Myceliophthora heterothallica in solid-state (SSC) and submerged (SmC) cultivation. Wheat bran and sugarcane bagasse were used for SSC and cardboard for SmC. Highest endoglucanase production in SSC occurred after 192 hours: 1,170.6 ± 0.8 U/g, and in SmC after 168 hours: 2,642 ± 561 U/g. The endoglucanases and beta-glucosidases produced by both cultivation systems showed slight differences concerning their optimal pH and temperature. The number of endoglucanases was also different: six isoforms in SSC and ten in SmC. Endoglucanase activity remained above 50% after incubation between pH 3.0 and 9.0 for 24 h for both cultivation systems. The effect of several chemicals displayed variation between SSC and SmC isoenzymes. Manganese activated the enzymes from SmC but inhibited those from SSC. For β-glucosidases, maximum production on SmC was 244 ± 48 U/g after 168 hours using cardboard as carbon source. In SSC maximum production reached 10.9 ± 0.3 U/g after 240 h with 1 : 1 wheat bran and sugarcane bagasse. Manganese exerted a significant activation on SSC β-glucosidases, and glucose inhibited the enzymes from both cultivation systems. FeCl3 exerted the strongest inhibition for endoglucanases and β-glucosidases.This work reports endoglucanase and beta-glucosidase production by the thermophilic fungus Cellobiose is considered as the smallest repetitive unit of cellulose and can be hydrolyzed into glucose residues [β-glucosidases. Endo-β-1,4-glucanases (EC 3.2.1.4) cleave the amorphous regions of the cellulose chain; exoglucanases or cellobiohydrolases (EC 3.2.1.91) attack the nonreducing or reducing chain ends and β-1,4-glucosidases (EC 3.2.1.21) hydrolyze cellobiose releasing glucose [Cellulose is the most abundant polysaccharide in the plant cell wall and consists in a linear chain composed of a varying number of nked by β → 4 glycresidues . Celluloresidues . CompletFungal cellulolytic enzymes have biotechnological applications in food, animal feed, wine, agriculture, biomass refining, and pulp and paper industries , 6. The The composition of the medium can influence the profile of enzymatic expression by the microorganism. Cellulases can be obtained more economically by microorganisms through submerged or solid-state cultivation. The low water activity in solid-state cultivation (SSC) shows a simple technique of high productivity, but it can affect the heat transfer to the substrate and thus could affect the growth of the microorganism . The useβ-glucosidases in solid-state and submerged cultivation media by thermophilic Myceliophthora heterothallica and to characterize the crude extracts obtained from both systems.The present study aimed to compare the production of endoglucanases and  Myceliophthora heterothallica F.2.1.4. was isolated from sugar cane bagasse compost and maintained at the Laboratory of Applied Biochemistry and Microbiology, UNESP.The thermophilic fungus M. heterothallica F.2.1.4. in 250 mL Erlenmeyer flasks with 50 mL medium composed of 1.0% sugarcane bagasse (ground to 1-2 mm); 0.14% (NH4)2SO4; 0.20% KH2PO4; 0.03% CaCl2; 0.02% MgSO2·7H2O; 0.50% beef peptone; 0.20% yeast extract; 0.03% urea; 0.10% micronutrients solution ; 0.2% glucose; and 2.0% agar, pH 5.0. All the solutions and the culture media were sterilized by autoclaving at 121°C for 20 minutes. This medium was inoculated with suspension obtained scrapping the mycelium from culture grown for 3 days at 45°C in 150 mL of nutrient solution (g/L): 3.5 (NH4)2SO4; 3.0 KH2PO4; 0.5 MgSO4·7H2O; 0.5 CaCl2; and 0.1% v/v Tween 80, pH 5.0.The inoculum was prepared by growing the fungusβ-glucosidase production in SmC was carried out in 250 mL Erlenmeyer flasks using cardboard as a carbon source, previously cut and ground in a blender. The mixture contained 1% cardboard and 20 mL of the nutrient solution described above was inoculated with 2 mL of the microorganism inoculum as described. The cultures were incubated on a rotary shaker (100 RPM) at 45°C for 10 days and samples were taken every 24 h, filtered through vacuum using Whatman paper number 1, and centrifuged at 6,000 ×g for 20 minutes. The supernatant was used as a crude enzyme solution.Endoglucanase and In order to evaluate the use of agroindustrial residues such as wheat bran and sugar cane bagasse as the substrate in SmC, they were washed, ground, and oven-dried (60°C for 48 h). The sugarcane bagasse was sieved, allowing selecting particles ranging from 1 to 2 mm. Subsequently, cultivation was carried out in 250 mL Erlenmeyer flasks containing 1% (1 : 1 w/w) of wheat bran and sugarcane bagasse, 20 mL nutrient solution, and 2 mL inoculum suspension. The medium was previously autoclaved as described. The cultures were incubated on a rotary shaker (100 RPM) at 45°C for 10 days and sampling was performed as explained above.The substrates used in the SSC were composed of used 5 grams of cardboard or a mixture of wheat bran and sugar cane bagasse (1 : 1). The substrates were inserted in polypropylene bags (12 × 20 cm) and sterilized at 121°C for 40 min. The fungus inoculation was carried out by adding 20 mL of the mycelium suspension. The moisture content of around 80% was reached with the addition of sterile distilled water. The fermentation was at 45°C for 14 days. Every 48 h, one bag was taken and the fermented material was mixed with 20 mL of distilled water per gram, stirred for 30 min, filtered, and centrifuged at 10,000 ×g, at 10°C. The supernatant was used as a crude enzyme solution.−1 sodium acetate buffer pH 5.0 containing 4% of carboxymethyl cellulose (CMC)  and 0.1 mL of crude enzyme solution. The reducing groups, expressed as glucose, released were assayed by using the DNS  method [μmol of reducing sugars per min under the assay conditions [Endoglucanase activity was measured at 60°C in a reactional mixture composed of 0.9 mL of 100 mM L) method . One uninditions .β-D-Glucosidase activity was measured in a mixture of 50 mm sodium acetate buffer, pH 5.0, 500 μL of 2 mM L−1 p-nitrophenyl-β-D-glucopyranoside (pNP-Glc) as substrate, and 50 μL of the enzyme solution incubated at 60°C for 10 min. The reaction was stopped by addition of 2 mL of 2 M Na2CO3 and the absorbance was read at 410 nm. One unit of β-glucosidase was defined as the amount of enzyme that releases 1 μmole of nitrophenol/min in the reaction conditions.All the enzyme assays were performed in triplicate.−1 NaCl until visualization of the bright spots corresponding to the enzyme [The endoglucanase solution was analyzed by electrophoresis on denaturing 10% sodium dodecyl sulphate (SDS) polyacrylamide gel using Tris-Glycine buffer pH 8.3. After electrophoresis, the enzyme was renatured with 1% triton X-100 for 30 minutes and subsequently the gel was overlaid with a mixture of 1% (w/v) CMC (Sigma-Aldrich) as substrate and 1% (w/v) agarose and incubated for 30 min at 50°C. For detection of endoglucanase activity, the gel was immersed in a 0.1% (w/v) Congo Red solution for 15 minutes and then washed with 1 M Le enzyme .β-glucosidase, the gel was incubated with 0.2 M L−1 acetate buffer pH 5.0 for 10 minutes at room temperature under gentle agitation. After that, the gel was treated with a solution of esculin 0.1% ferric chloride in 0.03% 0.2 M L−1 acetate buffer pH 5.0 until the appearance of bands [For of bands .The effect of pH on the enzyme activity was evaluated as described above, in the pH range from 2.0 to 11.0 using 0.1 M buffers: pH 2-3: sodium citrate; pH 3–5.5: sodium acetate; pH 6.0–7.5: HEPES; pH 8.0–9.5: glycine; and pH 10.0–11.0: CAPS. The effect of temperature was assayed incubating the reactional mixture in temperature range from 35 to 85°C in the optimal pH.β-glucosidase residual activity. Thermal stability was determined by incubating the crude enzyme from 40 to 70°C for 60 min, and residual activity was assayed as described above. For both experiments the data were compared to a control without preincubation, denoted as 100%.The effect of pH on enzyme stability was studied by incubating the crude enzyme solution in various buffers with pH ranging from 2.5 to 11.0 during 24 h at 25°C, followed by the determination of endoglucanase or t-test for independent samples using BioEstat 5.3 [The enzyme activity assays were done in the presence of metal ions and other compounds: ethanol, glucose, triton, and metal ions. The metal ions  were of analytical grade and were dissolved in ultrapure water (UPW). The extract was dialyzed against UPW before the assays. The results were compared by Student's stat 5.3 . Myceliophthora heterothallica by SSC and SmC using different lignocellulosic residues as substrates revealed that the extracellular enzyme production was highly dependent on the chemical composition of the tested culture media . When the ratio of 9 : 1 sugarcane bagasse/wheat bran (SSCSB)  was used, endoglucanase activity was 303.0 ± 1.7 U/g after 144 hours, and the production in SSC using cardboard (SSCP) achieved 182.7 ± 1.6 U/g. Changing the ratio of sugarcane bagasse/wheat bran to 1 : 1 (SSCWB), the production rose more than three times to 1,170.6 ± 0.8 U/g after 192 h , production increased to 67.3 ± 0.8 U/g after 288 h   was used, er 288 h . When usubstrate . On the The zymogram of beta-glucosidases  showed dAlthough by submerged cultivation (SmC) higher values of productivity were obtained in terms of U/g compared to cultivation in the solid state, two aspects must be considered: first, the volume of liquid obtained in the process. In SmC, the enzyme was diluted 100 times while in SSC it was diluted only 20 times. Since the aim of this project was the production of enzymes for use in their crude form, the initial concentration of the solution is important because it avoids a step to concentrate the enzyme, reducing the process costs and showing a frequent decreased yield of the enzyme due to nonspecific adsorption to membranes and concentrators, for example, an advantage of SSC. Aspergillus niger, who in fact verified a decrease in cellulase and pectinase production above a certain concentration of the carbon source.On the other hand, previous data from our group (unpublished) showed no linearity between the increase in concentration of the substrate (C source) from 1 to 2.5% and enzyme production, a fact also described by Kumar et al.  working In addition, the cultivation system also influences enzyme production since SSC has absence of water among the particles of the solid substrate which have been reported as an important factor that interferes in the extracellular enzymes secretion . The endA biochemical characterization was carried out for the highest SSC and SmC productions, namely, SSCWB and SmCP, respectively. The effect of pH on the endoglucanase activity was studied using CMC (4%) as substrate, and the endoglucanases produced by both systems showed maximum activity at pH 5.5 . Pycnoporus sanguineus in SmC showed optimum pH around 3.5–4.0 [ Trichoderma atroviride 676 and Penicillium sp. CR-316, respectively, cultivated in SmC, while others found a higher range, from 4.5 to pH 6.0, for the endoglucanases from the fungus Acremonium sp. produced in SSC [ M. thermophila M.7.7 exhibited an optimum pH of 5.0 [ A. niger in SSC exhibited higher activity at pH 4.0 [Reported values have been found for optimum pH in the crude extract in the range from pH 3.5 to 6.0, independent of the cultivation system , 27. The 3.5–4.0 , 29 obtad in SSC , and thoH of 5.0 . The endt pH 4.0 .The effect of the pH on the stability  was anal A. niger [ A. niger obtained by SSC have an optimum temperature of 60°C [ M. thermophila M.7.7 exhibited maximum activity at 70°C [The effect of temperature on endoglucanase activity  was analA. niger . Endoglu of 60°C , and tho at 70°C . P. sanguineus displayed an optimum temperature of 60°C [ T. atroviride 676 [ Penicillium sp. CR-316 [On the other hand, analyzing the enzymes obtained by SmC, those of of 60°C  and valuride 676  and simi. CR-316 . M. thermophila M.7.7 [Concerning the thermal stability of endoglucanases , the resla M.7.7 .2, which caused a strong inhibition for the endoglucanases obtained by SSC, while increasing the activity of the enzymes from SmC, suggesting that the “new” isoenzymes expressed in this cultivation system and described above would be significantly activated by that cation. Since EDTA inhibited the endoglucanases produced by both cultivation systems, it was expected that a divalent cation would increase the enzyme activity. Iron inhibition could be attributed to the effect of Fe3+ at the reducing ends of cellulose [The effect of several compounds on endoglucanase activity was analyzed using the crude extract produced by SSCWB and SmCP . The effellulose .β-glucosidase activity produced by both cultivation systems showed maximum activity at pH 5.0 (β-glucosidases fall into a wide range, 2–2.5 [β-glucosidases produced by SSC and SmC exhibited high activity above pH 3.5, up to 10.0. The residual activity falls sharply at more acid or alkaline pH values.The t pH 5.0 . Reportee, 2–2.5 , 4.5 [23e, 2–2.5 , 4.8 [35e, 2–2.5 , but mose, 2–2.5 . The effβ-glucosidase activity is shown in The effect of temperature on The thermal stability  was deteβ-glucosidase activity was analyzed using the crude extract produced by SSCWB and SmCP (2, which caused a strong activation for β-glucosidase obtained by SSC, while increasing the activity of those enzymes from SmC.The effect of several compounds on and SmCP . The eff M. heterothallica F.2.1.4. proved to be a great producer of endoglucanases, using both sugarcane bagasse and wheat bran by solid-state cultivation or using cardboard in submerged cultivation. Cardboard in SmC would be a residue of easy access and would be able to induce the synthesis of more isoforms of endoglucanases, which, when characterized in the crude extract, was shown to be thermostable and prompted us to perform further studies of biotechnological applications. The beta-glucosidases obtained by SmC showed higher stability.The thermophilic fungus


Cryptococcus neoformans. Gib2 contains a seven-bladed β transducin structure and is emerging as a scaffold protein interconnecting signalling pathways through interactions with various protein partners. Here, we present the crystal structure of Gib2 at a 2.2-Å resolution. The structure allows us to analyse the association between Gib2 and the ribosome, as well as to identify the Gib2 amino acid residues involved in ribosome binding. Our studies not only suggest that Gib2 has a role in protein translation but also present Gib2 as a physical link at the crossroads of various regulatory pathways important for the growth and virulence of C. neoformans.The atypical Gβ-like/RACK1 Gib2 protein promotes cAMP signalling that plays a central role in regulating the virulence of  We also present predictions of Gib2 residues involved in the association with ribosomes and discuss the role that Gib2-ribosome binding may play in the virulence of C. neoformans.Hence, Gib2, a scaffold protein interconnecting various cellular processes through binding a myriad of proteins, could also bind with the ribosome and function in ribosomal biogenesis and protein translation. By recruiting various proteins to ribosomes, Gib2 may act as a link between protein translation and other cellular processes. To better illustrate such functions, we here present the crystal structure of Gib2 and show its interaction with ribosomes of C. neoformans species includes two highly relevant but distinct varieties, var. grubii and var. neoformans. Gib2 was originally reported to have an essential function in C. neoformans var. neoformans (serotype D) because the knockdown of GIB2 by antisense suppression resulted in a severe growth defect, and no GIB2 deletion strains linked to the auxotrophic marker Ura5 could be recoveredGIB2 deletion strains could be readily recovered if dominant selective marker genes were usedGIB2 deletion strains displayed no reduction in the cAMP levels or apparent defects in melanin and capsule formation, suggesting that they are not directly linked to virulenceGIB2 deletion strain was reduced at 37°C but not at 30°C or 23°CGIB2 deletion strain survived nearly twice as long as those infected with the wild-type strainThe GIB2 disruption on the viability of C. neoformans, growth curves of the GIB2 deletion strain and its parental strain H99 were determined at 30°C and 37°C in rich YPD and nutrient-limiting YNB media. Although the two strains exhibited similar growth profiles at 30°C, the GIB2 deletion strain showed approximately a two-fold reduction in growth in YPD media at 37°C  Gib2 to 80S ribosomes purified from wild-type H99 and the GIB2 deletion strains. For comparison, the binding of human RACK1 to C. neoformans ribosomes was also tested. Following incubation with either Gib2 or RACK1, ribosomes were precipitated through a sucrose cushion by centrifugation, and the associated proteins were separated using SDS-PAGE. As a control, Gib2 and RACK1 proteins were loaded onto sucrose cushion in the absence of ribosomes. For reference, purified Gib2 and RACK1 proteins were directly loaded onto SDS-gel as well. The recombinant Gib2 and RACK1 were visualised by Western blotting using the anti His-tag and anti-RACK1 antibodies, respectively.Basic cellular functions, such as ribosomal biogenesis and protein translation, underlie the growth and differentiation of eukaryotic cells. Mammalian RACK1 and GIB2 deletion (Δgib2) strain  and the ) strain . Howeveribosomes , furtherC. neoformans Gib2 residues , except for Met1. The electron-density map is well defined apart from the first and last residue, as well as a short stretch of side chains between residues Gly276 and Arg282 in the extended loop linking blades six and seven S. cerevisiae Asc1 (PDB ID 3FRX)The structure of Although Gib2 crystallises as a monomer, Western blotting has revealed that it can form dimeric complexes under physiological conditionsS. cerevisiae ribosomes indicted that Asc1 is located in the head region of the 40S subunit close to the mRNA exit tunnel25253135S. cerevisiae ribosome (PDB: 3U5B and 3U5C)Tetrahymena thermophila 40S ribosomeC. neoformans ribosomes to other eukaryotic ribosomes is not known.Cryo-EM studies of C. neoformans eIF4A (eukaryotic translation initiation factor 4A) homologue was established previously by Wang et alThe D-E-A-D (Asp-Glu-Ala-Asp)-box containing RNA helicases are essential ribosomal components serving as initiation factors in protein translation in eukaryotic cellsC. neoformans is a fungal pathogen that causes life-threatening infections primarily in individuals with compromised immune systems. The virulence of C. neoformans is regulated by the cAMP-dependent signalling pathway456713GIB2 gene in C. neoformans serotype A, affected neither cAMP levels nor pigment and capsule formationGIB2 deletion strain infected mice had a longer survival than those infected by the wild-type strainC. neoformans.C. neoformans13C. neoformans Gib2 at a 2.2-Å resolution. The Gib2 structure features the β-propeller fold with each of the seven blades consisting of four antiparallel β-sheets  and Gib2 has been reported41C. neoformans. Other findings, such as the effect of phosphorylation of RACK1 on its interactions with β-integrins43C. neoformans. However, eukaryotic translation initiation factor 3 (eIF3) was reported to associate with ribosomes through binding to the one to three blade region of Asc146There are several examples of protein binding to the four to seven blade region of RACK1/Asc1 proteins. For instance, protein kinase C (PKC) can bind to blade six as revealed by peptide mapping studies38S. cerevisiae, the deletion of the ASC1 gene affected the phosphorylation of eukaryotic translation initiation factors 2 (eIF2) and 4A (eIF4A), affinity of eIF3 and eIF5 binding to 40S ribosomes, and assembly of the pre-initiation complex. These findings indicate an important regulatory role of Asc1 in general translation initiationExamples of the influence of ribosome-bound RACK1 on translation in different eukaryotes are accumulating. For instance, RACK1 stimulates translation by recruiting activated PKC to 40S subunits, and the PKC dependent phosphorylation of eIF6 on 60S subunits leads to subunit associationIn addition to affecting general translation, RACK1 and Asc1 were shown to regulate the translation of specific mRNAs51C. neoformans.An interactome analysis showed that Gib2 interacts with more than 50 proteinsC. neoformans. Gib2 interacts with G protein α subunit Gpa1 and assists its functions in the cAMP-signalling pathway to regulate virulence (melanin pigment and capsule formation) of C. neoformans13C. neoformans may face challenges in adjusting to changes in the living environment, leading to reduced fitness and virulence.Thus, Gib2 has both ribosome-independent and -dependent functions in C. neoformans. Solving the crystal structure of Gib2 sheds light onto its versatile functions as a ribosome-bound scaffold for numerous binding partners. It also provides a basis for future studies, such as the mutagenesis of Gib2, binding assays to identify/confirm its binding partners, the structural characterisation of these protein complexes to reveal the virulence mechanism, and drug target identification for antifungal therapy.Finally, Gib2 is emerging as a link between diverse cellular processes, virulence, and translation regulation in the widely spread and precarious human pathogen C. neoformans wild-type H99 and GIB2 deletion strains were grown in 5 L of YPD  medium at 30°C with shaking until OD600 reached 0.6–0.8. Cells were precipitated by centrifugation (3500 rpm for 15 min at 4°C), frozen in liquid nitrogen, and stored at −80°C.600 0.15 and grown either at 30°C or 37°C for 24 hours.To monitor growth, 100 ml of YPD or YNB (6.7 g of Yeast Nitrogen Base supplemented with 2% dextrose in 1 L of distilled water) medium was inoculated with a single fungal colony and grown overnight at 30°C. Three replicates of 100 ml YPD or YNB media were then inoculated with the overnight culture to ODThe pOPTHis-Lip-Gib2 plasmid was constructed for recombinant Gib2 expression, in which a TEV-cleavable His-tag was placed at the N-terminus. The human RACK1 gene was cloned, and the protein was purified as previously described4Cl, 10 mM magnesium acetate, 100 mM EDTA, 5 mM DTT, 0.2 mM PMSF, and 10% glycerol) and transferred in 1-ml aliquots into 2-ml cryo tubes containing 0.5 mg of glass beads (460 μm). Cells were lysed using a Precellys 24  tissue homogeniser .Three to five grams of fungal cells were re-suspended in 2 ml of buffer A  and centrifuged at 5,000 rpm for 10 min. The supernatant was overlaid on a 25% glycerol cushion in buffer B and centrifuged at 50,000 rpm for 3 hours. The pellet was re-suspended in buffer A with 10% sucrose and centrifuged at 5,000 rpm for 10 min. The supernatant was diluted two-fold with buffer A (without sucrose or glycerol), layered onto 10 to 30% sucrose gradient in buffer A and centrifuged at 19,000 rpm for 17.5 hours using SW 28 Ti type rotor (Beckman Coulter Inc. US).260, and 80S ribosome-containing fractions were pooled. Ribosomes were precipitated by centrifugation at 40,000 rpm for 20 hours, re-suspended in buffer G 2, and 2 mM DTT), and stored at −80°C.The ribosome profile was determined by continuous monitoring of A600 reached 0.3, the temperature was lowered to 16°C; when the OD600 reached 0.8–0.9, isopropyl β-D-1-thiogalactopyranoside  was added to induce Gib2 expression. Cells grown overnight were harvested by centrifugation and stored at −80°C.BL21(DE3)pLysS competent cells were transformed with the pOPTHisLip-Gib2 plasmid and grown in 5 L of 2YT media  supplemented with ampicillin and chloramphenicol. When the ODCells were re-suspended in lysis buffer  and lysed using a Panda homogeniser . The lysate was centrifuged at 20,000 rpm for 20 min. The supernatant was the loaded onto a HisTrap HP 5-ml column  equilibrated with the same buffer. Imidazole was used for protein elution. Gib2-containing fractions were pooled and dialysed overnight at 4°C against buffer containing 50 mM Tris-HCl pH 8.0, 50 mM NaCl, and 5 mM β-mercaptoethanol. The TEV protease  was added to remove the His-tag, and the purification process was repeated using 50 mM NaCl instead of 300 mM. The flow through was collected. Alternatively, dialysed Gib2 fractions were loaded onto a HiTrap Q 5-ml column and an increasing NaCl concentration was used for protein elution. Gib2-containing fractions were pooled and concentrated to 10 ml before loading onto a HiLoad 26/60 Superdex 75 pg column . Column pre-equilibration and protein elution were performed using buffer containing 20 mM Tris-HCl pH 8.0, 50 mM NaCl, and 5 mM β-mercaptoethanol. Gib2 appeared as a single peak and the corresponding fractions were pooled, concentrated, and stored at −80°C.80S ribosomes (1.5 μM) were incubated with 6 μM Gib2  or human RACK1 (~40 kDa) in buffer G in a final volume of 25 μl at 30°C for 25 min. The reaction mixture was filtered through a 0.22-μm filter, the volume was adjusted to 50 μl with buffer G, and layered onto 200 μl of 1.1 M sucrose cushion followed by centrifugation at 45,000 rpm for 18 hours at 4°C. For control, Gib2 and RACK1 proteins without 80S ribosomes were layered onto sucrose cushion in parallel. The pellet was washed once and dissolved in buffer G. Samples were analysed by SDS-PAGE and Western blotting analysis following standard protocols. The anti-His and anti-RACK1 antibodies were used for detecting Gib2 and RACK1, respectively.600 reached 0.5. IPTG  was added to induce protein expression at 25°C. After 8 hours, cells were harvested by centrifugation and stored at −70°C. To extract proteins, cells were suspended in lysis buffer  and lysed with sonication (2 sec pulses with 4 sec pauses for 2 min). Crude extracts were centrifuged  and supernatants were recovered. In case of GST-tagged Gib2, the supernatant was mixed with glutathione-sepharose resin  for 2 h. The slurry mixture was then centrifuged at 500 rpm for 2 min at 4°C, washed with Tris-NaCl buffer , and the protein was eluted by adding 15 mM glutathione. The eluted protein was dialyzed overnight against Tris-NaCl buffer using Slide-A-Lyzer cassette , recovered, and verified by SDS-PAGE and Western blotting analysis with the anti-GST antibody . Preparation of the His-tagged eIF4A protein was similar to the above except that crude protein extract was mixed with His-Select Nickel affinity gel , eluted with Tris-NaCl buffer containing 200 mM imidazole, and dialysed as above. Target protein was verified by Western blotting analysis with the anti-His antibody .Gib2 and eIF4A cDNAs were cloned into vectors pGEX4T-2 and pET32a, respectively, and transformed into Rosetta 2(DE3) cells . 1 L of LB medium was inoculated with 5 ml of overnight culture and grown at 37°C until ODFor binding, 500 μl of GST-Gib2 protein was added to glutathione-sepharose resin in Tris-NaCl buffer. 500 μl of His-eIF4A protein was then added and the mixture was incubated overnight with gentle rotation at 4°C. The resin was precipitated and washed three times with Tris-NaCl buffer. 100 μl of Tris-NaCl buffer and 25 μl of 5× protein sample buffer were then added to the resin before denaturing by boiling for 5 min. 4 μl of the sample was then separated by SDS-PAGE, transferred to the PVDF membrane, and visualized using the anti-GST and anti-His antibodies. For the negative control, the GST protein was used as an input.The TEV protease-cleaved Gib2 (~9.3 mg/ml) was used for crystallisation trials. Initial crystal hits were found using commercial screens: Crystal Screen, Index, and SaltRx ; and Wizard  using the Phoenix protein crystallisation robot  as sitting drops in 96-well plates. Crystals of ~400 × 100 × 50 μm grew at 18°C when 0.2 μl of the well solution (100 mM cacodylate (pH 6.5) and 1 M sodium citrate) was mixed with 0.2 μl protein sample. For cryo protection, 30% PEG3350 was added. Crystals were mounted and flash frozen in liquid nitrogen.S. cerevisiae Asc1 .Diffraction data were collected on the PXI beamline at the Swiss Light Source (SLS) at a 1-Å wavelength using a Pilatus 6 M detector  at 100 K. The data were processed using X-ray Detector Software (XDS)Accession codes: The coordinates and structure factors for Gib2 have been deposited in the PDB with accession code 4D6V at 2.2-Å resolution.


Hepatitis B virus (HBV) infection is a major risk factor which can lead to development of hepatocellular carcinoma (HCC). In this study, we aimed to explore the effects of toll-like receptor 4 (TLR4) downregulation on the growth and survival of HBV-related HCC cells and to examine the molecular mechanisms been involved.The expression levels of TLR4 were examined in a panel of HCC cell lines . The effects of TLR4 downregulation on the proliferation, apoptosis, and tumorigenicity of HBV-related HepG2.2.15 cells were determined. The effects of TLR4 downregulation on multiple signaling pathways were also measured. Co-immunoprecipitation and immunofluoresence staining assays were performed to investigate the interaction between TLR4 and HBV X protein (HBx).ERK1/2 approximately phenocopied the growth-suppressive effect of TLR4 downregulation on HepG2.2.15 cells. In addition, TLR4 showed a physical interaction with HBx.The mRNA and protein levels of TLR4 were significantly increased in HepG2.2.15 cells than those in the other cells which have been studied. Downregulation of TLR4 significantly decreased the proliferation and induced G2/M cell cycle arrest and apoptosis in HepG2.2.15 cells. TLR4 depletion inhibited HepG2.2.15 cell colony formation and tumor growth in nude mice. TLR4 silencing decreased the phosphorylation of ERK1/2 but not JNK1/2, p38, or NF-κB. Chemical inhibition of ERK1/2 activation and interaction with HBx. Therefore, TLR4 may be a potential therapeutic target for HBV-related HCC.Taken together, TLR4 plays a tumor-promoting role in HBV-related HCC cells, which is associated with regulation of  HBx), PreS2 activators, and hepatitis B spliced protein (HBSP) have been found to be implicated in hepatocarcinogenesis  containing aprotinin (2 mg/L), phosphatase inhibitor Leupeptin (5 mg/L) and phenylmethylsulfonyl fluoride . Protein concentrations were determined using the BCA protein assay . Equal amounts of proteins were subjected to 10 or 12 % SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes . The membranes were blocked with 5 % fat-free milk and incubated with primary antibodies at 4 °C overnight, followed by incubation with secondary antibodies for 2 h. Signals were visualized by chemiluminescence  and quantitated using Image J software. Primary antibodies used in this study were rabbit anti-TLR4 polyclonal antibody , rabbit anti-SAPK/JNK polyclonal antibody , rabbit anti-p44/42 MAPK (Erk1/2) polyclonal antibody , rabbit anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) polyclonal antibody, rabbit anti-p38 MAP Kinase polyclonal antibody , rabbit anti-Phospho-p38 MAP Kinase (Thr180/Tyr182) polyclonal antibody , rabbit anti-NF-kB p65 polyclonal antibody , and rabbit anti-phospho-NF-kB p65 (Ser536) polyclonal antibody  , and rabbit anti-JNK1 + JNK2 (phospho T183 + Y185) polyclonal antibody , and mouse anti-β-actin polyclonal antibody  . Anti-rabbit  and anti-mouse  IgGs were purchased from Proteintech .http://pga.mgh. harvard. edu/primerbank/): sense: 5′-AGA CCT GTC CCT GAA CCC TAT-3′; and anti-sense: 5′-CGA TGG ACT TCT AAA CCA GCC A-3′. The primers for amplification of GAPDH were as follows: sense: 5′-GTT GGA GGT CGG AGT CAA CGG A-3′; and anti-sense: 5′-GAG GGA TCT CGC TCC TGG AGG A-3′. All PCR amplifications were performed with an initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 62 °C for 30 s.Total RNA was extracted using TRIzol (Invitrogen) and cDNA synthesis was performed using the PrimeScript RT reagent Kit . qPCR was performed using the SYBR Premix Ex Taq ™II (Takara) on an Applied Biosystems 7300 Real-Time PCR System . The TLR4 primer sequence (PrimerBank ID: 373432602c1) came from PrimerBank  was added to each well and incubated for 1 h at 37 °C. The optical density (OD) was measured at a wavelength of 450 nm using a microplate reader.5 cells per well in 6-well plates. After treatment, cells were fixed in ice-cold 70 % ethanol. Cell cycle distribution was analyzed using the Cell Cycle Analysis Kit  and apoptosis was detected using the Annexin V-PE/7-AAD Apoptosis Detection Kit I  according to the manufacturer’s instruction. Stained cells were examined by FACSCalibur flow cytometry.Cells were seeded at a density of 5 × 10n = 5 for each group) to receive a s. c. injection of 5 × 106 HepG2.2.15 cells transduced with replication-defective lentivirus expressing TLR4-shRNA (Le-TLR4) or negative control shRNA (Le-NC) or normal saline (NS). Le-NC , Le-TLR4  or NS (40 μL/mouse) was injected intratumorally at several points every two days, with an accumulated dose of 1 × 108 TU. Tumor volumes were measured every 5 days with a caliper and calculated according to the formula: 0.5 × length × width2. At 18 days after the cell inoculation, mice were sacrificed. The same treatments were done to other 3 groups of nude mice received HepG2 cells. All experimental manipulations were undertaken in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Scientific Investigation Board of the Nanchang University, Nanchang, China.For tumorigenicity assays, 4- to 6-week-old male BALB/C nu/nu nude mice (16–18 g) were purchased from the Experimental Animal Center of Shanghai . Mice were randomly divided into 3 groups  for 30 min. Coverslips were incubated with mouse anti-HBx monoclonal antibody  or rabbit anti-TLR4 polyclonal antibody  in 1 % BSA at 4 °C overnight, followed by with fluorescence labeled secondary antibody  in 1 % BSA for 30 min at room temperature. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole . Images were captured using Nikon A1 Confocal Laser Microscope System and adjusted using NIS-Elements Viewer 4.0 .HepG2.2.15 cells were grown on 10-cm cell dishes. After culture for 36 h, cells were harvested and lysed in the immunoprecipitation lysis buffer  containing Aprotinin (2 mg/L), Leupeptin (5 mg/L) and PMSF (1 mmol/L). Total cell lysates were pre-cleaned with protein-A + G Sepharose Beads  for 3 h at 4 °C and centrifuged. Aliquots of the supernatant were used as input. The remaining supernatants were incubated with mouse anti-HBx or anti-TLR4 antibody or isotype control IgGs and then with protein A + G Sepharose Beads (50 % slurry) at 4 °C overnight. After centrifuging, the pellets were resuspended with the SDS sample buffer and boiled to remove Sepharose beads. Lysate inputs and immunoprecipitates were then subjected to SDS-PAGE and analyzed by Western blotting. Total cell lysates were used as input control.t test using SPSS 21.0 . P < 0.05 was considered significant.Data were presented as mean and standard deviation (SD) for normally distribution. Groups were compared by one-way Analysis of variance (ANOVA) and multiple comparisons by LSD-We firstly examined the mRNA and protein expression of TLR4 in a panel of HCC cells . As illustrated in Fig. TLR4-1), showing efficient knockdown of TLR4 , with less effect of TLR4 downregulation. However, TLR4 expression levels were little affected by the transfection of the TLR4-3 siRNA.To explore the biological significance of TLR4 in HBV-related HCC cells, we specifically knocked down its expression in HepG2.2.15 cells using siRNA technology. This cell line was chosen because of its high expression of TLR4 both at the mRNA and protein levels. The amounts of TLR4 mRNA and protein were significantly reduced in cells transfected with one TLR4 siRNA  reduced the proliferation rate of HepG2.2.15 cells, compared with those treated with LPS and SB203580, SP600125 or PDTC (Fig. To further confirm the mediating role of DTC Fig. .HBx, HBx was immunoprecipitated from HepG2.2.15 cells using anti-HBx monoclonal antibody and then subjected to Western blot analysis for the presence of TLR4. As shown in Fig. Immunofluorescence analysis revealed that TLR4 displayed a diffuse spotty distribution in the cytoplasm in HepG2.2.15 cells, and HBx had a consistent cytoplasmic staining Fig. . To confTLRs in cancer cells. For instance, Wang et al. [HBx gene results in upregulation of TLR4. Consistently, our data found that compared to the other HCC cell lines studied, HBV-related HepG2.2.15 cells had greater mRNA and protein levels of TLR4. These findings suggest that TLR4 expression is regulated in HCC cells at least partially by HBV infection, largely at the transcriptional level.TLR4 is dysregulated in many human cancers, such as gastric cancer , colorecg et al.  reportedg et al. . Wang etg et al.  showed tAccumulating evidence indicates that dysregulation of TLR4 is causally linked to tumor development and growth. TLR4 silencing has been found to suppress human NSCLC cell growth . Yuan etHBx plays a critical role in HBV-related HCC pathogenesis . Our bioHowever, we have to admit that NF-κB and MAPKs are limited research pathways. ERK1/2 may be the concerned signaling, and it shall not be the only one. In the future we will further investigate the possible pathways of TLR4 involved in HCC, and its relationship with HBx. Although HepG2.2.15 cell is just a single HBV-related cell line, its mRNA and protein expression level is highest among all 5 cell lines. Besides that, HepG2.2.15 cell carries HBV. Therefore, HepG2.2.15 cell has sufficient characteristics and representativeness for the research. We are conscious that if more cell lines are investigated, then as a result more sufficient evidence can certainly be provided to our experiement. We wish to achieve this in the upcoming experiments. Additionally, to further improve the accuracy of the experiments, we plan to upregulate TLR4 as positive treatment in the future.Our data show that TLR4 is required for the growth and survival of HBV-related HCC cells, which is mediated, at least partially, through ERK1/2 signaling. The interaction between TLR4 and HBx may be involved in the regulation of ERK1/2 activation by TLR4. TLR4 may thus represent a therapeutic target for HCC, especially HBV-related HCC.


Developmental processes are governed by a diverse suite of signaling pathways employing reversible phosphorylation. Recent advances in large-scale phosphoproteomic methodologies have made possible the identification and quantification of hundreds to thousands of phosphorylation sites from primary tissues. Towards a global characterization of proteomic changes across brain development, we present the results of a large-scale quantitative mass spectrometry study comparing embryonic, newborn and adult murine brain. Using anti-phosphotyrosine immuno-affinity chromatography and strong cation exchange (SCX) chromatography, coupled to immobilized metal affinity chromatography (IMAC), we identified and quantified over 1,750 phosphorylation sites and over 1,300 proteins between three developmental states. Bioinformatic analyses highlight functions associated with the identified proteins and phosphoproteins and their enrichment at distinct developmental stages. These results serve as a primary reference resource and reveal dynamic developmental profiles of proteins and phosphoproteins from the developing murine brain. Since its emergence two short decades ago, mass spectrometry-based proteomics has quickly matured to become not only a major force in discovery science, but also an important analytical tool in the testing of hypotheses formed in all manner of biochemical and biological disciplines . EssentiPreviously we conducted phosphoproteomic analyses of murine brain at embryonic day 16.5 (E16.5) using SCX ; neonata4P2O7, 1 mM Na3VO4, 50 mM β-glycerolphosphate, 10 µg/mL leupeptin, 5 µg/mL pepstatin A and 1 mM PMSF). The homogenate was sonicated on ice for six 30-s intervals, with 30-s rests on ice between blasts, using a Kontes 50 W sonication microprobe tip at 50% duty output. Insoluble cellular debris was removed by centrifugation at 7,000× g for 30 min at 4 °C. DTT  was added to 20 mL of the clarified lysate. The lysates were then incubated at 65 °C for 30 min. After cooling to room temperature, iodoacetamide was added to 12 mM and lysates were incubated for one hour at room temperature in the dark. Samples were diluted in 25 mM TRIS pH 8.0 to a urea concentration of 2 M. 750 µg of sequencing-grade of modified trypsin  was added to each 10 ml of lysate for overnight digestion at 37 °C. Peptides were acidified with trifluoroacetic acid (TFA) to 0.4% and insoluble material was spun down by centrifugation at 4,000× g for 20 min. The clarified supernatant from each developmental stage was applied to a separate, prewashed (100% acetonitrile (MeCN)) and equilibrated (0.1% TFA) tC18 (1 g) solid phase extraction Sep-Pak column . Peptides were washed with 0.1% TFA and eluted in 40% MeCN, 0.1%TFA. Eluates were frozen at −80 °C and lyophilized. For non-labeled peptides used in the anti-phosphotyrosine peptide immunoprecipitation experiment shown in 4P2O7, 1 mM Na3VO4, 1 mM PMSF, 10 µg/mL leupeptin and 5 µg/mL pepstatin A). The crude homogenate was clarified by centrifugation at 15,000× g and at 4 °C. Eleven hand-poured 7.5%–20% acrylamide gradient, preparative gels containing 6 mg protein each were cut into four distinct molecular regions. Similar regions were combined and subjected to in-gel tryptic digestion prior to peptide extraction and peptide immunoprecipitations. A detailed description of peptide preparation in this manner was described previously [Mice were procured and treated in accordance with an institutionally-approved IACUC protocol. Timed-pregnant or appropriately-aged CD-1 mice were ordered from Charles River Canada . Timed-pregnant (E16.5), P0 and P21 mice were sacrificed and dissected after a brief isoflurane administration. Several brains from each stage were pooled and the pooled brains of a given stage were quickly weighed and placed on ice immediately prior to lysis. Three P21 brains were pooled, 8 P0 brains were pooled and 15 E16.5 brains were pooled. For dimethyl-labeling experiments, pooled brain tissue from each stage (~750 mg) was dounce-homogenized in a total of 25 mL ice-cold urea lysis buffer  and 200 µL 600 mM NaCNBD3 (Cambridge Isotopes Laboratories) were added to both E16.5 and P21 peptides. 200 µL of 4% formaldehyde and 200 µL of 600 mM NaCNBH3 were added to P0 peptides. Reactions were allowed to proceed for 10 min, followed by a second 200 µL addition of each reagent and another 10 min incubation. Reactions were quenched by adding TFA to 10% and peptides were desalted over tC18 (0.5 g) solid phase extraction Sep-Pak columns as described above with the washing being 2.5% MeCN, 0.1% FA and the elution with 40% MeCN, 0.1% TFA. Peptide eluates were frozen and lyophilized. For the small-scale analysis shown in For the large-scale analysis, 10 mg (weighed) of dried tryptic peptides from each developmental stage were separately dissolved in 5.0 mL of 1 M HEPES, pH 7.5. 200 µL of fresh 4% D1H-dimethyl P0 peptides, 2H-dimethyl E16.5 peptides, 2H-dimethyl P21 peptides were dissolved in 1.4 mL of peptide immunoprecipitation buffer . 0.7 mL (5 mg) of 1H-dimethyl P0 peptides was mixed separately with 0.7 mL (5 mg) of 2H-dimethyl P21 peptides or 0.7 mL or 2H-dimethyl E16.5 peptides. Peptides were rocked at 4 °C for 30 min and insoluble remnants were removed by centrifugation in a microcentrifuge  at 4 °C for 15 min. 30 µL of a 50% slurry of immobilized α-pY100  was added to each mixed peptide solution. The solutions were rocked overnight at 4 °C and immune complexes were loaded onto a 200 µL gel-loading tip, pinched to arrest the resin. The resin was washed five times with PIPB and twice with water. The original flow through was collected for SCX-IMAC. Phosphotyrosine-harboring peptides were eluted with two applications of 40 µL of 0.15%TFA. The eluate was desalted on stage tips as previously described [2PO4 pH 2.65, 30% MeCN) and applied to a polysulphoethyl A solid phase extraction column  pre-washed with 80% MeCN and then with H2O, and then equilibrated with SCX buffer A for 30 min. Peptides were eluted fractionally with 6 mL each of SCX solvent B  adjusted with SCX solvent A to 0 mM, 10 mM, 25 mM, 40 mM, 60 mM, 90 mM and 150 mM KCl. Eluates were collected, frozen at −80 °C, lyophilized, and then desalted on tC18 (0.5 g) solid phase extraction Sep-Pak columns as described above and dried. The flow through from peptide IPs was subjected to SCX-IMAC as described previously ,3 but thDried peptides were suspended in 2.5% MeCN, 2.5% formic acid (FA) and loaded for nano-scale microcapillary LC-MS/MS in an LTQ-Orbitrap MS  fitted to a Finnigan Nanospray II electrospray ionization source, a Surveyor HPLC pump plus, and a Micro AS autosampler  essentially as described , allowing and at 4 °C. Normalized extracts were run on 7.5% or 10% (37.5:1 polyacrylamide/bis-acrylamide) SDS-PAGE gels to achieve separation. Immunoblotting was performed using 0.2 µm nitrocellulose membranes, blocked with 5% dry milk in Tris-buffered saline (TBST) essentially as described previously [Whole brains from E16.5, P0 or P21 CD-1 mice  to obtain Kyoto Encyclopedia of Genes and Genomes (KEGG) ,16 pathwEnrichments offer insight into active pathways, particularly when high protein counts (or high rank) are considered. We recognize that a simple rank order discounts various weighting options including weighting by protein counts or by fold enrichment. However, these approaches can also be conducted using the data in The KEGG analysis described above focused on enrichments found in entire datasets. However, given we had acquired quantitative mass spectrometry information, and given our primary focus on developmentally-regulated phosphorylation events, we next wanted to determine the nature of the phosphoproteins that we had identified and which had a relative abundance importantly higher in one developmental stage over another. To do this we took our Log2 fold change-sorted datasets described in Consistent with developmentally-dynamic phosphoproteomes these DAVID clustering results identify differential enrichment in gene ontology categories when analyzing the phosphopeptides that exhibit the largest changes in abundance between brain stages. Importantly, both the KEGG pathways and other DAVID clustering results are tied to distinct proteins and in some cases distinct phosphorylation sites which will facilitate the generation of hypotheses that lead to the characterization of proteins with putative roles in vertebrate brain development.We have conducted a large-scale, quantitative mass spectrometry study of murine brain at three developmental stages: E16.5, P0 and P21. The relative abundances for thousands of phosphorylation sites and proteins across these developmental stages were determined. Bioinformatic analyses identified phosphoproteins and proteins from each developmental stage with enrichments tied to distinct functionality. Several of these functionalities are consistent with nervous system development and development generally. However, much remains unexplored and it is anticipated that these results will serve as an important resource for developmental biologists as well as for cellular biochemists, particularly those studying the roles of phosphorylation on individual proteins or protein cohorts.


P < 0.0011, ADP EC50 1.8 µM). Similarly, an increase in ATP release was also observed with the P2Y agonist, UTP, up to a maximum of 70% of the hypotonic response (EC50 0.62 µM). Selective P2 receptor agonists, αβ-methylene-ATP, ATP-γ-S, and 2-methylthio-ADP had minimal effects on ATP release. ADP-stimulated ATP release was significantly inhibited by suramin . RT4 urothelial cells break down nucleotides (100 µM) including ATP, ADP, and UTP to liberate phosphate. Phosphate liberation was also demonstrated from endogenous nucleotides with approximately 10% of the released ATP broken down during the incubation. These studies demonstrate a role for P2Y receptor activation in stimulation of ATP release and emphasize the complexity of urothelial P2 receptor signalling.The release of ATP from the urothelium in response to stretch during filling demonstrates the importance of the purinergic system for the physiological functioning of the bladder. This study examined the effect of P2 receptor agonists on ATP release from two urothelial cell lines (RT4 and UROtsa cells). Hypotonic Krebs was used as a stretch stimulus. Incubation of urothelial cells with high concentrations of the P2Y agonist ADP induced ATP release to a level that was 40-fold greater than hypotonic-stimulated ATP release ( To date seven P2X receptors have been identified , with P2eceptors  expresseothelium , 6 as weothelium . It is hlium [3)  and myoflium [3)  and painlium [3) . Furtherlium [3)  but not 1 [2 [4 [11 [6 receptors are expressed on suburothelial myofibroblasts [P2X receptors are activated preferentially by ATP; thus urothelial derived ATP may have an autocrine action on urothelial P2X receptors. However the urothelium also expresses ectoATPase enzymes  and so i1 , 16, P2Y1 [2 , 16, 17,2 [4 [11  expresseroblasts  but theiroblasts , 19. Theroblasts .It is very clear that the complexity of urothelial P2 receptor signalling and the feedback of ATP breakdown and release have not been adequately explored to date. Therefore the aims of this study were to examine the effect of purine and pyrimidine nucleotides on urothelial cell ATP release. We hypothesise that purines and pyrimidines selective for P2Y receptors will modulate ATP release from the urothelium.2 in McCoy's 5A culture medium supplemented with 10% foetal bovine serum, 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 0.25 μg/mL of fungizone. When confluent, RT4 cells were passaged with 0.05% Trypsin-EDTA for 5 minutes and then plated onto T75 flasks for continuous passage or onto 24 well plates for use in experiments when confluent (approximately 3 to 5 days after passage).Human urothelial RT4 cells were obtained from the ECACC. Cells were grown at 37°C with 5% CO2 in Dulbecco's modified eagle's medium supplemented with 5% foetal bovine serum, 100 units/mL of penicillin, 100 μg/mL of streptomycin, 0.25 μg/mL of fungizone, and 1% glucose. UROtsa cells were passaged in the manner described above for RT4 cells.Human urothelial UTOtsa cells were a gift from Dr. Scott Garrett from the University of North Dakota. Cells were grown at 37°C with 5% CO3 25, KH2PO4 1.2, MgSO4 1.2, CaCl2 2.5, and D-glucose 11.7). The basal level of ATP release was then determined by a 10-minute incubation in 500 μL Krebs-Henseleit solution. These media were collected and followed with the experimental treatments. Cells were exposed to normal Krebs-Henseleit (control) or the indicated concentration of nucleotide. Cells were exposed to hypotonic Krebs-Henseleit (1 : 2 dilution of Krebs-Henseleit in distilled water), a stimulus commonly used to mimic stretch in cultured cells [μL) was collected and used for ATP determinations.ATP release was determined as we have described previously . Urothel−6 to 10-10 M) were mixed with the bioluminescence assay mix and the luminescence generated was measured immediately using a plate reader (BMG labtech Polarstar). The standard concentrations fell within the upper and lower limits of sensitivity of the ATP bioluminescence assay. The ATP concentration in the cell supernatant was calculated relative to the standard curve. Luminescence was also measured for all concentrations of the nucleotides used. The luminescence determined for each concentration of nucleotide was used as a blank for that treatment and was subtracted from the corresponding cellular luminescence before the ATP concentration was calculated. Treatments were carried out in triplicate and the mean ATP concentration (in nM) per treatment determined.ATP concentration in the supernatant was measured using the bioluminescence assay according to the manufacturer instructions. Equal volumes of the cellular supernatant or ATP standard solutions  [μM nucleotide. After 30 minutes the supernatant was collected and the phosphate concentration determined. In additional experiments confluent cells in 12 well plates were incubated in control or hypotonic (50% dilution in water) phosphate free media for 10 or 30 minutes and both ATP concentration and phosphate concentration determined.Confluent urothelial cells in 24 well plates were washed (three times) in phosphate free media  were mixed. After 15 minutes absorbance at 750 nm was measured in a plate reader (BMG labtech Polarstar). Phosphate concentration in the cell supernatant was determined relative to a standard curve . Treatments were carried out in triplicate and the mean phosphate concentration (in nM) per treatment determined.Phosphate liberated from nucleotides was determined as previously described . Equal vt-test. Concentration response relationships were examined using a sigmoidal concentration response curve. All statistics were performed using Graphpad Prims (version 6) .Results are nonnormally distributed and as such are expressed as median with interquartile range (IQR). Two different treatments were compared using a Mann-Whitney All cell culture reagents were purchased from Invitrogen . Bioluminescence ATP Assay kit and nucleotides were from Sigma-Aldrich . All other reagents were of high analytical grade.P = 0.0006). Incubation of RT4 cells with ADP (100 μM for 10 minutes) induced ATP release to a level that was 40-fold higher compared to the hypotonic stimulus (P < 0.0011). In the presence of AMP and adenosine, the level of ATP release was not significantly different to the control level (Hypotonic Krebs was used as a stretch stimulus and was seen to induce an approximate threefold increase in ATP release (ol level . The resol level .μM determined (n = 5). A concentration dependent inhibition of ATP release was seen with AMP (μM (n = 7). A concentration dependent increase in ATP release was observed with UTP, up to a maximum of approximately 70% of the hypotonic response (μM was determined (n = 7).When the concentration response effect of ADP induced ATP release was determined it was seen to only occur at high concentrations of ADP  with an with AMP  with an response . An EC50αβ-methylene-ATP, and P2Y agonist, ATP-γ-S, had no effect on ATP release . The more selective P2Y1 agonist, 2-methylthio-ADP, inhibited ATP release in a concentration dependent manner .In RT4 cells, concentration response relationships were also determined for P2 receptor agonists and antagonists. The P2X agonist, μM) had no effect on ADP-stimulated ATP release (88.4 (65.2–113.4)% of ADP-stimulated release, n = 8, P = 0.4) while suramin (100 μM) significantly inhibited ADP-stimulated ATP release (43.3 (27.6–41.7)% of ADP-stimulated ATP release, P = 0.002, n = 4; IC50 8.3 μM, n = 8).P2 receptor antagonists were investigated for their ability to inhibit ADP-stimulated ATP release. PPADS (100 P = 0.0087). Incubation of UROtsa cells with ADP (100 μM for 10 minutes) induced an almost 50-fold increase in ATP release compared to that seen with the hypotonic stimulus (P < 0.0022). UTP (100 μM) initiated ATP release similar to that seen with hypotonic stimulus while 2-methylthio-ADP (100 μM) elicited an ATP release response similar to the control level.The results observed in RT4 urothelial cells were also confirmed in UROtsa cells . SimilarμM), with the greatest amount of phosphate liberated from ADP and UTP (n = 8). After a 10-minute incubation the level of release in the control was approximately 40 nM and in hypotonic phosphate free media was approximately 80 nM which was comparable to that seen using Krebs-Henseleit solution (n = 8). Significantly more phosphate was detected in cells treated with hypotonic media for 10 and 30 minutes . After a 10-minute incubation the level of phosphate liberated indicated that approximately 10% (7 to 15%) of the endogenously released ATP was broken down during the incubations in control and hypotonic media, respectively. In the presence of the ectoATPase inhibitor ARL67156 (100 μM) the amount of ATP detected in cells treated with hypotonic phosphate free buffer increased 5-fold after both 10- and 30-minute incubations (n = 4).RT4 urothelial cells were shown to have the capacity to liberate phosphate from nucleotides  or uracil nucleotides  [1, P2Y12, and P2Y13 respond to ADP while P2Y11 receptors respond to ATP [1 or P2Y11 receptors. This is in agreement with previously reported EC50s at P2Y1 of 0.9–8 μM [2 or P2Y4 receptors, agreeing with previously reported EC50s (0.14–0.8 μM) at the P2Y2 receptor [4 receptor (2.5 μM) [1 agonist, 2-methylthio-ADP [It has been shown that P2Y1 , 16, P2Y1 [2 , 16, 17,2 [4 [11  receptord P2Y14) . Of the d to ATP . In the 0.9–8 μM –26. The receptor , 25 and (2.5 μM) , 25. In μM) [It is possible that the apparent ATP release stimulated by high concentrations of ADP was due to the conversion of ADP to ATP by cell membrane-bound adenylate kinase. While this enzyme is predominately intracellular , it has μM) . in vivo is unknown; however, nucleotides have recently been isolated from human urine samples [The source of ADP samples . In addi samples . The bas samples . Similar samples  but not  samples , 30. NTP samples . It is t samples .μM) [μM) [In the current study the activity of ectoATPase in urothelial RT4 cells was examined in three ways. Firstly, ectoATPase enzymes present on the RT4 urothelial cells were able to liberate phosphate from stretch-induced ATP release, indicating a capacity to generate mediators such as ADP from stretch-induced ATP release. Secondly, incubation of the ectoATPase inhibitor ARL67156 (100 μM)  resultedμM) [μM) . FinallyμM) [μM) .The results obtained in the current study demonstrate that P2 receptor signalling in the urothelium is a complex interaction between ATP release and breakdown. To add to the complexity, it now seems likely that products formed from breakdown of nucleotides such as ATP also have affects at P2 receptors located on the urothelium. Our understanding of this complex interaction between ATP release and breakdown has been shown schematically in 1, P2Y2, P2Y4, and P2Y6 being hypothesised. In addition, activation of P2Y2 receptors leads to release of the inflammatory mediators interleukins 8 and 6 from uroepithelial cells [2 receptor decreased in a feline model of interstitial cystitis [6 selective agonist induced bladder overactivity characterised by increased voiding frequency in a rat cystometry model in addition to increased ATP release into the voided fluid [Activation of urothelial P2Y receptors has been proposed to be responsible for a number of physiological functions of the urothelium. Exogenous application of P2Y receptor agonists, including ADP and UTP, has been shown to increase spontaneous activity in rat bladder sheets . Similaral cells , indicatcystitis . Intraveed fluid .Alterations in urothelial ATP release have been identified in bladder dysfunction including interstitial cystitis , painful


Pestivirus. BVDV infection is distributed worldwide and causes serious problems for the livestock industry. The thiosemicarbazone of 5,6-dimethoxy-1-indanone (TSC) is a non-nucleoside polymerase inhibitor (NNI) of BVDV. All TSC-resistant BVDV variants (BVDV-TSCr T1–5) present an N264D mutation in the NS5B gene (RdRp) whereas the variant BVDV-TSCr T1 also presents an NS5B A392E mutation. In the present study, we carried out twenty passages of BVDV-TSCr T1–5 in MDBK cells in the absence of TSC to evaluate the stability of the resistance. The viral populations obtained (BVDV R1–5) remained resistant to the antiviral compound and conserved the mutations in NS5B associated with this phenotype. Along the passages, BVDV R2, R3 and R5 presented a delay in the production of cytopathic effect that correlated with a decrease in cell apoptosis and intracellular accumulation of viral RNA. The complete genome sequences that encode for NS2 to NS5B, Npro and Erns were analyzed. Additional mutations were detected in the NS5B of BVDV R1, R3 and R4. In both BVDV R2 and R3, most of the mutations found were localized in NS5A, whereas in BVDV R5, the only mutation fixed was NS5A V177A. These results suggest that mutations in NS5A could alter BVDV cytopathogenicity. In conclusion, the stability of the resistance to TSC may be due to the fixation of different compensatory mutations in each BVDV-TSCr. During their replication in a TSC-free medium, some virus populations presented a kind of interaction with the host cell that resembled a persistent infection: decreased cytopathogenicity and viral genome synthesis. This is the first report on the stability of antiviral resistance and on the evolution of NNI-resistant BVDV variants. The results obtained for BVDV-TSCr could also be applied for other NNIs.Bovine viral diarrhea virus (BVDV) is the prototype  Pestivirus. This genus belongs to the family Flaviviridae, which also includes the genera Hepacivirus (hepatitis C virus) and Flavivirus .Bovine viral diarrhea virus (BVDV), along with classical swine fever virus and border disease virus, is a member of the genus BVDV infection is distributed worldwide and tends to be endemic in many countries BVDV is an enveloped single-stranded (+) RNA virus. Its genomic RNA is about 12.5 kb long and consists of a single open reading frame (ORF) flanked by 5′ and 3′ non-translated regions (NTRs). The ORF encodes a polyprotein of approximately 3,900 amino acids that is co- and post-translationally processed to mature viral proteins by cellular and viral proteases. The viral proteins are sequentially designated Npro, C, Erns, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B. The autoprotease Npro is a non-structural (NS) protein that plays a role in blocking IFN-α/β induction BVDV isolates are categorized into two biotypes according to their effect on cultured cells: noncytopathic (ncp) isolates, which infect permissive host cells without causing cell death, and cytopathic (cp) isolates, which produce rapid cytopathic effects (CPE) and kill cells 50 1.8±0.6.µM) r; T1–5) (TSC EC50>80.0 µM). All of them carry an NS5B N264D mutation, whereas BVDV-TSCr T1 also shows an NS5B A392E mutation The thiosemicarbazone of 5,6-dimethoxy-1-indanone (TSC) is a non-nucleoside polymerase inhibitor of BVDV  were obtained from wt BVDV p0 after ten passages in MDBK cells with increasing concentrations of TSC Madin Darby bovine kidney cells  were grown in Eagle's Minimal Essential Medium (EMEM), supplemented with 10% γ irradiated fetal bovine serum  (growing medium). BVDV type 1 NADL strain, cytopathic biotype  was provided by Dr Laura Weber, INTA Castelar, Argentina. Wild type (wt) BVDV p0 was obtained after three successive steps of biological cloning of BVDV NADL in MDBK cells. TSC-resistant BVDV (BVDV-TSCr T1–5 at a multiplicity of infection (MOI) of 0.01. Cell cultures were incubated in infection medium (infection medium: EMEM supplemented with 2.5% Donor Horse Serum -DHS- Gibco) in a 5% CO2 incubator at 37°C until cell monolayers presented 80–90% of viral CPE. The supernatant was collected and clarified by centrifugation for 10 min at 1,000×g at 4°C. After titration of the virus in MDBK cells, the virus was used to infect new cell monolayers to generate the next passage. This procedure was repeated twenty times, and the virus obtained from each BVDV-TSCr T1–5 was named BVDV R1–5.MDBK cells were infected with BVDV-TSC1–5, MDBK cells were seeded in growing medium at a density of 1.3×105 cells per well in a 24-well plate and incubated for 24 h at 37°C in a 5% CO2 incubator. Then, cells were infected with approximately 100 plaque-forming units (PFUs) of BVDV R1–5 or wt BVDV p0. Following 1 h of adsorption at 37°C, the inoculum was removed, cell monolayers were washed twice with phosphate-buffered saline (PBS), and 1 ml of overlay medium (2.5% DHS and 0.8% methylcellulose) containing 80 µM of TSC was added in each well. Mock-infected cells with and without TSC, and infected cells without TSC were included as controls. After 72 h of incubation at 37°C with 5% CO2, cells were fixed with formalin 10%, stained with 0.75% crystal violet, and the viral plaques counted.To evaluate the antiviral activity of TSC against BVDV R2 incubator at 37°C, cells were infected with wt BVDV p0 or R1–5 (MOI 0.01). Following 1 h of adsorption at 37°C, the inoculum was removed, cell monolayers were washed twice with PBS and 1 ml of infection medium was added in each well. Mock-infected cells were included as controls. After 48 h of incubation at 37°C with 5% CO2, apoptosis was measured in infected and mock-infected cell cultures.MDBK cells were seeded in a 24-well plate as described above. After 24 h in a 5% CO6 cells/ml) and 100 µl of each cell suspension was stained with 5 µl Annexin V (conjugated with Phycoerythrin- PE-) and 5 µl 7-Amino-Actinomycin (7AAD) and incubated for 15 min at room temperature in the dark. Finally, 400 µl of binding buffer was added and the cells were analyzed by flow cytometry . PE Annexin V and 7AAD fluorescence was collected at FL 2 (550–600 nm) and FL4 (640–700 nm) channels, respectively. Histograms and density plots of 20,000 events were obtained and analyzed with WinMDI 2.9 software For apoptosis measurements, PE Annexin V Apoptosis Detection Kit I  was used following the manufacturer's instructions. Briefly, supernatants from infected and mock-infected cells were harvested and cells in the monolayer were collected by trypsinization and resuspended in 0.5 ml of infection medium. Adherent and floating cells were pooled and pelleted by centrifugation at 500×g for 10 min. Pelleted cells were washed twice with 0.5 ml of cold PBS. Before the second centrifugation , cells were counted in a Neubauer chamber. To obtain the dead-cell control for apoptosis measurements, one mock-infected cell suspension was heated at 65°C for 30 min. Then, cells were resuspended in binding buffer (4×10A: N° undetected dead cells  =  NMIC − ° cells NIC° cells ). Then, the number of apoptotic cells in each infection (i.e. in the remaining adherent and floating cells) was quantified by AnV binding and flow cytometry as stated above (B: N° apoptotic cells  =  % AnVpos× NIC/100° cells ). Finally, the total cells that would die by each viral infection was estimated as % dead cells   =  (A+B) × 100/NMIC° cells .At 48 h p.i. some infected cultures already presented viral CPE, which is caused by cell apoptosis. The number of cells that would have died by this mechanism and could no longer be detected was estimated by the difference between the number of cells counted in a Neubauer chamber in the mock-infected cultures (MIC) and those counted in the infected ones (IC) . Following 1 h of adsorption at 37°C, the inoculum was removed, cell monolayers were washed twice with PBS, and 1 ml of infection medium was added to each well. The culture supernatant was collected every 24 h for 7 days and clarified by centrifugation for 10 min at 1,000×g at 4°C. The infectious virus in each sample was quantified by the PFU method in MDBK cells.To quantify the infectious virus produced by BVDV R4 and R5 showed CPE and levels of cell apoptosis similar to those of BVDV R1 and R2, respectively (see results section), they were not included in the following assays (unless otherwise indicated).Given that BVDV R5 cells per well containing a circular slide cover of 10 mm in diameter. After 24 h in a 5% CO2 incubator at 37°C, cells were infected with BVDV R1, R2, R3 or wt BVDV p0 (MOI 0.01). Following 1 h at 37°C, the inoculum was removed, cell monolayers were washed twice with PBS, and 1 ml of infection medium was added to each well. At 24, 48 and 72 h p.i., the cell culture medium was removed and cell monolayers were fixed with 4% formaldehyde/PBS for 15 min at room temperature. After washing three times with PBS, cultures were incubated with 0.5 ml of blocking buffer  for 30 min. Then, slide covers were removed and incubated overnight at 4°C with 30 µl of anti-BVDV NS3 Monoclonal Antibody , kindly provided by Dr Alejandra Capozzo . Slide covers were washed three times with PBS and incubated protected from light for 2 h at room temperature with Alexa Fluor 488 Goat Anti-Mouse antibody (Invitrogen) . Afterwards, cells were washed three times with PBS and the nucleus stained with 300 µl of DAPI (Invitrogen) (dilution 1∶100) for 5 min. The slides were mounted with FluorSave  and the cells were observed with a Nikon Eclipse 80i microscope with a 20× objective . The images were captured with a digital camera  and processed using ImageJ software.MDBK cells were seeded in a 24-well plate at a density of 1.3 ×102 incubator at 37°C, cells were infected with BVDV R1, R2, R3 or wt BVDV p0 (MOI 1). Following 1 h of adsorption on ice, the inoculum was removed, cell monolayers were washed twice with PBS, and 1 ml of infection medium was added to each well. After 24 h of incubation at 37°C, intracellular RNA was extracted with TRIzol reagent . RNA was retrotranscribed with random hexamers (Biodynamics) at 37°C for 90 min, using 0.5 U/μl M-MLV reverse transcriptase , and quantified by Real-time PCR.MDBK cells were seeded in a 24-well plate as stated above. After 24 h in a 5% COTo measure the intracellular viral RNA (ivRNA), a 108-bp fragment from the 5′NTR was amplified with the following oligonucleotides: 5′NTR forward 5′GAGGCTAGCCATGCCCTTAGT3′ and 5′NTR reverse 5′TCGAACCACTGACGACTACCCT3′. The reaction was carried out in 2X Power SYBR Green PCR Master Mix  in an ABI 7500 apparatus (Applied Biosystems), using the following experimental run protocol: 10 min of activation at 95°C, followed by 45 cycles of amplification and quantification  during which the SYBR GREEN signal was measured. To normalize target amplification data, a 91-bp fragment of bovine β-actin mRNA was amplified simultaneously in each sample and used as endogenous control (forward oligonucleotide: 5′CCCACACGGTGCCCATCTAT3′ and reverse oligonucleotide: 5′ CCACGCTCCGTGAGGATCTTC3′). Each sample was tested in triplicate and both positive and negative controls were included.1, R2 and R3 in relation with those infected with wt BVDV p0.Data were analyzed by means of the relative standard curve method r T1–5 and BVDV R1–5 virus suspensions obtained from clarified supernatants of infected cells. The RNA obtained was denatured at 70°C for 5 min and primed with the designed specific oligonucleotides  from wt BVDV p0, BVDV-TSCleotides . The revleotides  by PCR ap values were less than 0.050.The results were analyzed by Student's t test using InfoStat version 2011 1–5 were obtained after 20 passages of BVDV-TSCr T1–5 in the absence of TSC. BVDV R1–4 were not significantly inhibited by TSC, whereas wt BVDV p0 was completely inhibited  .BVDV R  0.003) . Howeverr T1–5 in the same conditions, some viral populations showed a delay in producing CPE. To reach 80–90% of CPE, the infections with BVDV-TSCr T2 and T5 required 72 h of incubation from the 1st to the 3rd passage and then 120 h of incubation from the 4th to the 20th passage. Similarly, BVDV-TSCr T3 required 120 h of incubation from the 13th to the 20th passage. This delay in producing CPE was also observed in the growth of viral plaques when the infected cultures were incubated in semi-solid medium. BVDV R2, R3, and R5 needed 72–96 additional hours of incubation to properly exhibit the viral plaques.The infection of MDBK cells with wt BVDV NADL cp at a MOI of 0.01 required 48 h of incubation at 37°C to reach 80–90% of CPE in the cell monolayer. However, along the passages of BVDV-TSC1 or R4 produced levels of apoptosis similar to those produced by wt BVDV p0 , whereas the infections with BVDV R2 and R5 produced lower level of apoptosis . BVDV R3 showed an intermediate value (9.1% of PE Annexin-positive cells) (BVDV cp isolates kill cells by triggering apoptosis e cells) .1 or R4 was lower than that in the cultures infected with BVDV R2, R3 or R5  and that in mock-infected cultures (6.4×105). Taking into account that apoptotic cells could be quantified only in the remaining adherent and floating cells, the total number of cells that would die by viral CPE in each infection was estimated . Having considered the differences in CPE between each viral infection, the results showed that the number of cells that would die by BVDV R2, R3 and R5 infections was markedly lower than that in wt BVDV p0 infection   .1 and R3–5) showed slightly higher virus titers (approximately three times higher) than wt BVDV p0, but, notably, BVDV R2 showed viral titers approximately 30 times higher.Although some viral strains produced no CPE, all viral populations showed the highest titers of infectious virus at 48 h p.i. . At that1 and R4 showed a marked decrease in viral titers at 72 h p.i., consistent with the lack of viable host cells as a result of the viral CPE. However, MDBK cells infected with R2, R3 and R5 continued producing infectious virus for at least four additional days.On the other hand, wt BVDV p0, R2 or R3 were still confluent until 72 h p.i and maintained a continuous production of NS3 . On the other hand, BVDV R3, which showed an intermediate level of decrease in cell apoptosis, also presented intermediate levels of ivRNA .The nucleotide sequence of Npro, Erns and the complete NS region of wt BVDV p0, BVDV-TSC1–5 conserved the mutations previously associated with the resistance to TSC 1–5 and C11,367A (A392E) in BVDV R1   .1: NS2 G5,207A (G472R); in BVDV R2: Erns T1,315Y (Y: mix of T and C), NS5A A8,731G (I9M), NS5A G8,860A, NS5A A9,617T (N305Y), and NS5B C11,987T; in BVDV R3: Erns T1,706G (C171G), NS4B A7,745R (R: mix of A and G) (E28G), NS5A A9,287R (K195E), NS5A G9,812R (E370K), NS5B A10,592G (I134V) and A10,975G (I261M); in BVDV R4: NS3 T6,912C, NS5A T9,285Y (M94T), NS5B C10,768T and NS5B G11,300C (K370N). Some other mutations  were conserved between BVDV-TSCr T1–5 and their respective BVDV R1–5: NS5B A10,723G in T1-R1 and NS5A T9,234C in T2-R2 (V177A). Notably, no new mutation was detected in BVDV R5 after 20 passages. However, whereas BVDV-TSCr T5 presented mixed population of T and C in nucleotide T9,234 (NS5A V177), BVDV R5 presented only C (A177)  . This sur T1–5) with mutations in NS5B  r T1–5 in the absence of TSC to analyze their genetic stability.We have previously reported the selection of TSC-resistant BVDV mutants (BVDV-TSC1–5). However, BVDV R1–5 produced higher titers of infectious virus than wt BVDV p0 and, along the passages in the antiviral-free environment, three out of the five viral populations  showed a delay in producing CPE.The resistance to TSC remained stable and mutations in NS5B associated with resistance were conserved in all the viral populations obtained , NS4B (BVDV R3), NS5A (BVDV R2–5) and NS5B (BVDV R3 and R4). Particularly, mutation NS5B I261M (BVDV R3) has been reported to compensate for the reduced fitness caused by the N264D mutation and to confer resistance to other BVDV NNI r were not replicated under a selective pressure, these additional mutations may have resulted from the genetic drift during the passages and could have also been detected in the wild type virus if it had been passaged in conditions similar to those of BVDV-TSCr.It is known that drug-resistant mutants may have fitness costs in the absence of the selecting drug, but the occurrence of compensatory mutations may allow them to partially or even completely regain fitness 2, R3, and R5 showed a delay in producing CPE. The cp biotypes of BVDV induce cell death by apoptosis 2, R3 and R5 showed a decrease in the induction of apoptosis and in the accumulation of viral RNA. However, BVDV R1–5 produced higher infectious virus titers than wt BVDV p0, and due to host cell survival, BVDV R2, R3 and R5 produced viral antigens and infectious viral progeny for at least four additional days.As mentioned above, BVDV R1–5 showed no deletion (or insertion) in the cIns or in any of the viral sequences. In addition, BVDV R2, R3 and R5 showed no amino acid changes in NS2 or NS3, whose bond cleavage is required for RNA replication and is linked to BVDV cytopathogenicity 2, R3 and R5 in producing CPE may not result from an alteration in the production or function of these viral proteins. On the other hand, BVDV R3 showed a mutation in NS4B (E28G). This protein would also play a role in BVDV cytopathogenicity BVDV NADL cytopathogenicity is determined by an in-frame insertion in the NS2 coding region of a 270-nucleotide cellular mRNA sequence , which modulates NS3 production and upregulates RNA replication 2 and R3 and the only one detected in BVDV R5 were in NS5A. BVDV NS5A is a zinc metalloprotein essential for the formation of a functional replicase complex 2, V177A in BVDV R5 and K195E in BVDV R3) and domain II (N305Y in BVDV R2 and E370K in BVDV R3), both critical for RNA replication Interestingly, most of the additional amino acid changes found in BVDV RTo our knowledge, this is the first report on the stability and evolution of non-nucleoside polymerase inhibitor-resistant BVDV. The study of the TSC-resistant viruses that showed reduced CPE and prolonged virus production may be an interesting tool to learn more about the virus-cell interaction, viral cytopathogenesis, attenuation and persistence. Further studies and reverse genetic experiments would be needed to identify the specific phenotype of each of the additional mutations observed.


DCC and UNC5C, are frequently downregulated in many cancers. We hypothesized that downregulation of DCC and UNC5C has an important growth regulatory function in gastric tumorigenesis.The gene expressions of netrin-1 dependence receptors, DCC and UNC5C were performed in a Japanese cohort of 98 sporadic gastric cancers and corresponding normal gastric mucosa specimens. Loss of heterozygosity (LOH) analyses and microsatellite instability (MSI) analysis was applied to determine chromosomal instability (CIN) and MSI phenotypes, respectively. More than 5 % methylation in the DCC and UNC5C promoters were found in 45 % (44/98) and 32 % (31/98) gastric cancers, respectively, and in 9 % (9/105) and 5 % (5/105) normal gastric mucosa, respectively. Overall, 70 % (58 of 83 informative cases) and 51 % (40 of 79 informative cases) of gastric cancers harbored either LOH or aberrant methylation in the DCC and UNC5C genes, respectively. In total, 77 % (51 of 66 informative cases) of gastric cancers showed cumulative defects in these two dependence receptors and were significantly associated with chromosomal instability. Both DCC and UNC5C were inactivated in 97 % of CIN-positive gastric cancers and in 55 % of CIN-negative gastric cancers.In the present study, a series of genetic and epigenetic analyses for DCC alterations are apparent in the early stages, and UNC5C alterations escalate with the progression of the disease, suggesting that the cumulative alterations of netrin-1 receptors was a late event in gastric cancer progression and emphasizing the importance of this growth regulatory pathway in gastric carcinogenesis.Defect in netrin receptors is a common feature in gastric cancers. The online version of this article (doi:10.1186/s13148-015-0096-y) contains supplementary material, which is available to authorized users. Helicobacter pylori (H. pylori) infection. Previous reports indicated a 93.1–100 % infection rate for H. pylori in patients with gastric cancer, while only 1.2–2.8 % of individuals infected with H. pylori develop gastric cancer  among gastric cancer tissues that displayed over 1.0 % methylation in the DCC promoter and 4.9 %  in the corresponding normal gastric mucosa specimens that displayed over 1.0 % methylation  and <5.0 % methylation as methylation-negative (unmethylated)). Using this criterion, we observed DCC methylated cases in 44/98 gastric cancers (45 %) and in 9/105 normal gastric mucosa (9 %).We investigated DCC promoter methylation and various clinicopathological and genetic features. DCC methylation status was significantly associated with MSI status. MSI-positive gastric cancers were significantly more frequently associated with DCC methylation than with DCC unmethylation  at D18S35, 17/55 (31 %) at D18S69, and 21/58 (36 %) at D18S58  levels by reverse transcription quantitative polymerase chain reaction (RT-qPCR) using a primer set previously described [DCC expression and CpG methylation status in the DCC promoter region in 10 gastric cancer cell lines  and one normal lung fibroblast cell line (NHLF). All cell lines, except for MKN74, GCIY, OGUM-1, MKN1, and NHLF cells, showed decreased expression of DCC gene transcripts, and their promoters were methylated  staining results are shown in Fig. Next, we investigated the association between DCC protein expression and genetic and epigenetic alterations in the DCC promoter methylation and 18q LOH. We found that among the cancers with complete loss of DCC expression, 5/7 cancers (71 %) demonstrated both DCC promoter methylation and 18q LOH. In contrast, 8/33 cancers (24 %) showed focal loss of DCC expression and both methylation and LOH, 5/33 cancers (15 %) showed LOH alone, and 12/33 cancers (36 %) displayed methylation alone. Among the cancers that were positive for DCC expression, only 2/34 cancers (6 %) demonstrated both methylation and LOH, 13/34 (38 %) cancers LOH alone, and 8/34 cancers (24 %) methylation alone  and in cancers with 18q LOH alone , intermediate in cancers with DCC methylation alone , and lowest in cancers negative for DCC alterations . When we categorized gastric cancers with LOH ratios higher than 0 as CIN-positive, 12/17 gastric cancers (71 %) with both DCC methylation and 18q LOH, 13/17 gastric cancers (76 %) with 18q LOH alone, 12/22 gastric cancers (55 %) with DCC methylation alone, and 6/25 cancers (24 %) negative for DCC alterations were categorized as CIN-positive . Our data suggest that DCC alterations caused by both epigenetic and genetic alterations were significantly associated with gastric cancers exhibiting the CIN phenotype.Among the 58 cancers with UNC5C methylation status was examined in a cohort of 98 gastric cancers and 105 normal gastric mucosa specimens. Location of the UNC5C gene and a panel of representative COBRA results are depicted in Fig. UNC5C methylation levels as continuous variables. We found that 39/98 gastric cancers (40 %) and 16/105 normal gastric mucosa specimens (15 %) displayed more than 1 % methylation in the UNC5C promoter. Of the samples that showed more than 1 % methylation in the UNC5C promoter, mean methylation levels of UNC5C were significantly higher in gastric cancers compared with their corresponding normal gastric mucosa specimens . Using this cutoff value, 31/98 gastric cancers (32 %) and 5/105 normal gastric mucosa specimens (5 %) were diagnosed as UNC5C-methylated. Next, we examined associations between UNC5C promoter methylation status and the clinicopathological and genetic features of gastric cancers. UNC5C methylation showed a significant association with MSI status. MSI-positive gastric cancers were significantly more frequent in gastric cancers with UNC5C methylation compared with those without UNC5C methylation  by RT-PCR  and one normal lung fibroblast cell line (NHLF), we found that only two cell lines, GCIY and NHLF, expressed all five splicing variants of UCNC5C messenger RNA. In nine gastric cancer cell lines, there was no expression of any of the five splicing variants of UCNC5C mRNA  of UNC5C unmethylated cell lines lacked UNC5C mRNA expression . Thus, UNC5C gene transcript expression was significantly associated with promoter methylation level. Additionally, we tried to perform IHC staining for UNC5C protein expression. Unfortunately, we were unable to analyze UNC5C protein expression due to lack of appropriate antibodies.Before examining UNC5C expression and methylation status in the UNC5C were 29 % (11/38 informative cases) at D4S2380, 23 % (10/44) at D4S470, and 28 % (12/43) at D4S1559 .The frequencies of 4q21–23 LOH at each microsatellite marker for P < 0.0001; Table P = 0.0003; Table KRAS/PIK3CA mutations were found in 4q LOH-negative gastric cancers with a non-significant difference (P = 0.08).Similar to 18q LOH, we found a strong correlation between 4q LOH and CIN phenotype. The LOH ratio calculated for the remaining seven loci was significantly higher in gastric cancers with 4q LOH compared with those without 4q LOH  for stages I and II vs. 63 % (26/41) for stages III and IV, P = 0.02, Pearson’s chi-square test) and in advanced categories for lymph node metastasis (25 % (5/20) for N0 vs. 59 % (35/59) for N1–N3, P = 0.008, Pearson’s chi-square test).Next, we investigated the relationship between UNC5C alterations, 10 cancers showed alterations both in terms of UNC5C methylation and 4q LOH, 13 cancers showed 4q LOH alone, and 17 cancers showed UNC5C methylation alone. The LOH ratio calculated for the remaining seven loci was significantly higher in cancers with both UNC5C methylation and 4q LOH  and in those with 4q LOH alone (0.37 ± 0.25), intermediate in cancers with UNC5C methylation alone (0.24 ± 0.29), and lowest in cancers negative for UNC5C alterations . When we categorized the cancers that showed LOH ratios higher than 0 as CIN-positive, 8/10 gastric cancers (80 %) with both UNC5C methylation and 4q LOH, 11/13 gastric cancers (85 %) with 4q LOH alone, 9/17 gastric cancers (53 %) with UNC5C methylation alone, and 11/39 cancers (30 %) negative for UNC5C alterations were categorized as CIN-positive . Similar to the DCC alterations in gastric cancer, our data highlight that UNC5C alterations caused by both epigenetic and genetic events were significantly associated with CIN-positive gastric cancers.Among the 40 cancers with UNC5C and/or DCC defects and TNM stage in the 98 gastric cancers that were informative for both UNC5C and DCC genetic/epigenetic results  for stages III and IV) than in earlier-stage cancers (24 % (8/33) for stages I and II, P = 0.001, Pearson’s chi-square test; Fig. UNC5C or DCC and their relationship with tumor stage, UNC5C alterations were found in 18 % (2/11 informative cases), 48 % (12/25 informative cases), 63 % (19/30 informative cases), and 64 % (7/11 informative cases) of stages I, II, III, and IV cancers, respectively, showing that UNC5C alterations gradually developed according to the progression of the TNM stage , 76 % (19/25 informative cases), 66 % (19/29 informative cases), and 77 % (10/13 informative cases) of stages I, II, III, and IV cancers, respectively  of stage I, 19 of 25 (76 %) of stage II, 19 of 29 (66 %) of stage III, and 10 of 13 (77 %) of stage IV cancers  of stage I, 12 of 25 (48 %) of stage II, 19 of 30 (63 %) of stage III, and 7 of 11 (64 %) of stage IV cancers  and 5/105 normal gastric mucosa specimens (5 %) exhibited UNC5C methylation. Therefore, we performed UNC5C IHC on the clinical specimens to examine the two-hit theory in which aberrant promoter methylation and allelic losses were the key factors determining lack of DCC. However, we were unable to perform UNC5C IHC on the clinical specimens because of the lack of an appropriate antibody for tissue staining.The other netrin-1 receptors, UNC5A, UNC5B, and UNC5C, were also discovered as putative tumor suppressor genes in various tumors , 27. AmoDCC and UNC5C share the same netrin ligand and are colocalized in the gut [DCC or UNC5C may not be sufficient to promote tumor development in the stomach. In this study, we found that 97 % of gastric cancers with CIN and 55 % of those without CIN showed simultaneous alterations in both DCC and UNC5C, supporting our hypothesis that inactivation of both receptors may be required in the development of gastric cancer. Our finding that dysregulation of DCC predominantly occurs in the early phase of gastric cancer whereas UNC5C alterations occur later suggests that inactivation of these receptors is not a random process but occurs in a statistically predictable, sequential manner.Because both  the gut , 26, 40,H. pylori in the human gastric mucosa is a well-known inducer of chronic inflammation and gastric cancers and is associated with a high incidence of aberrant DNA methylation [H. pylori infection by recovering the cagA repeat sequence from gastric cancer specimens as well as normal gastric mucosa samples. Of 98 cancers, 70 cancers were positive for the cagA sequence. However, there was no association between the presence of the cagA sequence in cancer tissue and clinicopathological and molecular features, specifically not DCC nor UNC5C methylation incidence. Conversely, with respect to normal counterpart gastric mucosa, a total of 102 normal gastric mucosa samples were available for analyzing the presence of the cagA sequence in this study. Of 102 gastric mucosa samples, the cagA sequence was successfully recovered in 79 (74 %). Interestingly, when we defined DCC and UNC5C methylation at 1 % or more as a continuous variable  and <1.0 % methylation as methylation-negative (unmethylated)), only UNC5C methylation was significantly associated with the presence of the cagA sequence in normal gastric mucosa (data not shown), suggesting that inflammatory processes associated with H. pylori infection causes aberrant methylation in UNC5C promoter CpGs but not in the DCC promoter. Thus, our results suggested that H. pylori infection does not induce aberrant methylation randomly but in target loci by a particular signal cascade. To address questions underlying H. pylori infection and epigenetic changes, further investigations are warranted.hylation , 30, 29.DCC and UNC5C receptors. Such alterations are apparent in the early stages and continue to escalate in both receptor types with disease progression, emphasizing the importance of this growth regulatory pathway in gastric carcinogenesis.We provide previously unrecognized and novel evidence that most gastric cancers, particularly those with CIN, possess alterations in both We collected tissue specimens of primary gastric cancer and matched normal gastric mucosa from 105 patients who had undergone surgery at the Okayama University Hospital . Of 105 gastric cancer patients, seven did not have sufficient tumor tissue for analysis. Thus, in this study, a total of 98 gastric cancer specimens and 105 matched normal gastric mucosa tissues were analyzed. All normal gastric mucosa specimens were obtained from sites adjacent to the tumor but at least 5 cm away from the tumor site. All patients provided written informed consent, and the study was approved by the ethics committee of the Okayama University Hospital. All patients also gave informed consent for usage of their data for future analyses. The histological diagnosis was established according to the World Health Organization International Histological Classification of tumors, with subclassification into two histological categories: differentiated type (well and moderately differentiated tubular adenocarcinoma) and undifferentiated type (poorly differentiated adenocarcinoma and mucinous adenocarcinoma). The pathological stage was determined according to the International Union Against Cancer TNM classification (Seventh edition).2.A total of 12 human gastric cancer cell lines , three human colorectal cancer cell lines , one human colon fibroblast cell line (CCD18Co), and one human lung fibroblast cell line (NHLF) were purchased from American Type Culture Collections , Japanese Collection of Research Bioresources Cell Bank , Health Science Research Resources Bank , RIKEN , or Cambrex Bio Science Walkersville, Inc. . All cell lines were cultured in appropriate culture medium supplemented with 10 % fetal bovine serum, penicillin (100 IU/mL), and streptomycin (100 μg/mL) at 37 °C in a humidified incubator with 5 % COGenomic DNA from the cell lines was extracted using QIAamp DNA Mini Kit . All gastric cancers and normal gastric mucosa samples were fresh-frozen tissue specimens, from which DNA was extracted using standard procedures that included proteinase-K digestion and phenol–chloroform extraction. Total RNA from six cultured cell lines  was obtained using the TRIzol reagent . Total RNA from 10 gastric cancer cell lines  and a normal lung fibroblast cell line, NHLF, was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions.UNC5C (including primers for the detection of splicing variants), DCC, and beta-actin . The expression level of each target gene was analyzed based on the ΔCT method, and beta-actin was used as an endogenous control to normalize the amount of total RNA in each sample.The first-strand complementary DNA synthesis was performed using the Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies Inc.) and miScript II RT Kit (Qiagen) with a total of 1.0 μg RNA. RT-PCR was performed using specific primer pairs for DCC and UNC5C promoters in gastric tissues and cell lines was analyzed by combined bisulfite restriction analysis , the hMSH2 locus on 2p16 (D2S123), the APC locus on 5q21 , the UNC5D locus on 8p12 (D8S87), and the p53 locus on 17p13  [UNC5C and DCC loci. When a tumor showed a LOH ratio higher than 0, the tumor was categorized as CIN-positive.In addition, to examine the association between netrin-1 receptor disorders and CIN phenotype, we analyzed additional seven sets of polymorphic microsatellite sequences that are tightly linked to known tumor suppressor genes and DNA mismatch repair genes, including the 0, TP53) . Of 98 gMSI status was analyzed for all 98 gastric cancer patients using three mononucleotide repeat markers  as described previously . When atKRAS and BRAF mutation status was analyzed in all 98 patients as described previously [PIK3CA exon 9 and 20 mutation status was also analyzed by direct sequencing. PCR and sequencing were performed using PIK3CA exon 9- and 20-specific primer pairs  repeat sequence in the cagA protein, which binds to the Src homology 2 domain-containing protein tyrosine phosphatase, SHP-2, on gastric epithelial cells. The cagA was recovered by PCR amplification performed on genomic DNA templates from tumor tissues. We modified the primer design to develop PCR products shorter than the PCR products described previously [To determine eviously , 43. PCRDCC protein expression analysis. Staining was carried out manually with formalin-fixed paraffin-embedded tissues. Thin (5 μm) sections of representative blocks were deparaffinized and dehydrated using gradient solvents. Following antigen retrieval in citrate buffer (pH 6.0), endogenous peroxidase was blocked with 3 % H2O2. Thereafter, slides were incubated overnight in the presence of a purified mouse anti-human DCC monoclonal antibody . A further incubation was carried out with a secondary antibody and the avidin-biotin-peroxidase complex  and then incubated with biotinyltyramide followed by streptavidin-peroxidase. Diaminobenzidine was used as a chromogen, and hematoxylin as a nuclear counterstain. Tissue sections with obvious nuclear staining were considered positive. The only foci of neoplasia that were scored as negative were those for which there was definite evidence of unspecific positively staining admixed or surrounding non-neoplastic cells such as normal colonic mucosal cells, lymphocytes, or stromal cells.A total of 89 gastric cancers from 98 patients were available for IHC staining for DCC and UNC5C methylation levels were analyzed as continuous variables, as were computed means, standard errors of the means, and standard distributions. Next, the methylation status of the DCC and UNC5C promoter was analyzed as a categorical variable . Differences in frequencies were evaluated by Pearson’s chi-square test or the Wilcoxon/Kruskal–Wallis test. Whenever the Kruskal–Wallis test indicated differences among these subgroups, further pairwise comparisons for each of the subgroup was performed by a non-parametric multiple comparison method using the Steel–Dwass test. All reported probability (P) values were two-sided, and a P value of less than 0.05 was considered statistically significant.Statistical analyses were performed using JMP software . First,