios  to stone-head cabbage significantly decreased the production of root and shoot biomass and raised the ratio of the shoot to the root. Copper contamination in the root environment generally results in retarded production of root and shoot biomass and a reduced photosynthetic activity. Moreover, it causes chlorosis, necrosis and bleaching of pigments . Toxic metal-induced hindered root growth also reduces the uptake of essential nutrients .Brassica species increase their demand for sulfur supply (2+ there was a significant decrease in the shoot nitrate contents. The reduced production of plant biomass at ≥5 µM Cu2+ could be attributed to reduced supply of nitrate in the shoot. The decrease in nitrate: sulfate ratio in the shoot of stone-head cabbage under elevated Cu levels may be attributed to enhanced sulfate contents, however the link between uptake rates of nitrate and sulfate is not evident yet (Plants have evolved a tightly-controlled mechanism for the absorption, allocation and assimilation of sulfate under normal conditions. . Enhancer supply . Exposurdent yet . Since bdent yet .Roots accumulated a slightly raised level of water-soluble non-protein thiols when compared with that of the shoot. Only a small proportion of the rise in thiol content might be attributed to a Cu-induced synthesis of phytochelatins in Chinese cabbage . Previou2+ negatively affected the plant biomass production and pigment contents. Furthermore, elevated Cu content considerably affected the concentration of sulfate and water-soluble non-protein thiol of stone-head cabbage which might be due to the induction of phytochelatins to detoxify excess copper.This investigation has shown that the elevated copper levels in the root surroundings proved toxic for stone-head cabbage. Copper exposure at ≥2 µM Cu10.7717/peerj.1119/supp-1Supplemental Information 1Click here for additional data file.