Overexpression of mutant p53 is a common finding in most cancers but testicular tumours accumulate wild-type p53 (wtp53). In contrast to the accepted concept that p53 homozygous mutant mice do not accumulate mutant p53 in normal cells, our study on a mutant p53 mouse model of Li-Fraumeni syndrome harbouring the hot-spot p53R172H mutation described an elevated level of mutant p53 in non-cancerous mouse tissues. Here we use detailed immunohistochemical analysis to document the expression of p53R172H in mouse testis. In developing and adult testes, p53R172H was expressed in gonocytes, type A, Int, B spermatogonia as well as in pre-Sertoli cells and Leydig cells but was undetectable in spermatocytes and spermatids. A similar staining pattern was demonstrated for wtp53. However, the intensity of wtp53 staining was generally weaker than that of p53R172H, which indicates that the expression of p53R172H can be a surrogate marker of p53 gene transcription. Comparing the responses of wtp53 and p53R172H to irradiation, we found persistent DNA double-strand breaks in p53R172H testes and the formation of giant spermatogonia (GSG) following persistent DNA damage in p53R172H and p53-null mice. Strikingly, we found that p53R172H promotes spontaneous formation of GSG in non-stressed p53R172H ageing mice. Two types of GSG: Viable and Degenerative GSG were defined. We elucidate the factors involved in the formation of GSG: the loss of p53 function is a requirement for the formation of GSG whereas DNA damage acts as a promoting trigger. The formation of GSG does not translate to higher efficacy of testicular tumorigenesis arising from mutant p53 cells, which might be due to the presence of delayed-onset of p53-independent apoptosis. It is not clear why tumour cells with p53 mutations are not selected for in the development of this cancer.Testicular malignancy is the most common cancer type in males aged 15–34 years and is still on the rise.6 The ability to detect p53R172H protein in morphologically normal tissues provides an avenue to study the regulation of p53R172H expression throughout the different stages of mouse development under non-stressed conditions and in the presence of cellular stressors (for example irradiation), which further helps us to understand the function and distribution of wtp53 in normal mouse tissues.The level of p53 protein is tightly regulated by Mdm2, which promotes proteasomal degradation of p53 to maintain its low basal level in the absence of cellular stressors. Our previous study on a mouse model of Li-Fraumeni syndrome with a missense mutation at codon 172 of the p53 gene  described an elevated level of p53R172H in cycling/proliferating but non-cancerous morphologically normal mouse tissues. Similar to wtp53, p53R172H is also degraded by Mdm2 and responds to irradiation.9 Testicular cells are sensitive to DNA damage; thus, the testis is a suitable organ to study expression patterns of p53R172H and its response to DNA damage. The progression of spermatogonial stem cells passes through 12 stages of seminiferous epithelial cycles,10 with each stage being characterized by a combination of testicular cell types. Residing on the basement membrane of seminiferous tubules, undifferentiated spermatogonia (Aundiff or Asg) proliferate to form differentiating spermatogonia (Adiff) type A1 at stage VIII, and type A2, A3, A4, Int, B spermatogonia during stage IX–VI. Aundiff become quiescent at stages III–VII. Inactivated preleptotene spermatocytes can be found in a more adluminal position at stage VII, and further differentiate to active preleptotene spermatocytes at stage VIII, which enter the prophase of meiosis (leptotene and zygotene) at stages IX–XII.12 In addition, there are supportive cells, for example Leydig cells that reside at highly vascularized interstitium, and Sertoli cells, which are in close proximity to spermatogonia and basal lamina.Spermatogenesis progresses through a series of cycling cell lineage development relying on the activities of spermatogonial stem cells and transient amplifying spermatogonia.p53−/− mice and compared this to p53R172H/R172H mice to check for any gain-of-function of p53R172H. We are particularly interested in the formation and clearance of giant spermatogonia (GSG), which might be associated with subsequent development13 or elimination of testicular malignancy after p53 mutation.In this study, we investigated the expression of p53 proteins – wtp53 and p53R172H mutant (mutp53) through a detailed immunohistochemistry (IHC) analysis of morphologically normal embryonic, postnatal and adult mouse testis. We looked for any difference in the way mutant testicular cells respond to DNA damage induced by γ-irradiation. We also studied the effect of loss of p53 function using p53R172H/R172H mice exhibit morphologically normal testes with well-developed seminiferous tubules and interstitium. There was detectable and heterogeneous mutp53 expression in spermatogonia and Leydig cells in p53R172H/R172H mice , high amounts of p53 mRNA in spermatogonia and Leydig cells of both p53+/+ and p53R172H/R172H mice were detected -neomycin fusion protein (βGeo) was inserted into intron 1 of the p53 gene sequence, and was under the control of the endogenous p53 promoter. β-galactosidase activity indicated p53 gene transcription and could be visualized by X-gal staining. β-galactosidase was detected in spermatogonia and Leydig cells of staining . Homozyg18 At E13.5, gonocytes uniformly expressed mutp53. However, at E16.5, gonocytes stopped expressing mutp53 but pre-Sertoli cells marked by Sox9 expressed strong mutp53 , primordial germ cells are enclosed by somatic cells (pre-Sertoli cells). Primordial germ cells become gonocytes and proliferate until E15.5 before becoming quiescent. Gonocytes renew proliferation shortly after birth to give rise to Asg.g mutp53 . At postg mutp53 . Strong g mutp53  revealing mutp53 . This su19 These cells expressed both mutp53 and cyclin D1 revealing their proliferative properties , zygotene (Z), pachytene (P) spermatocytes could be found at P10, P14 but mutp53 was undetectable in these cell types. Mutp53 was also undetectable in secondary spermatocytes at P21 and P28 (data not shown). We also detected the expression of wtp53 in postnatal testes of The data from developing embryonic/postnatal to adult testes further indicate that mutp53 protein can be detected in proliferating type A, Int and B spermatogonia, a subset of Leydig cells, proliferative pre-Sertoli cells during embryonic stages, proliferating gonocytes and PriA in embryos and neonate.2 Therefore, it is important to investigate if the DNA damage response in p53 wild-type testes and mutant testes differs. p53+/− and p53R172H/− mice were subjected to 2 Gy irradiation and testes were harvested at different time points including 3 h, 6–8 h, 1 day, 2–3 days and 6 days post irradiation. The induction of p53, p21 and DNA double-strand breaks (DSBs), the states of proliferation and apoptosis were compared in irradiated p53+/− and p53R172H/− mice testes by staining of p53, p21, 53BP1, γH2AX, PCNA and cleaved caspase 3.As described earlier, mutp53 is not necessary for testicular tumorigenesis.p53+/− and p53R172H/− mice testes were low .The basal levels of p53 in were low ; howeverwere low . The numadiation . p21 wasp53+/− and p53R172H/− mice with and without irradiation. In non-irradiated control mice testes, we observed distinct levels of pan-nuclear distribution of 53BP1 in a subset of spermatogonia (mostly A spermatogonia) representing a pre-existing pool of 53BP1 , 53BP1 and γH2AX in testes from of 53BP1 . Condensof 53BP1  and irraof 53BP1 .p53+/− and p53R172H/− mice testes at 3 and 6–8 h time points  cell cycle progression is impaired differently in mice of distinct genotypes. In addition, this enables us to verify any loss-of-function and/or gain-of-function of p53R172H mutant.To further determine the long-term effects of DSBs induced by irradiation, we subjected p53R172H/R172H mice showed loss of spermatogonia. However, there was an interesting appearance of some dysplastic cells, named ‘Giant spermatogonia' (GSG) lying on the basal compartment of the different stages of seminiferous tubules  indicating they were no longer actively proliferating.22 We then stained for cyclin A2 and Aurora B, which were immunopositive in mutp53 highly expressed G2/M phase spermatogonia  compared to p53+/+ mice. Although we could not find significant differences in the number of GSG between p53R172H/R172H and p53−/− mice 14/21 days post irradiation, the sharply reduced number of GSG at 21 days compared to that at 14 days post irradiation indicates those GSG might have been eliminated from system by programmed cell death : (1) Viable GSG expressed high level of mutp53, cyclin A2, pan-nuclear 53BP1 distribution and condensed H3p/Aurora B. Furthermore, viable GSG did not co-express cleaved caspase 3 and thus were not apoptotic  p53R172H/R172H and p53−/− mice but absent in p53+/+ mice indicated by the immunostaining of cyclin A2, Aurora B and mutp53  (p53R172H/R172H mice was significantly higher when compared with that in p53−/− mice (P<0.05), indicating that p53R172H could possibly promote the formation of GSG.The formation of GSG was not limited to irradiated d mutp53 . Since >(P<0.05) . At 5-mop53+/+, p53R172H/R172H and p53−/− mice testes by 53BP1 and γH2AX immunostaining. Although DSBs were rare in younger 2-mo mouse testis, there was a general increase in spermatogonia and Sertoli cells of 5-mo p53+/+, p53R172H/R172H and p53−/− mice  and performed in compliance with IACUC regulations. The murine Trp53 knockout and p53R172H mice and the irradiation methods to those mice were previously described.oach see . Briefly34 The experiments were repeated three times. The antibodies used for IHC/immunofluorescence staining are listed in 34Mice testes for IHC and immunofluorescence staining were proceeded as described previously.in situ detection using the RNAscope Detection Kit and Trp53 (NM_010195) probe according to the manufacturer's instructions .Tissue sections were processed for RNA 35 and see Mice testes for X-Gal staining were fixed and stained for X-Gal  (Invitrogen) as described previouslySee P-values were analysed using Graphpad software (Prism) and calculated by 1-way ANOVA.Four to six mice per group were used for histological analysis. Cyclin A2 and cleaved caspase 3 stained testis slides were scanned by VSViewer system and 20 views of × 10 images were captured for the counting of GSG and apoptosis cells. The


Entamoeba histolytica, the etiological agent of amoebiasis, follows a novel pathway, which includes nuclear division without the nuclear membrane disassembly. We report a nuclear localized Ca2+-binding protein from E. histolytica (abbreviated hereafter as EhCaBP6), which is associated with microtubules. We determined the 3D solution NMR structure of EhCaBP6, and identified one unusual, one canonical and two non-canonical cryptic EF-hand motifs. The cryptic EF-II and EF-IV pair with the Ca2+-binding EF-I and EF-III, respectively, to form a two-domain structure similar to Calmodulin and Centrin proteins. Downregulation of EhCaBP6 affects cell proliferation by causing delays in transition from G1 to S phase, and inhibition of DNA synthesis and cytokinesis. We also demonstrate that EhCaBP6 modulates microtubule dynamics by increasing the rate of tubulin polymerization. Our results, including structural inferences, suggest that EhCaBP6 is an unusual CaBP involved in regulating cell proliferation in E. histolytica similar to nuclear Calmodulin.Cell cycle of  E. histolytica, the etiological agent of amoebiasis, is a protozoan parasite responsible for around 100,000 deaths per year in developing nations. Though the organism has been identified more than 100 years back, there is not much understanding about the biology of this organism. Calcium signaling plays an important role in the biology of this organism. Here we show structure-functional relationship of one of the Ca2+-binding proteins (abbreviated as EhCaBP6) and suggest its involvement in cell division in this parasite. EhCaBP6, a nucleo-cytosolic Ca2+-binding protein, is a microtubule end binding protein and overexpression of its gene induces an increase in number of microtubular assemblies in E. histolytica. Cell division cycle in E. histolytica occurs along the microtubular structures without disruption of nuclear envelope. Occurrence of multinucleated cells in culture suggests duplication and reduplication of nuclear DNA without cytokinesis. Although Kinesin like protein (Klp1), Formin1 and EhCaBP6 were shown to be part of the microtubular assembly, their role in regulation of the cell cycle is not yet documented. Further, E. histolytica does not have a typical CaM like protein. However, the 3D structure of EhCaBP6 with two Ca2+-binding sites is similar to CaM, in spite of their low sequence similarity. Here, we demonstrate that EhCaBP6 regulates cell cycle specifically by facilitating DNA synthesis, transition from G1 to S phase and cytokinesis. The structural and functional similarity between EhCaBP6 and CaM suggests EhCaBP6 to be a functional homologue of nuclear CaM with important roles in regulation of cell cycle. Apo-formembrane .2+]2-EhCaBP6 were recorded on a JASCO J-810 spectropolarimeter equipped with a Peltier temperature controller. Protein concentration of 20 μM was used in recording the far-UV CD spectra for characterizing the secondary structure of the protein at different pH values ranging from 5 to 9.The CD spectra of  HSQC and 2D [13C-1H] HSQC using pulsed-field gradients for coherence selection [15N-1H] HSQC (τm = 60 ms) [15N-1H] HSQC (τm = 80 ms), 3D NOESY-[13C-1H] HSQC (τm = 100 ms), 3D HNHA, GFT D HNHA, 3D HNHB, GFT D HNHB and 3D HCCH COSY [13C/15N doubly labeled samples for backbone and side-chain resonance assignments were as described earlier [1H-1H]-NOESY in 2H2O with a mixing time (τm) of 100 ms. All the spectra were processed using TopSpin 3.1 and analyzed by TATAPRO 2-EhCaBP6 were carried out with CeCl3 (Sigma Aldrich) to monitor Ca2+-displacement by Ce3+. The protein concentration was 0.5 mM in a buffer containing 50 mM Tris-HCl (pH = 6.8) and 50 mM NaCl. For each titration, an aliquot of 0.5 μL CeCl3 of the stock solution (50 mM) was added to the NMR tube containing the protein solution, mixed, equilibrated and followed by recording 2D [15N-1H] HSQC at 25°C. The HSQC spectra thus recorded were processed with identical processing parameters. Integral volumes for the individual 15N-1H cross peaks in the resultant spectra were measured using Topspin 3.1.Paramagnetic titration experiments with [Ca2+-ligand coordination (described below in the Ca2+-Ligand Distance Constraints section) using standard simulated annealing and torsion-angle dynamics protocol with the program Cyana 3.97 [1H-1H]-NOESY-[15N-1H]-HSQC,3D-[1H-1H]-NOESY-[13C-1H]-HSQC and 2D-[1H-1H] NOESY spectra were identified, assigned, and linked in the NOESY spectra using the program CARA. A total of 1003 distance constraints, which included 465 intra-residue, 428 inter-residue , 59 medium-range, and 51 long-range distance constraints, were used in the structure calculation. Hydrogen bond constraints were added for residues that are involved in α–helical and β–sheet conformation. These secondary structural elements were characterized by the chemical shift indices and 3J(1HN-1HA) values derived from GFT D HNHA, and were further supported by calculating deuterium exchange rates. A total of 78 H-bond constraints were used during the structure calculation. The dihedral angle constraints were derived from TALOS using the backbone 1HA, 15N, 13CA, 13CB, and 13CO chemical shift values. A total of 252 (φand ψ) dihedral angle constraints were used in the structure calculation of EhCaBP6 in its Ca2+-bound state.The 3D structure of EhCaBP6 was calculated using the information derived from the solution NMR data, which included nOe distance restraints, backbone torsion-angle restraints, restraints due to hydrogen bonds, and Caana 3.97 ,55. The 2+-binding loop there is a strong hydrogen bond formed between the side-chain carboxyl oxygen of Asp at position 1 and the backbone 1HN of the Gly residue at position 6. In the present case, the 1HN of Gly-28 and Gly-101 are involved in such hydrogen bonding, causing a downfield chemical shift in their spectral signatures. Further, this led to the appearance of corresponding 15N-1HN correlations 2D [15N-1H] HSQC in a non-overlapping region. It has also been found from crystallographic studies on several EF-hand CaBPs that Ca2+ coordinates directly with the highly homologous residues at positions 1, 3, 5, 7, and 12 of individual Ca2+-binding loops. In the present case, the coordinating residues in EF-I and EF-III could be confirmed by paramagnetic titration experiments, where in Ca2+ is displaced by a trivalent Ce3+ in [Ca2+]2-EhCaBP6, which results in the broadening of the spectral signatures of the coordinating residues. The information thus derived was used in the final stages of structure determination and refinement. The upper bound distance constraints between the Ca2+ and residues at positions 1, 3, 5, 7, and 12 were thus varied from 3 Å to 4.5 Å to fix the Ca2+ within the two Ca2+-binding loops EF-I and EF-III.Crystallographic data on several EF-hand proteins suggest that in a canonical Ca2+]2-EhCaBP6 has been deposited in the PDB (pdb id: 5B7X). The corresponding structural statistics for NTD and CTD separately are given in All these constraints taken together (on an average 9 constraints per residue), five hundred structures were calculated, from which 20 structures with the lowest target function values were selected for structural analysis individually for both the N- and C-terminal domains (NTD and CTD) and also for the full length protein. The program PROCHECK-NMR available on PSVS-1.5 web server was used to estimate the quality of the lowest energy structures of NTD and CTD and validated them. The structures were visualized using VMD .The coorE. histolytica HM-1:IMSS strains were maintained and grown in TYI-S-33 medium containing 250 μg/ml benzyl penicillin and 0.25 mg/ml streptomycin per 100 ml of medium. The cells were then grown for 48 h (60–70% confluent) prior to carrying out experiments.The transformed cells containing tetracycline inducible vector were grown in the presence of 10 μg/ml of Hygromycin B. For experiments, cells were grown for 48 h in the presence of 10 μg/ml Hygromycin B. The cells were serum starved for 24 h with incomplete media containing 0.5% of adult bovine serum. Post 24 h serum starvation; cells were replenished with complete media containing 15% adult bovine serum. 10 μg/ml of Hygromycin B as well as 30 μg/ml tetracycline was added and the cells were harvested at different time points (0–12 h).E. histolytica. In short, log phase culture was harvested and washed with 1X PBS pH 8.0. The harvested cells were washed twice with incomplete cytomix buffer (10 mM K2HPO4/KH2PO4 (pH 7.6), 120 mM KCl, 0.15 mM CaCl2, 25 mM HEPES (pH 7.4), 2 mM EGTA, 5 mM MgCl2). The cells were then resuspended in 0.8 ml complete cytomix buffer (incomplete cytomix buffer containing 4 mM ATP and 10 mM reduced Glutathione). A 100 μg of plasmid DNA was added to the cells and subjected to two consecutive pulses of 3,000 V cm− 1 (1.2 kV) at 25 mF . The transfectants were initially allowed to grow without drug. However, after 48 h, 5 μg/ml Hygromycin B was added. Concentration of the drug was increased gradually to 10 μg/ml till a stable cell line was obtained.Electroporation was used for transfection of pEhHYG-tetR-O-CAT shuttle vector was used for cloning of sense and antisense constructs . The CATCaBP6 Sense Fp: 5’ CGG GGT ACC ATG TCT ATG GAA ATA GAA GC 3’CaBP6 Sense Rp: 5’ GCG GGA TCC TTA AAG AGC AAC AAG TTT AA 3’CaBP6 Antisense Fp: 5’ CGC GGA TCC ATG TCT ATG GAA ATA GAA GC 3’CaBP6 Antisense Rp: 5’ GCC GGT ACC TTA AAG AGC AAC AAG TTT AA 3’The constructs were then transfected as mentioned previously .The gene encoding EhCaBP6 was cloned in the BamH1 restriction site of pEh-Neo-GFP vector. The vector has been previously constructed by cloning the GFP mut3 allele of GFP  in the uTo obtain synchronous culture at G0/G1 phase, the cells were serum starved for 24 h with incomplete media containing 0.5% adult bovine serum. The cells were released from the G0/G1 arrest by transferring the culture to complete medium containing 15% adult bovine serum. In order to obtain cells at different phases, cells were harvested at different time point after serum replenishment. In case of transfectants, the cells were induced with 30 μg/ml of tetracycline after serum replenishment and were harvested at different time point.E. histolytica cells, which were briefly resuspended in warm incomplete TYI-33 medium, were transferred onto acetone wiped cover slips placed in a 35 mm petri dish and allowed to adhere for 10 min at 37°C. The culture medium was removed and cells were fixed with 3.7% pre-warmed paraformaldehyde (PFA) for 30 min. After fixation, the cells were permeabilized with 0.1% Triton X-100/PBS for 3 min. The fixed cells were then washed with 1X PBS and neutralized for 30 min in 1X PBS containing 50 mM NH4Cl. The cover slips were blocked with 1% BSA/PBS for 1 h followed by incubation with primary antibody at 37°C for 1 h. The cover slips were washed with 1X PBS followed by 1% BSA/PBS before incubation with secondary antibody for 1 h at 37°C. Antibody dilutions were used as follows: Anti-m-EhCaBP6 at 1:300, anti-r-Eh β-tubulin at 1:50, anti-mice Alexa 555 at 1:300 and anti-rabbit Alexa 488 (Molecular Probes) at 1:300. The preparations were further washed with 1X PBS and stained with Hoechst (20 μg/ml) for 10 min at 37°C. The cells were washed thoroughly with 1X PBS and mounted on a glass slide using DABCO . The antibody coupled to CNBr-Sepharose beads was washed thrice with coupling buffer followed by blocking the unbound sites with 0.1 M Tris Glycine (pH 9.0). The antibody coupled CNBr beads were used to pull-down the antigen and the proteins binding to the antigen by incubating with the precleared lysate for an overnight at 4°C in a reaction volume of 200 μl. Immune complexes were centrifuged at 3000 g for 5 min followed by three washes each with buffer 1 (10 mM Tris-Cl (pH 7.5), 150 mM NaCl, 0.1% ovalbumin (w/v), 0.1% TritonX-100 (w/v), 0.05% sodium azide (w/v)) followed by buffer 2  and buffer 3 (60 mM Tris-Cl (pH 6.8)). The pellet was resuspended in 50 μl of SDS PAGE buffer (125 mM Tris-Cl (pH 6.8), 2% SDS, 0.1% DTT, 30% glycerol, 5% β-mercaptoethanol, and bromophenol blue), and boiled for 5 min. The bound proteins were separated from beads by centrifugation for 5 min and the supernatant was analyzed by SDS-PAGE.The HM1 total lysate (2 mg) was spun to remove cell debris and further pre-absorbed on Protein A-Sepharose beads (Preclearing). The purified anti-mice EhCaBP6 antibody was conjugated to CNBr-Sepharose beads in coupling buffer  membrane and processed using standard methods. The antigens were detected with polyclonal antibodies raised in rabbit and mice followed by secondary anti-rabbit and anti-mice immunoglobulin conjugated to HRPO . ECL reagents were used for visualization (Millipore). The concentration of protein in the sample was estimated by bicinchoninic acid (BCA) assay using BSA as a standard. SDS–PAGE analysis was carried out in 10–12% acrylamide gel under reducing conditions.2, 1 mM EGTA, and 100 mM PIPES-KOH (pH 6.9)) in the presence and absence of 1 mM GTP and 20 μM Taxol at 37°C for 1 h. After incubation, the samples were centrifuged at 100,000 g for 1 h at 37°C. The supernatant and pellet were separated and brought to equal volumes in SDS-PAGE buffer. The supernatant and pellet fraction were analyzed using 14% SDS-PAGE and visualized by staining gels with Coomassie Brilliant Blue R-250. EhCaBP6 alone and bovine β–tubulin alone were used as controls.Co-sedimentation assay was carried out following the protocol from Tonglin Mao et. al . The bin2+. Each titration consisted of injecting 2/3 μL aliquots of 10 mM Ca2+ solution  into 100–200 μM protein solution (1.7 mL) after every 3 minutes, to ensure that the titration peak returned to the baseline prior to the following injection. A total of 20 injections were carried out. Aliquots of concentrated ligand solutions were injected into the buffer solution (50 mM Tris-Cl (pH 7.0), 100 mM NaCl without the protein) in a separate ITC run, to subtract the heat of dilution. Three sets of titration were carried out in apo as well as holo form of the protein. The ITC data thus obtained was baseline corrected and analyzed using the software ORIGIN (supplied with VP-ITC). The amount of heat released per addition of the titrant was fitted to different models to find out the number of binding sites and the metal binding affinities of the protein. Repeating the experiment thrice with different concentrations of protein sample could reproduce the experimental data.Isothermal titration calorimetry measurements were performed on a Microcal VP-ITC titration calorimeter at 25°C. Protein samples were decalcified, centrifuged and degassed prior to the titration with CaDictyostelium, bovine and E. histolytica was done  and 10 μM was immobilized on a gold chip in 10 mM sodium acetate (pH 5.0) according to manufacturer’s amine coupling kit. After protein immobilization, the surface was blocked with 1 M ethanolamine (pH 8.5), followed by regeneration using 50 mM NaOH. The interaction experiments were performed using a buffer containing 10 mM HEPES (pH 7.4), 150 mM NaCl and 0.2 mM calcium chloride. We also performed interaction experiment in the absence of Ca2+ and supplementation with 5 mM of EGTA in the above buffer. The binding experiments were carried out with different concentrations  of EhCaBP6 in running buffer and injected at a rate of 20 ml/min. The association kinetics was monitored for 300 s and dissociation kinetics was monitored for another 300 s. The data were recorded at 25°C and data analysis was performed using Biacore T2000 SPR Kinetics evaluation software.To characterize the interaction between Ehβ-tubulin and EhCaBP6, multiple sequence alignment of β–tubulin from human, was done . The seqGrowth kinetics was studied by inoculating equal number of cells (5000 cells/ml). The cells were allowed to grow at 35°C and harvested at 12, 24, 36, 48, and 72 h post inoculation. The cells were harvested in 1X PBS pH 7.2 by chilling on ice followed by centrifugation at 1500 g for 5 min. The cell pellet was resuspended in 1 ml 1X PBS pH 7.2. The cells were counted on Cell counter by mixing cells with 0.4% Trypan blue in a ratio of 1:1. At every time point, the cells were harvested and counted as mentioned above. Each experiment was performed in triplicate and the experiment was performed thrice. The standard error bars were calculated and represented.E. histolytica. Therefore, to study the effect of EhCaBP6 on rate of DNA synthesis, thymidine incorporation assay was done according to protocol mentioned in Nishant et al. -HSQC spectrum in going from (A) apo to (B) holo-EhCaBP6.2D [(TIF)Click here for additional data file.S3 FigThe sequential occupancy was monitored by the sequential appearance of Gly peak corresponding to EF-I (G28) followed by EF-III (G101).(TIF)Click here for additional data file.S4 Fig15N-1H]-HSQC spectra of mutant  (A) In the presence of Ca2+ and (B) In the presence of EDTA. (C) An overlay of 2D-[15N-1H]-HSQC spectra of mutant in the presence of Ca2+ (blue) and in the presence of EDTA (red).2D-[(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 FigE. histolytica. (A) EhCaBP1 and EhCaBP5 are enriched near phagocytic cups (marked by a *), EhCaBP3 is found during the closure of phagocytic cups and in mature phagosomes (marked by an arrow). EhCaBP6 was neither found near phagocytic cups nor in mature phagosomes. The secondary antibody used was anti-mice or rabbit- Alexa Fluor 488 (green) in a ratio of 1:300. (B) Densitometry analysis of EhCaBPs at phagocytic cup, in phagosome and nucleus plotted as a function of percentage relative intensity. A total of five regions of interest (ROI) were taken for analysis in each cell in the respective compartments. The sample size contained 50 cells per experiment. Each of these experiments was repeated three times.Immunostaining of cells undergoing erythrophagocytosis with antibodies against EhCaBP1, EhCaBP3, EhCaBP5 and EhCaBP6 from (TIF)Click here for additional data file.S8 FigE. histolytica and proteins present in the precipitated material were identified by specific antibodies in western blots as indicated. Whole cell lysates were prepared in presence of either CaCl2 or EGTA. Prebleeds of indicated antibodies were used for immunostaining as control (lane I and IV). Lanes II and V represent immunoprecipitation in the presence of CaCl2 and lane III and VI show immunoprecipitation profile in the presence of EGTA. The total input lysate was also probed for the presence of EhCaBP6 and Eh β-tubulin by their respective antibodies (Lane VII and VIII). Anti-m-EhCaBP6 and anti-R- Eh β-tubulin were used at a dilution of 1:2000 and 1:300, respectively.Antibody against EhCaBP6 was used to pull down EhCaBP6 and its binding partner Eh β-tubulin from whole-cell lysate of (TIF)Click here for additional data file.S9 Fig2 and (B) 5 mM of EGTA.In the presence of (A) 1.5 mM of CaCl(TIF)Click here for additional data file.S10 Fig(A) Immunostaining of EhCaBP6 in BAPTA-AM untreated and treated cells. EhCaBP6 (green) was probed with anti-m-EhCaBP6 and anti-m-Alexa-Flour 488 secondary antibody. The nucleus was stained with DAPI. (B) Densitometry analysis of EhCaBP6 in nucleus and cytosol. A total of five random regions of interest (ROI) were chosen from nucleus and cytosol from each cell and the intensity was determined. The sample size contained 50 cells per experiment. Each of these experiments was repeated three times. This panel shows the relative intensities (%) of EhCaBP6 present in nucleus and cytosol. (C) Subcellular fractionation of normal HM1 cells and cells treated with 500 μM BAPTA-AM. Total lysate from BAPTA-AM untreated and treated cells were fractionated into nuclear, cytosol and membrane fractions. The fractions were probed with anti-m-EhCaBP6. The blots were also probed with antibodies against Eh-fibrillarin (I), Eh-coactosine (II), and Eh-TMK9 (III) as markers for nuclear, cytosolic and membrane fractions, respectively.(TIF)Click here for additional data file.S11 Fig2+-binding to the double negative mutant of EhCaBP6. The protein concentration was 145 μM in 50 mM Tris-HCl (pH = 7.0) containing 100 mM NaCl.Thermogram of Ca(TIF)Click here for additional data file.S12 Figin vivo upon induction with varying G418 concentration. The total lysate was probed with anti-GFP antibody. E. histolytica trophozoites transfected with GFP vector alone was used as control. (B) Immunostaining of GFP constructs  in Paraformaldehyde fixed cells with anti-GFP antibody (1:300) and anti-EhCaBP6 antibody (1:300). The fluorescence conjugated secondary antibody (Alexa-488 (green), Alexa -555 (red)) were used to probe the primary antigen. (C) Quantitative analysis of the relative intensity in nucleus and cytoplasm using NIS-Elements Analysis software (Nikon) by taking into consideration 10 region of interest (ROI) in the nucleus and cytoplasm. The experiment was performed thrice. The representative data is an average of ROI from three independent experiments.(A) Expression analysis of GFP-native EhCaBP6 and GFP-DNM6 (TIF)Click here for additional data file.S13 FigDictyostelium, Bovine and E. histolytica done by ClustalW. (B) Percent identity matrix as determined by ClustalW.(A) Multiple sequence alignment of β–tubulin from Human, (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


NCR1gfp/gfp) mice in comparison to OVA immunized wild type (NCR1+/+) and adjuvant immunized mice. Histological analysis of OVA immunized NCR1gfp/gfp mice revealed no preservation of the lung structure and overwhelming peribronchial and perivascular granulocytes together with mononuclear cells infiltration. OVA immunized NCR+/+ mice demonstrated preserved lung structure and peribronchial and perivascular immune cell infiltration to a lower extent than that in NCR1gfp/gfp mice. Adjuvant immunized mice demonstrated lung structure preservation and no immune cell infiltration. OVA immunization caused an increase in PAS production independently of NCR1 presence. Bronchoalveolar lavage (BAL) revealed NCR1 dependent decreased percentages of eosinophils and increased percentages of lymphocytes and macrophages following OVA immunization. In the OVA immunized NCR1gfp/gfp mice the protein levels of eosinophils’ (CCL24) and Th2 CD4+ T-cells’ chemoattractants  in the BAL are increased in comparison with OVA immunized NCR+/+ mice. In the presence of NCR1, OVA immunization caused an increase in NK cells numbers and decreased NCR1 ligand expression on CD11c+GR1+ cells and decreased NCR1 mRNA expression in the BAL. OVA immunization resulted in significantly increased IL-13, IL-4 and CCL17 mRNA expression in NCR1+/+ and NCR1gfp/gfp mice. IL-17 and TNFα expression increased only in OVA-immunized NCR1+/+mice. IL-6 mRNA increased only in OVA immunized NCR1gfp/gfp mice. Collectively, it is demonstrated that NCR1 dampens allergic eosinophilic airway inflammation.The function of NCR1 was studied in a model of experimental asthma, classified as a type 1 hypersensitivity reaction, in mice. IgE levels were significantly increased in the serum of OVA immunized NCR1 deficient ( Allergic asthma, classified as type 1 hypersensitivity reaction, is a chronic inflammatory disease of the airways characterized by reversible airflow obstruction, bronchial hyper-responsiveness, airway inflammation and production of allergy specific immunoglobulin E (IgE) . StudiesIn predisposed individuals, initial exposure(s) of professional antigen-presenting cells (APCs) to an allergen leads mainly to the activation of allergen-specific T helper 2 (Th2) cells and IgE synthesis, which is known as allergic sensitization . The polThe earliest contact between an antigen and the innate immune cells is thought to direct the subsequent antigen-specific T-cell response. Thus, cells of the innate immune system, such as NK cells, NKT cells, and γδT cells might regulate the development of allergic airway disease. The ability of NK cells to produce cytokines and chemokines in response to direct or indirect stimuli helps them regulate multiple immune responses. NK cells can rapidly produce IFNγ along with myriad of cytokine and chemokines that attract DCs and macrophages to the area of inflammation. This co-localization of DCs and NK cells results in a cross talk, which can influence the maturation and activation of both cells. Additionally, NK cells demonstrate contact-dependent co-stimulation. NK cells express several co-stimulatory ligands allowing them to provide direct co-stimulation to T and B cells .+CD56+ NK2 cells in PBMCs of allergic rhinitis patients were shown . How. How30].+ T-cells to the site of inflammation[NCR1gfp/gfp mice in comparison to NCR1+/+ mice, suggesting that the absence of NCR1 caused an increase in eosinophil and Th2 cells recruitment to the lungs. The increase in CCL24 level in the lung following OVA immunization correlates with the increased percentage of eosinophils observed in the absence of NCR1. However, the increase in CCL17 does not correlate with the percentages of lymphocytes in the lung of OVA immunized NCR1 non-expressing mice, which did not differ from that observed in OVA immunized NCR1 expressing mice. The observed increase in the CCL22 in OVA immunized NCR1gfp/gfp mice did not reach significance in comparison to OVA immunized NCR1+/+ mice. Thus, there might be additional mediators that control the migration of lymphocytes to the site of inflammation following OVA immunization [CCL24 chemoattractants secreted from lung epithelial cells or DC’s, direct eosinophils, while CCL17 and CCL22 direct Th2 CD4lammation, 56–64. nization –68.NCR1gfp/gfp and NCR1+/+ mice.In order to better understand the mechanism of regulation, we screened Th1, Th2, Th17 cytokine mRNA and CCL17 chemokine in the mouse lungs following OVA immunization. The strong elevation of IL-4, IL-13 and CCL17 mRNA following OVA immunization demonstrates an intense allergic airway response, which was NCR1 independent. However, CCL17 protein expression in the lung BALF was significantly increased in the absence of NCR1, suggesting that an increase in CCL17 protein production occurs albeit similar mRNA expression in the OVA immunized IL-6 increased only in absence of NCR1 in the mice following immunization with OVA suggesting either no involvement of NCR1 or NCR1 dampens IL-6 expression. Indeed, no alteration in Th1 cytokines (IL-2 and IFNγ) were observed upon allergic airway inflammation as expected.NCR1 has no role in regulating Th1 cytokines as no increase in Th1 cytokines and chemokine expression levels following OVA immunization could be observed. Th1 cytokines were originally regarded as potentially protective against asthma, which was considered a purely Th2 response . In a diin vivo in mice.NK cells were suggested as a target for asthma therapy. In several studies, NK cells were suspected to augment allergic inflammation. In other studies, NK cells were shown to enhance neutrophil and eosinophil apoptosis, which may lead to the resolution of the inflammation. In agreement with the latter studies in the current study, we demonstrate for the first time that NCR1 receptor dampens the allergic response in the OVA model of asthma S1 FigNCR1+/+ and NCR1gfp/gfp C57Bl/6 mice were i.p. immunized with either OVA/Alum or adjuvant only on days 0 and 14. Ten days after the second immunization, mice were challenged twice intranasally with OVA at days 24 and 27. Twenty four h following the second challenge, serum was drawn for measurement of the total IgE levels. A representative experiment is presented . *p<0.05; **p<0.01 compared with the NCR1+/+ OVA-immunized group ±SEM (1 way ANOVA multiple comparison GraphPad).(TIF)Click here for additional data file.S2 Figp<0.01, ***p<0.001 compared with the NCR1+/+ adjuvant treated group (two-tailed Student t-test). Results are summarized from two different independent experiments.The bar graph shows the average RQ of MUC5A/C and Gob5 mRNA level for each group of mice. Each bar represents mean ±SD of n = 3 to 7. ** (TIF)Click here for additional data file.S3 FigNCR1+/+  and NCR1gfp/gfp  mice were immunized with either OVA or adjuvant, as described in the Materials and Methods section. The BAL was lavaged from each mouse, stained with anti CD45 and PI, and analyzed by flow cytometry for live immune cell count (A). The number of eosinophils (B) macrophages (C) was determined by a differential staining. Percent dead cells in the BAL (D). These results are from a representative experiment. Statistical analysis: two-tailed Student t-test. *p < 0.05; ***p < 0.001 ****p < 0.0001(TIF)Click here for additional data file.S4 Fig+GR1- cells (myeloid DCs), 2. CD11c++GR1- cells (lung DCs), 3. CD11c+GR1+ cells (plasmacytoid Dc), 4. CD11c-GR1+ cells, 5. CD11c-GR1int cells. The populations were then gated on Sigelic F marker and SSC to distinguish between eosinophils and neutrophils. CD11c-GR1+ cells were divided into two sub-populations: 52% were sigelic F-SSClow cells and 38% were sigelic F-sschigh cells (neutrophils). CD11c-GR1int cells were 27% Sigelic F+SSChigh cells (eosinophils). Ly94-Ig analysis was performed on each of the populations (1–5).Single cells were gated to exclude doublets by size. CD45 positive and 7AAD (a marker for dead cells) negative cells were then further gated on CD11c and GR1 markers. Five populations were gated according to these markers: 1. CD11c(TIF)Click here for additional data file.S5 FigNcr1+/+ and Ncr1gfp/gfpC57Bl/6 mice were i.p. immunized with either OVA or adjuvant on days 0 and 14. Ten days after the second immunization, mice were challenged twice intranasally with OVA at days 24 and 27. 24 h following the second challenge, BALF was taken from each mouse and used in ELISA to detect levels of (A) CCL24 (n = 8 to 11) and (B) CCL17 (n = 9 to 10) and (C) CCL22 (n = 9–10). **p<0.01, ***p<0.001 (One tail ANOVA multiple comparison).(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


DPYD genotyping and phenotyping. The goal of this prospective observational study was to perform exhaustive exome DPYD sequencing and to examine relationships between DPYD variants and toxicity in advanced breast cancer patients receiving capecitabine.Deficiency in dihydropyrimidine dehydrogenase (DPD) enzyme is the main cause of severe and lethal fluoropyrimidine-related toxicity. Various approaches have been developed for DPD-deficiency screening, including DPYD exome, along with flanking intronic regions 3’UTR and 5’UTR, were sequenced on MiSeq Illumina. DPD phenotype was assessed by pre-treatment plasma uracil (U) and dihydrouracil (UH2) measurement.Two-hundred forty-three patients were analysed (88.5% capecitabine monotherapy). Grade 3 and grade 4 capecitabine-related digestive and/or neurologic and/or hemato-toxicities were observed in 10.3% and 2.1% of patients, respectively. in silico). Combined analysis of deleterious variants *2A, I560S (*13) and D949V showed significant association with grade 3–4 toxicity  71.4%, relative risk (RR) 6.7, p<0.001) but not with grade 4 toxicity. Considering additional deleterious coding variants D342G, S492L, R592W and F100L increased the sensitivity to 26.7% for grade 3–4 toxicity , and was significantly associated with grade 4 toxicity , suggesting the clinical relevance of extended targeted DPYD genotyping. As compared to extended genotype, combining genotyping (7 variants) and phenotyping (U>16 ng/ml) did not substantially increase the sensitivity, while impairing PPV and RR.Among the 48 SNPs identified, 19 were located in coding regions, including 3 novel variations, each observed in a single patient  related to capecitabine, as compared to conventional genotyping restricted to consensual variants *2A, *13 and D949V.Exploring an extended set of deleterious  Since its launch in 1998, the 5FU oral prodrug capecitabine has gradually become a major drug and is currently considered as a standard of care for advanced breast cancer. Capecitabine is ultimately metabolized by thymidine phosphorylase which produces 5FU at target cell level. Next, intracellularly-produced 5FU enters either the anabolic or the catabolic route. Most 5FU is deactivated into fluorodihydrouracil by ubiquitous dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme of 5FU catabolism, expressed in various human tissues as well as in human cancer cells . ConsequDPYD, a large gene spanning 950 kb on chromosome 1p22 (23 exons comprising 4399 nucleotides)  was previously associated with loss of enzyme activity in vitro. A discrepancy was observed between in silico and in vitro functionality for variant T65M (proficiency not assessable by means of the UMD-Predictor tool).For the 19 exonic SNPs, results of terature ,16 for kFig 3illustrates the distribution of UH2/U and U plasma concentrations according to DPYD variants with very deficient  or moderately deficient (D949V) enzyme activity in vitro, as well as M166V and HapB3. Only variant *2A and D949V were associated with a low UH2/U ratio . Only variant D949V was associated with an elevated U concentration (p = 0.005).For the 205 patients with validated phenotypic data, mean pre-treatment plasma UH2/U ratio was 11.1  and mean plasma U was 10.9 ng/ml . The sum of variant alleles did not impact UH2/U or U concentrations. DPYD variant on toxicity showed that only variant D949V and *2A were significantly associated with an increased risk of developing grade 3–4 toxicity, with similar performance level: 66.7% (2/3) toxicity in wt/var patients vs 11.8% (28/239) in wt/wt patients . D949V and *2A variants had no significant impact when focusing on grade 4 toxicity. Of note, the only patient carrying the S492L allele and the patient carrying the F100L allele, both developed grade 4 toxicity (Table 2).Analysis of the impact of each individual DPYD variants in combination was next examined (Table 4). The presence of one deficient allele among consensual variants *2A, D949V or I560S was significantly associated with grade 3–4 toxicity  but was not predictive of grade 4 toxicity. We then considered the 11 in silico pathogenic or probably pathogenic variants . All these variants had very low MAF (<1%) and were only observed in heterozygous state. This set of 11 in silico deleterious variants was significantly associated with both grade 3–4  and grade 4 hemato-digestive-neurotoxicity . Finally, we considered together variants *2A, D949V and I560S associated with all variants having shown very deficient (<25%) or moderately deficient (25–60%) in vitro enzyme activity relative to wild-type . Taking into account the 7 above-cited in vitro deleterious DPYD alleles  improved the performance of the genotyping test relative to both the 3 consensual variants and the 11 in silico deleterious variants, on both grade 3–4  and grade 4 toxicities .Predictive value of th percentile = initial hypothesis) were significantly prone to develop grade 4 toxicity (Fisher Exact test p = 0.047), however significance was not reached when regarding grade 3–4 toxicity. Best uracilemia cutoff was 91st percentile: elevated uracilemia above 16 ng/ml was significantly associated with a RR of 20.6 to develop grade 4 toxicity  (Table 4). As compared with genotyping of either 3 consensual variants, or 7 in vitro deleterious, or 11 in silico deleterious variants, the combined genotype-phenotype approach  did not improve toxicity prediction. For instance, with the best combined approach (7 in vitro deleterious variants) sensitivity dropped from 26.7% to 25%, PPV from 72.7% to 27.3%% and RR from 7.6 to 2.7 for grade 3–4 toxicity; for grade 4 toxicity sensitivity increased from 60% to 66.7%, and PPV dropped from 27.3% to 9.1% and RR from 31.4 to 16.4 (Table 4).In patients with validated phenotypic data, distribution of UH2/U was not different according to toxicity. In contrast, uracilemia was higher in patients developing grade 4 toxicity relative to patients who did not (Mann-Whitney test p = 0.016). Patients with uracilemia above 14 ng/ml . In vitro functionality has already been reported for 11 presently-observed missense variations, with conflicting results for M166V variant only  [The main objective of this prospective study was to examine relationships between ure data . The fouure data . The nov100 [FS] , stronglTable 4). In order to perform variant combinations as objectively as possible, two combination approaches were tested, one based on in silico prediction using the UMD-Predictor system [in vitro functional data reporting DPD enzyme activity in transgenically-expressed missense DPYD variants [in vitro deleterious variants (Table 4). As compared to the 3 consensual variants, adding in vitro deleterious variants D342G, S492L, R592W and F100L increased the sensitivity to 26.7% for grade 3–4 toxicity, with a similar RR . Moreover, the combination of the seven in vitro deleterious variants was significantly associated with grade 4 toxicity  (Table 4). Present results show that extended DPYD genotyping to known in vitro deficient variants clearly improves the performance of consensual DPYD genotyping for pre-emptive identification of patients at-risk to develop severe fluoropyrimidine-related toxicities. An alternative strategy, although probably more time-consuming, may be to perform exome DPYD sequencing, and consider in silico prediction of coding variations. Even though the present study shows this latter approach to be less effective, both strategies deserve to be validated in a future prospective study.Sensitivity of genotyping considering the 3 consensual variants *2A, D949V and I560S together was 16.7% for grade 3–4 toxicity  and was not significant on grade 4 toxicity  were significantly prone to develop grade 4 toxicity relative to patients with U below 16 ng/ml . Finally, identifying at-risk patients based on either deficient phenotype (U>16 ng/ml) and/or deficient genotype did not substantially increase sensitivity as compared to genotype alone, while impairing toxicity predictive value and relative risk (Table 4). Present results suggest that the best approach for preventing grade 3–4 toxicity would be extended DPYD genotype, while prevention of more severe grade 4 toxicity may be based on plasma uracil only. The weak association between indirect DPD phenotyping  and grade 3–4 capecitabine-related toxicity may reflect the lack of correlation reported between PBMC-DPD enzyme activity and plasma UH2/U ratio, while a significant but weak correlation was observed between plasma U and PBMC activity [st percentile). These observations fully justify the need to implement DPD-deficiency screening based on indirect or direct DPD phenotyping approaches for preventing lethal toxicities. An alternative possible relevant phenotyping approach is to explore uracil metabolism after administration of a loading dose of uracil that results in temporary DPD enzyme saturation [MTHFR, CDA, TYMS or ENOSF1 [Previous studies have suggested that pre-treatment plasma UH2/U or U is associated with fluoropyrimidine toxicity . A recenactivity ,12,31, oactivity . In lineturation ,38. Toxituration , or to pr ENOSF1 ,41,42 thDPYD *2A genotyping (dose reduced by >50% in heterozygous carriers) was feasible, cost-effective, and improved safety [DPYD variants and emphasize their potential impact on the toxicity of capecitabine-based treatments. A next step would be to establish whether upfront fluoropyrimidine dose-adjustment based on an extended DPD-deficiency screening approach could further improve safety without impairing treatment efficacy, while remaining cost-effective.In conclusion, DPD-deficiency is recognized to be a leading cause of severe fluoropyrimidine toxicity ,17 and ad safety . PresentS1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.


Some latency-reversing agents (LRAs) inhibit HIV-specific CD8+ T cell responses. In a prior study of protein kinase C (PKC) agonists, we found that bryostatin-1 inhibited elite controller/suppressor (ES) CD8+ T cell suppressive activity whereas prostratin had no effect. Ingenol-B is another PKC agonist with potent LRA activity both by itself and in combination with the bromodomain inhibitor JQ1; however its effect on CD8+ T cell mediated control of HIV-1 replication is unknown.CD8+ T cells were isolated from ES and treated with bryostatin-1, prostratin, ingenol-B, and JQ1 as well as a combination of each PKC-agonist with JQ1. The cells were then tested in the viral suppression assay. To assess possible mechanisms of inhibition, CD8+ T cells were treated with the LRAs and analyzed for the expression of various immune cell markers.Ingenol-B had no effect on the ability of ES CD8+ T cells to suppress viral replication, however, the combination of ingenol-B and JQ1 caused a modest, but significant decrease in this suppressive capacity. The mechanism of the inhibitory effect of the JQ1 and ingenol-B combination relative to ingenol-B alone was unclear but the effect appeared to be dose dependent.Ingenol-B does not inhibit HIV-specific CD8+ T cell responses in vitro. These responses are however modestly inhibited when 100 nMingenol-B is combined with JQ1. Since HIV-specific CD8+ T cell activity may be essential for the eradication of reactivated latently infected cells, the potency of latency-reversal activity of drug combinations must be balanced against the effects of the combinations on HIV-specific CD8+ T cell responses. Shock and kill strategies have been proposed as a possible mechanism for HIV-1 eradication . The strRecent studies have indeed shown that different classes of LRAs inhibit the responses of natural killer (NK) cells ,8, T celEuphorbiaceatirucalli plant [Ingenol-B is a relatively new addition to the gradually increasing list of potential candidates for latency reversal , 16. It li plant . While ili plant , its derli plant , 20 and li plant ,21, and li plant . This syGiven their efficacy at latency reversal both alone and in combination with other drugs such as JQ1, ingenol derivatives appear to be promising candidates for the shockandkill cure agenda. However, while much has been done to assess its potency as an LRA, no studies have assessed the effect of the drug on the ability of HIV-specific CD8+ T cells to directly inhibit viral replication. This question is addressed in this study with a focus on the effect of the ingenol derivative ingenol-B on CD8+ T cells when used either alone or in combination with the bromodomain inhibitor JQ1.We also assessed the effects of other PKC agonists, and their combinations, on CD8+ T cell function, using CD8+ T cells form ES, since the cells of these patients have been shown to have superior, HIV specific suppressive capacities , 29, 30.HIV-1 positive and HIV-1 negative blood samples were obtained from donors with written, informed consent and handled according to a Johns Hopkins University IRB approved protocol. Elite suppressors are patients who have maintained undetectable viral loads without antiretroviral therapy. Chronic progressors (CPs) are patients on antiretroviral therapy who have maintained undetectable viral loads for more than 1 year. Healthy donors (HDs) are HIV negative donors. The clinical characteristics of the ES subjects are summarized in env replaced with gfp and whose expression is controlled by the HIV promoter. At the conclusion of the six-hour drug treatments, the CD8 + T cells were washed and addedat a 1:1 effector:target ratio to the spinoculated CD4 + T cells. The cells were co-cultured for three days prior to Flow cytometry (FACS) analysis  were obtained from whole blood via Ficoll-Paque PLUS gradient centrifugation .CD8 + T cells were isolated from PBMCs of elite suppressors by positive selection with Miltenyi beads. CD4+ T cells were isolated from the CD8+ T cell negative cells by negative selection using Miltenyi beads. CD8 + T cells from elite suppressor patients were treated for six hours with either media, DMSO , JQ1 , ingenol-B , bryostatin-1 at two concentrations , prostratin, or PMA . In some experiments, the PKC-agonists were combined with JQ1 at the same concentrations. All drug concentrations were selected based on either physiological relevance in clinical studies (bryostatin at 1nM) or previous work that demonstrated them to be optimal for latency reversal , 31. Bulanalysis .6/well in a 48 well plate and treated for 6 hours at 37°C with the individual LRAs or LRA combinations listed above. Cells were washed three times after drug treatment and re-seeded into 96 well plates at 200,000 cells/well/treatment type and assessed for immune marker expression via FACS at four time points: 6 hours (immediately after drug treatment), 24 hours, 48 hours and 72 hours. We also explored the potential of select LRAs/LRA combinations, to either induce proliferation, or increase cell death, in CD8+ T cells isolated from healthy donor PBMCs. In this assay, CD4+ and CD8+ T cells were isolated from donor PBMCs and combined at a 1:1 ratio. They were washed with pre-warmed PBS and exposed to 1uM CFSE  at a 1x106cells/ml of CFSE with gentle vortexing. Cells were then incubated for 10mins at 37°C and CFSE quenched with 5 times original volume of cold media followed by a5 minute incubation at 4°C. Cells were washed one more time with media and treated for 6 hours with select LRAs or LRA combinations in 96 well plates at 200,000 cells/well/treatment. Drug combinations are the same as used a prior, except for the addition of ingenol-B at 10nM and PMA/Iono at 50ng/ml and 1uM respectively. At the end of this incubation, cells were extensively washed, and cultured for an additional 3 days, after which cell death and proliferation were assessed by FACS. For Ki-67 staining, cells were treated with drugs for 6 hours prior to cell culture. On day 3, antibodies to surface markers were added and the cells were then fixed with 70% cold ethanolfollowed by intracellular staining with the Ki-67 antibody (Biolegend).PBMCs were isolated from HDs or CPs, seeded at 1x10For the suppression assay, the following antibodies were used: CD3 , CD8  and CD69 . The percent infection was assessed by quantifying GFP expression using the following formula:The following markers were used to assess cell death, immune activation, and exhaustion: CD3 , CD8 , CD69 , 7AAD , Annexin V  and PD1 CD25 , HLA-DR  and Ki67 . Proliferation was assessed using the CellTrace cell proliferation kit from Invitrogen, and read in the FITC channel.All statistical analysis and associated graphs were generated in GraphPad Prism 7. For each parameter studied, statistical significance was assessed using a one-way ANOVA with repeated measures. The Geisser-Greenhouse Correction was also employed to account for violations of sphericity. In experiments that entailed replicates, standard deviations were used to determine variances from corresponding means. In all cases, the Dunnett’s Multiple Comparison’s test was used to assess variations of treatment groups from the DMSO control. Multiplicity adjusted P values were reported to assess significance with the following p-value demarcations: ns (p>0.05), * (p<0.05), ** (p<0.01), *** (p<0.001), **** (p<0.0001).To determine the effect of individual drugs on the suppressive capacity of HIV-specific CD8+ T cells, we treated CD8+ T cells isolated from elite suppressors for 6hrs with three different doses of ingenol-B that have been tested in vitro, and then co-cultured them (after extensive washes) over a 3-day period with autologous CD4+ T cells spinoculated with a lab strain HIV pseudovirus. There was no inhibition of CD8+ T cell suppressive capacity with 10 nM, 100nM or 1000nM of the drug compared to the DMSO control . We therAs previously described , bryostaIn order to address the different effects of the inhibitory (bryostatin-1) and non-inhibitory (prostratin and ingenol-B) PKC-agonists, we measured the amount of immune activation they induced on CD8+ T cells from healthy donors . With thIngenol-B treatment resulted in a modest but significant increase in annexin V expression at 2 days, but no significant increase in cell death was observed at any other time point .The percPrior studies have shown that ingenol-B causes a significant downregulation of surface CD4 expression , 20 and In order to determine whether the modest inhibitory effect of the combination of ingenol-B with JQ1 was dependent on the dose of ingenol-B, we performed experiments with 10nM of the drug in combination with the same dose of JQ1. As shown in Ingenol derivatives have been shown to function efficiently both as monotherapy and in combination with other drugs such as JQ1 and vorinostatin vitro , 20, 21 While some ingenol derivatives are thought to function via alternative pathways , it is gA recent study has shown that the reversal of latency by itself is not sufficient to eliminate latently infected CD4+ T cells, and that HIV-specific CD8+ T cells may also be needed for the eradication of these cells . This fiIn this study, we screened ingenol-B to determine whether it inhibited HIV-specific CD8+ T cell responses. We used CD8+ T cells from ES since these patients have potent HIV-specific CD8+ T cell responses that are probably responsible for their control of HIV-1 infection , 29, 30.Besides CD3 modulation, we also looked at the expression levels of other cell surface markers such as CD8, annexin V and PD1, to determine if these could explain the difference in suppressive capacities of CD8+ T cells treated with either ingenol-B or its combination with JQ1. We found that ingenol-B alone and in combination with JQ1 does not significantly modulate CD8 or PD1expression on T cells. We also found that treatment with ingenol-B or ingenol-B/JQ1 resulted in minimal cell death which confirms studies by Abreu et al .Our data are also consistent with a recent study that showed that treatment of peripheral blood mononuclear cells from healthy donors and HIV-infected patients with ingenol-B increased the number of antigen specific CD8+ T cells that expressed perforin and CD107a .However,In summary, our data suggest that drug combinations should be tested before being used in clinical trials because even LRAs that do not inhibit HIV-specific CD8+ T cell responses by themselves may have significant effects when they are used in combination with each other. Furthermore, while certain combinations of LRAs may be more potent at inducing viral transcription than individual LRAs, the advantages of this enhanced potency may be neutralized by the inhibitory effects of the drug combinations on HIV-specific CD8+ T cell responses.S1 FigPBMCs from 6 CPs were treated with LRAs for 6 h, washed, then cultured for up to three days and examined for CD69 expression on CD8 + T cells. Mean expression ± standard error is indicated for each treatment at (A) 6 hours, (B) day 1, and (C) day 3. Symbols directly above treatments indicate differences from the DMSO control. * p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Cells were either non treated (NT) or treated with DMSO (0.1%), bryostatin-1 at 10nM (BRYO), ingenol-B at 1000, 100 or 10nM , PMA at 50ng/mL (PMA) and JQ1 at 1uM (JQ1). In some experiments the PKC-agonists were combined with JQ1 at the same concentrations . PMA and ionomycin  were used as a positive control.(TIF)Click here for additional data file.S2 FigPBMCs from 6 CPs were treated with LRAs for 6 h, washed, then cultured for up to three days and examined for CD25 expression on CD8 + T cells. Mean expression ± standard error is indicated for each treatment at (A) 6 hours, (B) day 1, and (C) day 3. Symbols directly above treatments indicate differences from the DMSO control (* p< 0.05). Cells were either non treated (NT) or treated with DMSO (0.1%), bryostatin-1 at 10nM (BRYO), ingenol-B at 1000, 100 or 10nM , PMA at 50ng/mL (PMA) and JQ1 at 1uM (JQ1). In some experiments the PKC-agonists were combined with JQ1 at the same concentrations . PMA and ionomycin  were used as a positive control.(TIF)Click here for additional data file.S3 FigPBMCs from 6 CPs were treated with LRAs for 6 h, washed, then cultured for up to three days and examined for CD38 and HLA-DR co-expression on CD8 + T cells. Mean expression ± standard error is indicated for each treatment at (A) 6 hours, (B) day 1, and (C) day 3. Symbols directly above treatments indicate differences from the DMSO control (* p< 0.05). Cells were either non treated (NT) or treated with DMSO (0.1%), bryostatin-1 at 10nM (BRYO), ingenol-B at 1000, 100 or 10nM , PMA at 50ng/mL (PMA) and JQ1 at 1uM (JQ1). In some experiments the PKC-agonists were combined with JQ1 at the same concentrations . PMA and ionomycin  were used as a positive control.(TIF)Click here for additional data file.S4 FigPBMCs from 6 CPs were isolated and treated with LRAs for 6 h, washed, then cultured for up to three days and examined for cell death as measured by percent annexin V expression. Mean expression ± standard error is indicated for each treatment at (A) 6 hours, (B) day 1, and (C) day 3. Symbols directly above treatments indicate differences from the DMSO control . Cells were either non treated (NT) or treated with DMSO (0.1%), bryostatin-1 at 10 nM (BRYO), ingenol-B at 1000, 100 or 10nM , PMA at 50ng/mL (PMA) and JQ1 at 1uM (JQ1). In some experiments the PKC-agonists were combined with JQ1 at the same concentrations  or with PMA and ionomycin .(TIF)Click here for additional data file.S5 FigPBMCs from 8 HDs were treated with LRAs for 6 h before being washed and cultured for up to three days and examined for cell exhaustion as measured by percent PD1 expression. Mean expression ± standard error is indicated for each treatment at (A) 6 hours, (B) day 1, (C) day 2 and (D) day 3. Symbols directly above treatments indicate differences from the DMSO control. * p< 0.05, ** p < 0.01, *** p <0.001, **** p < 0.0001. Cells were either non treated (NT) or treated with DMSO (1%), bryostatin-1 at 10 or 1 nM (BRYO 10 and BRYO 1 respectively), prostratin at 0.3 uM (PRO), ingenol-B at 100nM (ING), PMA at 50ng/mL (PMA) and JQ1 at 1uM (JQ1). In some experiments the PKC agonists were combined with JQ1 at the same concentrations .(TIF)Click here for additional data file.S6 FigPBMCs from 6 CPs were isolated and treated with LRAs for 6 h, washed, then cultured for up to three days and examined for cell death as measured by percent annexin V expression. Mean expression ± standard error is indicated for each treatment at (A) 6 hours, (B) day 1, and (C) day 3. Symbols directly above treatments indicate differences from the DMSO control (* p< 0.05). Cells were either non treated (NT) or treated with DMSO (0.1%), bryostatin-1 at 10 nM (BRYO), ingenol-B at 1000, 100 or 10nM , PMA at 50ng/mL (PMA) and JQ1 at 1uM (JQ1). In some experiments the PKC-agonists were combined with JQ1 at the same concentrations  or with PMA and ionomycin .(TIF)Click here for additional data file.S7 FigPBMCs were isolated from 6 healthy donors and CD4+ and CD8+ T cells enriched. Cells were combined at a 1:1 ratio, co-cultured and treated with LRAs for 6hrs after which cells were extensively washed and incubated for an additional 3 days before examining for CD8+ T cell death by percent annexin V expression. Mean expression ± standard error is indicated for each treatment at day 3. No significant differences in cell death were observed, relative to the DMSO control. Cells were either non treated (NT) or treated with DMSO (1%), ingenol-B at 100nM or 10nM (ING 100 or ING 10), PMA at 50ng/mL (PMA), JQ1 at 1uM (JQ1) and ingenol-B/JQ1 combinations at the same concentrations.(TIF)Click here for additional data file.S8 Fig(A) PBMCs were isolated from 6 HD PBMCs and CD4+ and CD8+ T cells enriched. Cells were combined at a 1:1 ratio and exposed to CellTrace CFSE. They were treated with LRAs for 6hrs incubated for an additional 3 days before examination for CD8+ T cell proliferation by percent CFSE expression. Mean expression ± standard error is indicated for each treatment at day 3. Cells were either non treated (NT) or treated with DMSO (1%), ingenol-B at 100nM or 10nM (ING 100 or ING 10), PMA at 50ng/mL (PMA), PMA/Ionomycin , JQ1 at 1uM (JQ1) and ingenol-B/JQ1 combinations at the same concentrations. (B) PBMCs were isolated from 6 CPs and treated for 6 hours with LRAs. They were then extensively washed and incubated for an additional 3 days before intracellular staining with the Ki-67 monoclonal antibody. Mean expression ± standard error is indicated for each treatment.(TIF)Click here for additional data file.S9 FigPBMCs from 6 CPs were isolated and treated with LRAs for 6 h, washed, then cultured for up to three days and examined for CD3 expression. Mean expression ± standard error is indicated for each treatment at (A) 6 hours, (B) day 1, and (C) day 3. Symbols directly above treatments indicate differences from the DMSO control . Cells were either non treated (NT) or treated with DMSO (0.1%), bryostatin-1 at 10 nM (BRYO), ingenol-B at 1000, 100, or 10nM , PMA at 50ng/mL (PMA) and JQ1 at 1uM (JQ1). In some experiments the PKC-agonists were combined with JQ1 at the same concentrations  or with PMA and ionomycin .(TIF)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.S2 Dataset(XLSX)Click here for additional data file.S3 Dataset(XLSX)Click here for additional data file.S4 Dataset(XLSX)Click here for additional data file.S5 Dataset(XLSX)Click here for additional data file.S6 Dataset(XLSX)Click here for additional data file.S7 Dataset(XLSX)Click here for additional data file.S8 Dataset(XLSX)Click here for additional data file.S9 Dataset(XLSX)Click here for additional data file.


Interestingly, S100A14 blocked the store-operated Ca2+ influx by suppressing Orai1 and STIM1 expression, leading to FAK expression activation, focal adhesion assembly and MMP downregulation. Taken together, our results indicate that S100A14 may have a role in the induction of differentiation and inhibition of cell metastasis in GC.S100A14 is a calcium-binding protein involved in cell proliferation and differentiation as well as the metastasis of human tumors. In this study, we characterized the regulation of S100A14 expression between biological signatures and clinical pathological features in gastric cancer (GC). Our data demonstrated that S100A14 induced the differentiation of GC by upregulating the expression of E-cadherin and PGII. Moreover, S100A14 expression negatively correlated with cell migration and invasion in  Hence, searching for pathological diagnosis and metastasis-related biomarkers is necessary for the clinical prediction and assessment of GC.Gastric cancer (GC) is the third most important cause of global cancer mortality.4 Accumulating evidence has indicated that the dysregulation of S100 family members correlates with tumor progression in various types of cancers, including breast cancer, liver cancer and colorectal cancer.8 Specifically, S100A2,9 S100A410 and S100A611 are associated with tumor differentiation and promoted tumor growth. In addition, S100A4,13 S100A8/A9,14 S100P15 and S100A1316 have been shown to be involved in tumor invasion and metastasis. In our previous study, we explored and identified a panel of differentially expressed genes between intestinal type and diffuse type GC, including genes encoding S100 protein family members, by gene microarray and experimental studies of GC.17 We further identified the varied expression of seven S100 members in GC tissues and cell lines, including S100A2, S100A6, S100A10, S100A11, S100A14, S100P and S100B, based on our previous microarray screening.18 Interestingly, the effect of S100A14 expression on tumor behavior and progression was controversial in different tumors, and its role in GC has not yet been clarified.The S100 protein family has been reported to contribute to multiple biological processes, such as growth, cell motility, signal transduction, transcription, cell survival and apoptosis, which are related to normal development and tumorigenesis.18 Hence, we will illustrate the previously unknown tumor-related effect of S100A14 on tumor differentiation, cell proliferation and metastasis in GC.Our previous work showed that decreased expression of S100A14 was associated with poor prognosis in GC.P=0.313, To clarify the clinical significance of S100A14, we first used immunohistochemistry to screen the expression of S100A14 in 485 cases of primary GC tissues and 289 cases with matched adjacent normal tissues by immunohistochemistry. Our results confirm that there was no significant difference in S100A14 expression between normal tissues  and tumoP<0.001) and differentiation (P=0.044), but negatively correlated with tumor depth (P=0.014), lymph node status (P=0.011) and distant metastasis  was significantly shorter than that for patients with high S100A14 expression   . These rr=0.981, P<0.001; r=0.885, P<0.001; To understand the mechanism whereby S100A14 promotes the differentiation of GC, we determined the expression of E-cadherin and PGII, which are gastric mucosa differentiation and mature markers, under altered S100A14 expression levels. We observed that the localization of S100A14 and E-cadherin protein expression was overlapped on the cell membrane in most well-differentiated GC tissues  and thatIn addition, we established S100A14 downregulated stable transfectants in AGS cells and S100A14 overexpressed stable transfectants in BGC823 cells . IntriguWe next investigated whether S100A14 has an important role in GC cell migration and invasion. As shown in P<0.01; Moreover, we used a mouse multiple pulmonary carcinoma metastasis model to further investigate the role of S100A14 in inhibiting GC metastasis. Notably, compared with BGC823-vector cells, the total cell foci of BGC823-S100A14 that metastasized to the lungs was reduced by 64.0% P<0.01;  as shown2+ homeostasis. To understand the underlying mechanisms involved in the suppression effects of S100A14 on metastasis in GC, we first investigated the effect of S100A14 on the intracellular calcium levels. As shown in 2+ was increased in AGS-Ri2 and AGS-Ri3 cells compared with AGS-vector cells. Consistent with this finding, S100A14 overexpression in BGC823 cells blocked the store-operated Ca2+ influx  could significantly inhibit AGS-Ri2 and AGS-Ri3 cell migration and invasion induced by S100A14 deficiency  and the calcium-dependent protease calpain in focal adhesions.19 Transwell assays showed that calpeptin  and EGTA decreased the migration and invasion induced by interfering with S100A14 expression in AGS cells , MMP9  and MMP11  expression  and filaggrin (FLG) expression.28 In our study, S100A14 significantly correlated with the differentiation of gastric mucosa and the statuses of the mature markers PGII33 and E-cadherin34 in GC tissue, and PGII and E-cadherin expression was altered with S100A14 expression in GC cells. Another previous study showed that S100A14 expression resulted in G1-phase cell cycle arrest and inhibited cell growth but failed to induce cell apoptosis during the process of promoting the terminal differentiation of ESCC cells.28 However, our cell growth assay and animal model data showed that S100A14 had no effect on GC cell growth in vitro and in vivo  but also downregulates the expression of MMPs via decreasing cellular Ca2+ levels. Therefore, the molecular mechanism by which the Ca2+ signal induced by the loss of S100A14 affects the expression of MMPs should be further studied in the future.Matrix metalloproteinases (MMPs) have a vital role in the tumor invasion process by degrading multiple elements of the extracellular matrix (ECM).2+ influx, leading to FAK activation and decreased MMP expression in GC cells  were used to construct a tissue microarray. In total, tissue samples from 440 cases of primary gastric tumor and 289 cases with matched normal tissues were obtained from Peking University Cancer Hospital, and 212 cases of GC had follow-up data. All tissue specimens were from the patients without radiation therapy and chemotherapy.18 The slides were incubated with polyclonal anti-S100A14 antibody  at 4 °C overnight. Diaminobenzidine  staining was used for detecting immunoreactivity. The intensity of immunoreactivity was graded 0, 1+, 2+ and 3+ for no staining, weak, medium and strong staining, respectively. Scores of 0 and 1+ were regarded as low expression; scores of 2+ and 3+ were considered high expression.Immunohistochemistry was performed using a standard technique with the avidin-biotinylated peroxidase complex as described previously.2O2, the slides were incubated with blocking reagent  for 30 min at room temperature. Antigens were then successively detected using the Opal protocol: each primary antibody was incubated at 4 °C overnight, followed by detection using HRP-conjugated secondary antibody  and TSA-fluor , after which the primary and secondary antibodies were thoroughly eliminated by heating the slides in citrate buffer (pH 6.0) for 10 min at 95 °C. In a serial fashion, each antigen was labeled with distinct fluorophores. Nuclei were subsequently visualized with DAPI , and the slides were coverslipped using ProLong Gold Antifade Mountant . This Opal protocol of MMP2 , MMP9 , MMP11 , Pepsin C , E-cadherin  and S100A14  was applied to the GC tissue microarrays. The multiple fluorophore-antibody staining on the same tissue was photographed and analyzed by PerkinElmer.Slides loaded with gastric cancer tissue microarrays were deparaffinized in xylene and rehydrated in a graded alcohol series. Antigen retrieval was performed in citrate buffer (pH 6.0) at 95 °C for 20 min and cooled down at room temperature. After the quenching of endogenous peroxidase in 3% H2.The gastric cancer cell lines BGC823, MGC803, SGC7901, AGS and N87 and gastric epithelial cells transfected with SV40 (GES-1) were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum , 100 units/ml streptomycin, and 100 units/ml penicillin. All the cell lines were maintained at 37 °C in 5% COμg/ml puromycin (Genechem) for 2 weeks to form stable clones post-transfection . The pcDNA3.1-S100A14 and pcDNA3.1-vector plasmids29  were transfected into BGC823 cells containing 400 μg/ml G418 (Gibco) to form stable clones . The efficacy of transfection was identified by real-time PCR, western blotting (WB) and immunofluorescence.The specific RNAi sequences of S100A14 were designed by Invitrogen (RNAi-2:5′-GGAGUUUCUGGGAGCUGAUTT-3′ RNAi-3:5′-GAGCUGGGCUAUGGGAAAUTT-3′) and inserted into the GV248 plasmid , then the plasmids and vector were transfected into AGS cells. And the cells were seeded in selection medium containing 2 Total RNA was extracted from the cell lines using TRIzol  and subjected to RT-PCR and real-time PCR. The primers used are listed in μg protein was subjected to 12% SDS-PAGE, transferred to a PVDF membrane  and incubated with the following antibodies: S100A14 antibody , Pepsin C antibody , E-cadherin antibody , Orai1 antibody , STIM1 antibody , FAK antibody , MMP2 antibody , MMP9 antibody  and MMP11 antibody . The membrane was then incubated with HRP-conjugated goat anti-mouse IgG  or HRP-conjugated goat anti-rabbit IgG . β-Actin antibody  was used as a control. The protein level was detected by ECL . Chemiluminescence was visualized using a SmartChemi image analysis system .Proteins were extracted from the cells for western blot analysis. In total, 50 The cells were grown on glass slides, washed with PBS, and fixed in 4% paraformaldehyde for 10 min. After 1 h blocking with 5% BSA , the slides were incubated with anti-S100A14  and anti-Vinculin  followed by goat anti-mouse-TRITC-conjugated IgG  or goat anti-rabbit-Cy5-conjugated IgG . After the slides were washed, they were studied using a confocal fluorescence imaging microscope .μm pores in a polycarbonate membrane . The invasion assay was performed using a Matrigel Invasion Chamber (Corning). The cells (5 × 104) were suspended in serum-free DMEM and seeded in triplicate in each insert. DMEM supplemented with 10% FBS was used as a chemoattractant. After 12~24 h incubation, the cells were fixed immediately with 4% formaldehyde and stained with 0.1% crystal violet. The cells that penetrated the membrane were imaged and counted.The migration assay was performed using Transwell plates with inserts containing 8 5 cells were suspended in 1 × Hank’s buffer (Macgene) and injected into the tail veins of female NOD/SCID mice . Five mice were included in each group. The lung tumor specimens were filled with Bouin's fixative, immersed in formalin, embedded in paraffin and subjected to H&E staining and IHC staining for S100A14 6 weeks later. The lung metastasis nodes from all mice were photographed and analyzed.Animal experiments were carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the Ethics Committee of Peking University Cancer Hospital and Institute. For the experimental pulmonary metastasis model, 5 × 1050 The cells were plated onto gelatin-coated 30 mm plates 1 day before the experiments. The cells were washed with Tyrode's solution and incubated with 4 μM Fluo-4 AM (Invitrogen) or Fura-4 AM (Invitrogen) for 30 min at room temperature. The cells were then washed with Tyrode's solution. To measure store-operated Ca2+ influx, 4 mM EGTA and 4 mM thapsigargin (TG) (Abcam) were added to deplete internal calcium stores. Ca2+ influx was induced by the subsequent addition of 2 mM Ca2+ (free) after store depletion. The change in calcium fluorescence was monitored using a confocal fluorescence imaging microscope , which was performed in Professor Heping Cheng’s laboratory at the Institute of Molecular Medicine, Peking University.The calcium assay was performed as described previously.χ2-test). Kaplan–Meier survival analysis and the log-rank test were used to evaluate the prognosis of the patients. For in vitro and in vivo assays, statistically significant differences were determined by Student’s t-test. P⩽0.05 was considered statistically significant in all the cases.All statistical analyses were conducted using SPSS 20.0 software . The data are expressed as the means±S.D. Correlations between protein expression and clinical features from patients were performed using the chi-square test (


E1-modified oncolytic Ads have shown that this therapeutic platform was safe, but with limited efficacy, indicating the necessity of targeting other viral genes for manipulation. To improve the therapeutic efficacy of oncolytic Ads, we treated the entire Ad genome repeatedly with UV-light and have isolated AdUV which efficiently lyses cancer cells as reported previously . In this report, we show that no mutations were observed in the early genes (E1 or E4) of AdUV while several mutations were observed within the Ad late genes which have structural or viral DNA packaging functions. This study also reported the increased release of AdUV from cancer cells. In this study, we found that AdUV inhibits tumor growth following intratumoral injection. These results indicate the potentially significant role of the viral late genes, in particular the DNA packaging genes, to enhance Ad oncolysis.Adenoviruses (Ads) have been extensively manipulated for the development of cancer selective replication, leading to cancer cell death or oncolysis. Clinical studies using  E1b-deleted Ads, such as dl1520 and H101, have been shown to replicate within tumors while only inducing fever in a dose-dependent manner beyond those symptoms observed from cancer chemotherapy [Human adenoviruses (Ads) which have been modified for cancer selective replication, known as oncolytic virotherapeutics, comprise a very promising cancer therapeutic platform for the treatment of solid tumors . Cancer otherapy ,4,5. Theotherapy ,7. Howevotherapy ,10,11,12E) and late (L) genes based upon the expression of these genes across time [E1a, E1b, E2, E3 and E4. The development of oncolytic Ads has primarily focused on the genetic manipulation of E1a and E1b. The proteins encoded by E1a are produced immediately after infection to modulate the cell cycle, recruit cellular proteins, and regulate the expression of cellular and viral genes [E1b gene encodes two major polypeptides of 55,000 kDa (55K) and 19,000 kDa (19K). The expression of E1b55K, but not E1b19K, has been shown to cooperate with E1a to transform rodent cells [E1a expression and is involved in the induction of cyclin E required for Ads to efficiently replicate [The Ad genome, composed of linear double-stranded DNA of approximately 36 kilobases (kb), is divided into early , for example, is a gene-attenuated oncolytic virus with an 827-bp deletion and a nonsense point mutation in E1b that generates a premature stop codon to prevent the complete translation of the E1B55K protein [dl1520 has shown promising oncolytic efficacy in preclinical and clinical trials. Several hundred cancer patients were treated with dl1520 via various routes of injection in phase I and phase II clinical trials [dl1520 and chemotherapy treatment was suspended due to limited therapeutic efficacy [E1a, using cancer selective promoters [E1a controlled by cancer-selective promoters, such as OBP-301 (Telomelysin®) driven by human telomerase reverse transcriptase (hTERT) promoter [E1a is regulated by the human cyclin E promoter [E1a augmented the oncolytic efficacy of Ads [Two major strategies have been applied for the development of oncolytic Ads. One is to delete or attenuate viral genes, such as  protein ,29. Ad dl trials . Howeverefficacy . Anotherromoters . Oncolytpromoter  and CV70promoter , have prpromoter ,35. Cyclpromoter . We havepromoter ,20, thery of Ads .E1b deletions or E1a regulated by cancer selective promoters have achieved some success in human clinical trials, the efficacy of oncolytic Ad virotherapy overall has been disappointing [Although Ads with pointing ,31,38,39pointing ,39. Therpointing . More thdl309 (Ad5) viral particles which were repeatedly treated with UV type-C irradiation [E1, E4) in AdUV, but several mutations were found within the late genes encoding Ad structural proteins involved in viral DNA packaging. AdUV was shown to effectively lyse cancer cells while safely and efficiently suppressing A549 tumor xenograft growth in nude mice.We have previously developed AdUV, which was isolated from a pool of the adenovirus serotype 5 adiation . AdUV wa2 incubators at 37 °C.HEK293 (ATCC no. CRL-1573) human embryonic kidney, MRC5 (ATCC no. CCL-171) human non-cancerous lung fibroblast, HBEC (ATCC no. CRL-4051) human non-cancerous human bronchial epithelial cells, A549 (ATCC no. CCL-185) human lung carcinoma and Saos-2 (ATCC no. HTB-85) human osteosarcoma cells were all purchased from the ATCC . A549, MRC5 and HEK293 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM). Saos-2 cells were maintained in McCoy’s 5A media. DMEM and McCoy’s 5A media were supplemented with 10% fetal bovine serum, L-glutamine, and penicillin/streptomycin (100 U/mL). HBEC cells were cultured in airway epithelial cell basal medium (ATCC PCS-300-030) supplemented with bronchial epithelial cell growth kit (ATCC PCS-300-040). All other cell culture reagents were obtained from VWR . All cells were cultured and maintained in humidified 5% COdl309 is E1-wildtype virus [E3, preventing the expression of E3B 10.4K, 14.6K, and 14.7K genes, but not the E3A glycoprotein (gp) 19K gene [E1b-deleted vector similar to dl1520 and H101 and was used as a cancer selective control in these studies [E1 (E1a and E1b) deleted, with green fluorescent protein (GFP) expression driven by the cytomegalovirus (CMV) promoter [E1 and E4 unless otherwise specified, and are based upon Ad5 backbone sequences (GENEID# AC_000008.1).Ad5 pe virus  and was 19K gene . Adhz60  studies . AdGFP ipromoter . AdGFP, promoter . All Adsdl309 was treated with UV irradiation for 5 min, inactivating approximately 90% of virus particles. The UV-light source was a germicidal lamp, USEG30T3 , fitted with a 30 watt G30T8 UV-light bulb , which produces UV type-C irradiation. UV irradiation was measured with a model 25X UVX radiometer . Using these conditions, 213 μW/cm2 UV type-C was produced, generating a total UV dose of 639 J/m2 after 5 min exposure. Irradiated viruses were then used to infect Saos-2 cancer cells, in which Ads cannot replicate as well as in HEK293 and A549 cells [To select Ads with increased spread, Ad5 49 cells . Four ho49 cells .3 cells per well and infected with virus samples serially diluted tenfold. The presence or absence of CPE in HEK293 cells was recorded, after a minimum of seven days, to calculate the virus titer.Virus titer was determined using the median tissue culture infective dose (TCID50) method ,22,44. H5 and 1.5 × 105 cells per well, respectively. A549 cells were then infected with Ad5, Adhz60 or AdUV at a multiplicity of infection (MOI) of 1. The cells and cell culture samples were then collected at 6, 24, 36, 48, 72, 96 and 120 h post-infection. The cells were separated from the cell culture media and discarded from each sample following centrifugation at 2000 RPM (350 RCF) at 4 °C for 5 min using a micromax RF refrigerated microcentrifuge equipped with an IEC 851 rotor . Cell free media samples were titered to detect the concentrations of Ads released.To determine the release kinetics of AdUV, A549 and Saos-2 cells were seeded onto 12-well plates at a density of 1 × 104 (A549) or 4.5 × 104 (Saos-2) cells per well onto 24-well plates and allowed to adhere overnight. Cytotoxicity was assessed by crystal violet staining after five days [Cells were seeded at a density of 3 × 10ive days . SuspendDNA sequencing was performed at the University of Louisville  KBRIN bioinformatics core facility using Ion PGM Sequencing 200 kit v2 and Ion PGM system. An Ion 314 v2 Chip was used for loading the enriched Ion One Touch 200 ISP samples. Open source integrated genomics viewer (IGV) software was downloaded to view these DNA sequencing data. Mutations with low DNA sequencing error rates were listed in 6 A549 lung cancer cells into the right flanks of NCr-nu/nu mice at six weeks of age. Ads were titrated on HEK293 cells using the plaque assay to determine the plaque forming units (PFU) for AdUV and AdGFP prior to intratumoral injection as described previously [3, mice were randomized into two treatment groups to receive 5 × 108 PFU of AdGFP (n = 8) or AdUV (n = 7) in 50 µL PBS every three days for a total of four treatments (2 × 109 PFU) from days 6 to 15. Tumor volumes were determined by externally measuring these tumors in two dimensions with a caliper and calculated based upon the following equation, V = (L × W2)/2, where L is length and W is the width of the tumor as described previously [Female athymic nude mice (NCr-nu/nu) were obtained from the National Cancer Institute . Tumors were formed following the subcutaneous injection (SC) of 5 × 10eviously ,47. Onceeviously . Eight mTumors were harvested seven days following the fourth treatment and embedded in optimal cutting compound , and stored at −20 °C. Briefly, slides were air-dried and fixed in ice-cold methanol at 4 °C for 20 min. The endogenous tissue peroxidase activity was blocked by in a 0.3% hydrogen peroxide 3% methanol solution for 30 min according to the Vectastain ABC protocol . These tissues were then blocked in SuperBlock  for 45 min at RT. Sections were incubated with goat-anti-adenovirus polyclonal antibody , diluted 1:800, overnight at 4 °C. The signals were amplified by a biotinylated anti-goat IgG diluted 1:200 in conjunction with Vectastain avidin-biotin complex method kit . Visualization was achieved using 3,3-diaminobenzidine-tetrahydrochloride (DAB) for 5 min at RT; the reaction was stopped by dipping the slide in water . All antibodies were applied and diluted in SuperBlock. Slides were washed three times with PBS following the application of SuperBlock, primary antibody, secondary antibody and the ABC reagent. Biotinylated secondary antibody and ABC reagents were both applied to slides for 1 h at RT. Slides were counterstained with hematoxylin and photographed at 200 × and 400 × magnifications using an Olympus BX53 microscope .p-value less than 0.05. Statistical significance was assessed using the two-way analysis of variance analysis (ANOVA) test and the log-rank sum test. Multiple comparisons made using two-way ANOVA were corrected for by Bonferroni’s method; all p-values reported were therefore adjusted using this approach. Multiple comparisons were not made for the log-rank sum test, therefore this reported p-value was not adjusted. All statistical tests were conducted using GraphPad Prism 5 software .All experiments were repeated at least three times. Quantification of results was reported as means of three independent experiments plus or minus (±) the standard deviation (SD). Statistical significance was set at dl309 (Ad5), an E1-wildtype and E3-deleted Ad [E1 and E3 sequences of AdUV were shown to match the Ad5 genome, validating Ad5 as the parental strain of AdUV. Twenty-five AdUV mutations with a low error rate of sequencing (≤15%) are listed in AdUV was isolated following the repeated UV irradiation of leted Ad ,48. The leted Ad . The E1 There are several mutations in genes associated with virus packaging  and 17387. The mutation at 17387 changed the endogenous glycine amino acid to an arginine residue . Glycine and p32 ,60. The  and p32 . There a and p32 . These p and p32 . This mu and p32 . TherefoA mutation was also observed at 26727–32 which deleted six DNA nucleotides, removing two encoded alanine residues from 33K . The 33KThe effect of these mutations may co-operate to bind and package Ad DNA more effectively and stimulate the rapid progression of AdUV from the early to late stages of Ad replication via enhanced Ad major late promoter transactivation.pIII at 15829 resulted in a non-sense mutation which changed the encoded glycine to an alanine and also produced a premature stop codon, preventing the translation of twelve amino acids  into the pIII peptide  with fewer phosphorylation sites.There were also several mutations in other virus structural proteins  and AdUV  of AdUV or the negative control AdGFP every three days for a total of four treatments  survived until day 125  while only two AdGFP treated mice (n = 8) survived to that time point (The efficacy and safety of AdUV was further tested in vivo using an A549 subcutaneous athymic nude mouse (NCr-nu/nu) xenograft model. Once tumors grew to 30 mmeatments A. The leme point B. Represme point C. Furtheme point D. These E1a and E1b, E2, and E4, remained unchanged however, some critical Ad late genes were mutated and are worthy of additional study. These AdUV mutations likely enhanced Ad-mediated cancer cell lysis and cancer selectivity. Twelve of these mutations in the AdUV genome of interest were shown to alter the encoded amino acids, five of these mutations occurred within Ad capsid structural genes  pathway and immune cell–cell interaction, respectively. The absence of mutations in the early viral genes, E1 and E4, was surprising as these genes have important roles in an array of virus–host interactions. By far, the development of cancer selective Ads has focused upon the modification of the Ad early genes by the deletion of the E1b genes, E1b-19K and E1b-55K, or the regulation of E1a gene expression using cancer-selective promoters [The goal of this study was to investigate which genes in Ad genome may affect Ad oncolytic efficacy, and, therefore, may be modified and combined with other approaches in the development of future oncolytic Ads. The Ad early genes in AdUV, romoters ,77. Howeromoters . This hyromoters .In this report, we describe mutations in six different Ad proteins with known roles to package viral DNA during virion maturation. These Ad capsid proteins include IVa2, pTP, 33K, pV, 52K and pVIII. In particular, pV, IVa2, pVI and pVIII interact with and anchor Ad DNA onto the capsid . The mutIVa2 and 33K mutations found in AdUV may have increased the formation of this complex to induce early to late phase transition during Ad replication. Furthermore, there exists indirect evidence that IVa2 and 33K can also form a complex with DNA-binding protein (DBP) on one of the Ad capsid vertexes facilitating Ad DNA packaging [We previously reported that AdUV was shown to rapidly transition from early to late phase gene expression . IVa2 anackaging . These mE1b-wildtype Ad. In early oncolytic studies in the 1950s, wildtype Ads were the first viruses used in clinical trials [Our results have also shown that AdUV can be safely used in animals and suppress tumor growth. AdUV is an l trials . HoweverE1b by AdUV also likely increased its oncolytic potential. Several other E1b-wildtype oncolytic vectors have been described with increased cancer therapeutic efficacy containing other modes of cancer selectivity such as cancer selective promoters, or partial E1a deletions [E1b expression is not essential for Ad replication in cancer cells [E1b expression may enhance the efficiency of Ad replication in cancer cells, likely via cyclin E induction [E1a expression and is involved in the induction of cyclin E which is required for efficient Ad replication [E1b upregulates autophagy [E1b expression supports autophagy induction by Ads to promote virus replication and cancer cell lysis by generating nutrients for building viral particles [E1b-wildtype status.The expression of eletions ,82. Evener cells , E1b expnduction  and autonduction . We havelication ,21,22,23utophagy . Autophautophagy ,85,86. Iarticles ,83. TherE1b expression and mutations in the Ad late proteins, especially those associated with genome packaging, may augment Ad release and spread within tumor tissue, leading to enhanced oncolytic therapy efficacy. One of the limitations of this study is that we do not know the specific effects of each of these mutations upon oncolytic replication at this time; it is possible that several of these mutations may cooperate to enhance oncolytic virotherapy efficacy.These findings taken together provide additional insight into the construction of oncolytic Ads. It appears that