The two gels showed good antiviral therapeutic indexes . MZC showed greater anti-simian-human immunodeficiency virus reverse transcriptase (SHIV-RT) activity than TFV 1% gel in rhesus macaque vaginal explants. MZC protected mice from vaginal herpes simplex virus 2 (HSV-2) challenge (P < 0.0001), but the TFV 1% gel did not.We compared the preclinical safety and efficacy of tenofovir (TFV) 1% gel with that of MZC gel [containing 50 μM MIV-150, 14 mM Zn(O Recent clinical trials like CAPRISA 004   have invexplants . Herein,explants  in a sidexplants .MZC, TFV 1%, and 3% CG gels were formulated as previously described . MZC, li5/ml) or activated PBMCs (2 × 106/ml) were treated for 1 h with dilutions of gels (triplicates) before adding 100 focus forming units (FFU) or one hundred 50% tissue culture infective doses (TCID50) of virus, respectively. TZM-bl cells were incubated for 72 h before staining with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside  to count FFU. The supernatant was replaced for PBMCs with fresh stimulation medium on days 1 and 4 postinfection. The p24 level in the supernatant was determined on day 7 after infection by p24 enzyme-linked immunosorbent assay (ELISA) . The 50% cytotoxic concentration  of each gel formulation was estimated using XTT and CyQuant by running the antiviral assay in the absence of virus (50s) were calculated based on gel dilution factor in order to compare the efficacies of the two gels, each containing a different active pharmaceutical ingredient (API). By comparing the EC50s based on gel dilution, we observed how MZC with only 0.002% of MIV-150 can achieve better or similar antiviral activity than TFV gel containing 1% of the API. MZC gel was generally more potent than TFV 1% gel in blocking HIV-1 infection in TZM-bl or PBMC with the clear exception of one multidrug-resistant (MDR) strain [OL-1/4(II)d4] containing 2 mutations (K101E and Y181I) associated with decreased susceptibility of viruses to NNRTI (50s for two strains (71361-1 and 56252-1) containing the 65R amino acid change associated with HIV resistance to TFV. Although NNRTIs are known to select resistant viruses rapidly, MIV-150 seems to select resistance at a slower pace compared to other NNRTIs and requires two or more mutations in a single genome to decrease HIV susceptibility  vaginal explant model using cell-free virus inoculum and also cell-associated virus.Cell-based assays are excellent tools for screening potential microbicides, for testing antiviral properties against a variety of isolates/MDR strains, and for monitoring the stability of formulations. However, the testing of a lead formulation in the explant model allows for assessment of preclinical safety and efficacy in a more relevant HIV target cell and architectural context. We tested MZC and TFV 1% gel in our ex vivo cell-free simian-human immunodeficiency virus reverse transcriptase (SHIV-RT) challenge was performed as described in Barnable et al. (4 TCID50/explant) 24 h or 4 days after gel exposure. Following 18 h of viral challenge, the tissues were washed and cultured for 14 days in the presence of IL-2. Simian immunodeficiency virus (SIV) p27 release was measured at 0, 3, 7, 11, and 14 days of culture by ELISA. Cell-associated SHIV-RT infection was performed following a different model  for ∼18 h, washed, and maintained in culture medium in the presence of IL-2 (100 U/ml) (versus 10 μM lamivudine [3TC] control). Mitomycin-C-treated, SHIV-RT-infected PBMCs cultured alone were included as controls. The SHIV-RT p27 concentration was determined in supernatants during 14-day culture. Soft endpoint (SOFT) analysis demonstrated that MZC and TFV 1% gels significantly reduce SHIV-RT infection from cell free (P < 0.0001). Similar results were observed with cumulative analysis (not shown).Macaque vaginal mucosal samples (biopsy specimens or necropsy tissues) were collected and transported overnight as previously described . Histoloe et al.  in an imnt model  where maell free  and cellell free  challengin vitro and in vivo) against HSV-2 (3 PFU/mouse) and were examined and scored daily for 21 days as previously described (We have previously shown that the combination of CG and zinc acetate (as in the MZC formulation) results in antiviral synergy (st HSV-2 . We explst HSV-2 . Mice weescribed . Despiteescribed .A possible explanation for these divergent results (compared to CAPRISA 004 results in humans) is that TFV phosphorylation and/or TFV uptake may be less efficient in mice than in human cells. In fact, subtherapeutic (below a lower limit of quantification [LLOQ] of <100 ng/g) TFV diphosphate (TFV-DP) levels were found in murine cervicovaginal tissue, and even TFV-only levels were low  as determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) .in vitro and in vivo anti-HPV activities make this formulation a very appealing MPT candidate targeting three noncurable viral sexually transmitted infections (STIs) (MZC's potent s (STIs) . Howevers (STIs)  (a simil


In vivo, intravenous (i.v.) injection of liposomal C8 significantly inhibited HepG2 xenograft growth in severe combined immuno-deficient (SCID) mice, and mice survival was significantly improved. These preclinical results suggest that liposomal C8 could be further studied as a valuable anti-HCC agent.Hepatocellular carcinoma (HCC) remains a global health threat. The search for novel anti-HCC agents is urgent. In the current study, we synthesized a liposomal C8 ceramide, and analyzed its anti-tumor activity in pre-clinical HCC models. The liposomal C8 (ceramide) potently inhibited HCC cell  survival and proliferation, more efficiently than free C8 ceramide. Yet, non-cancerous HL7702 human hepatocytes were resistant to the liposomal C8 treatment. Liposomal C8 activated caspase-dependent apoptosis in HCC cells, and HCC cytotoxicity by liposomal C8 was significantly attenuated with co-treatment of caspase inhibitors. At the molecular level, we showed that liposomal C8 activated ASK1 -JNK  signaling in HCC cells. On the other hand, JNK pharmacological inhibition or dominant negative mutation, as well as ASK1 shRNA-knockdown remarkably inhibited liposomal C8-induced apoptosis in HCC cells. Further studies showed that liposomal C8 inhibited AKT-mTOR  activation in HCC cells. Restoring AKT-mTOR activation by introducing a constitutively-active AKT alleviated HepG2 cytotoxicity by liposomal C8.  Hepatocellular carcinoma (HCC) is a global health threat ,2,3. It Ceramides, a family of lipid molecules composed of sphingosine and fatty acid, are found in high concentrations within cell membranes ,8. CeramThe cell-permeable short-chain ceramides  have displayed significant anti-tumor activity against a variety of cancer cells (reviewed in ). These in vitro and in vivo. The underlying signaling mechanisms were also studied.Recent studies have shown that C6 ceramide and liposomal C6 ceramide could significantly inhibit HCC cell growth ,14,15. IHuman HCC cell lines, HepG2, SMMC-7721 and Huh-7, were purchased from the Cell Bank of Shanghai Biological Institute CAS . HCC cells were maintained in DMEM/RPMI-1640 medium supplemented with 5% fetal bovine serum (FBS) at 37°C. The human HL-7702 hepatocytes were obtained from the Fudan University IBS center , and were cultured in RPMI-1640 containing 10% FBS. Cells were changed to serum-free medium 24 h before the assay. All cell culture reagents were obtained from Gibco Life Technologies .sn-glycero-3-phosphoethanolamine/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxyPEG(2000)]/N-octanoyl-sphingosine-1-[succinyl(methoxyPEG(750))]/C8-ceramide (4.9:2.6:1.2:1:3 M ratio), dried under nitrogen, and hydrated with sterile isotonic 0.9% NaCl solution. Liposomes were prepared by sonication and extrusion of the solution through 100-nm polycarbonate membranes. Ceramide loading in liposomes was 14% (w/w). The liposomes were kept at 4°C in saline at a concentration of 25 mg/mL (containing 3.5 mg/mL C8 ceramide) before use. All lipids were purchased from Avanti .Ceramide liposomes and control liposomes (no C8 ceramide) were prepared, briefly, lipids were solubilized in chloroform, combined in a specific molar ratio DSPC/1,2-dioleoyl-The caspase-3 inhibitor Z-VAD-fmk and the caspase-9 inhibitor Z-LEHD-fmk were purchased from Calbiochem . The two JNK  inhibitors, SP600125 and JNK inhibitor IX (JNKi-IX), were purchased from Selleck . p-AKT (Ser 473) antibody (9271), AKT1 antibody (2938), p-S6 ribosomal protein  antibody (2211), S6 antibody (2317), p-p70 S6 kinase 1  antibody (9209), S6K1 antibody (9202), p-SAPK/JNK1 (Thr183/Tyr185) antibody (9251), Jun N-terminal protein kinase (JNK1) antibody (3708), p-ASK1 (Thr 845) antibody (3765), ASK1 antibody (3762), cyclin D1 antibody (2922), HIF-1α antibody (3716), c-Jun antibody (9165) and (β-)Tubulin (D2N5G) antibody (15115), cleaved-caspase-3 antibody (9661) and β-actin antibody (3700) were all purchased from Cell Signaling Tech..3 cells/well) were seeded onto the 96-well tissue culture plate. Following indicated treatments, twenty μL of MTT (5 mg/mL) was added to the wells, and incubated for additional 2 h. Absorbance was recorded at 590 nm using an Eliza Plate Reader.Cell proliferation was analyzed by 3--2,5-diphenyl tetrazolium bromide  assay. Briefly, cells  was mixed with an equal volume of trypan blue, and incubated for 5 min at room temperature. Cell death ratio was calculated by the number of trypan blue positive cells divided by the total cell number × 100%.Following indicated treatment, cell death ratio was calculated by counting the cells in a Neubauer chamber. An aliquot of the total cell suspension , or caspase-9 substrate (Ac-LEHD-AFC). Enzymatic activity was measured at excitation wavelength of 380 nm and emission wavelength of 440 nm.Cells were lysed in the buffer containing 5 mM Tris (pH 8), 20 mM EDTA, and 0.5% Triton-X100. Reaction mixture contained 20 mM HEPES (pH 7), 10% glycerol, 2 mM dithiothreitol, 30 Following applied treatment, TUNEL  In Situ Cell Death Detection Kit (Roche) was utilized to test cell apoptosis according to the manufacturer’s instructions. Cells were also stained with 4’,6’-diamino-2-phenylin-dole  to visualize cell nuclei. Cell apoptosis percentage was calculated by the TUNEL percentage (TUNEL vs. DAPI) detected under a fluorescence microscope (Zeiss). At least 500 cells in 10 random views (1: 200 magnification) for each condition were included to calculate TUNEL percentage. Representative images were also shown.2, 1 mM MgCl2, and 4% BSA), with 5.0 μg/mL Annexin V-FITC (Invitrogen) for 15 min under dark. Cells were also washed with PBS and resuspended in binding buffer with 2.0 μg/mL propidium iodide (PI) (Invitrogen). A total of 10,000 cells per sample were analyzed by flow cytometry in a FACS . Annexin V percentage was recorded as a quantitative apoptosis measurement.After applied treatment, the floating and attached cells were collected. Cells were then washed with cold PBS, and incubated with 0.5 mL of binding buffer  were separated by electrophoresis in SDS-PAGE gels. Proteins were then transferred to the PVDF membranes, and detected with the specific primary antibodies. The immuno-reactive proteins after incubation with corresponding secondary antibodies were detected with the enhanced chemiluminescence (ECL) system . Band intensity was quantified through ImageJ software, and was normalized to the corresponding loading control. All the blot data in this paper were repeated four times, and similar results were obtained.HepG2 cells were seeded onto the six-well plate with 50% confluence plus polybrene (Sigma). ASK1-shRNA (h) lentiviral particles  (10 μL/mL medium) were directly added to cultured HepG2 cells for 12 h. Cells were then cultured in fresh medium for additional 24 h. Control cells were incubated with same amount of scramble shRNA lentiviral particles  (10 μL/mL medium). Stably cells were selected by culturing in the presence of puromycin (2.5 μg/ml) for 10–12 days. ASK1 expression in stable cells was verified by Western blots.The dominant negative JNK1 (dn-JNK1), achieved by switching Lys in the ATP-binding domain to Arg, was designed and synthesized by Genechem . The cDNA was inserted into pSuper-puro plasmid (a gift from Dr. Dongfeng Xu’s Lab). The dn-JNK1 plasmid or the empty vector (pSuper-puro) was then transfected into HEK-293 cells together with viral packaging protein plasmids VSVG and Hit-60 (Promega) with the Lipofectamine 2000 (Invitrogen) reagent. The virus-containing supernatants were collected and filtered, and were then added to HepG2 cells (seeded onto the six-well plate with 50% confluence with polybrene). Stably cells were selected by culturing in the presence of puromycin (2.5 μg/ml) for 10–12 days. JNK1 expression in stable cells was verified by Western blots.The adenoviral vector expressing a constitutively active AKT1 (ca-AKT1) and the empty vector expressing green fluorescence protein (Ad-GFP) were gifts from Dr. Teng , and wers.c.) with a significant amount of HepG2 cells (1×107 per mouse). After 7–10 days, the tumors reached the average volume of 100 mm3, the mice were divided into four groups: Saline, low-dose of liposomal C8 ceramide , high-dose of liposomal C8 ceramide , the empty liposomes (no ceramide), with 10 mice per group. Saline or liposomes were injected intravenously (i.v.) every 2 days, for a total of 30 days. The tumor volumes were recorded every 10 days, calculated using formula: π/6 × larger diameter × 2. For the mice survival studies, humane endpoints were utilized to minimize mice suffering. Animals were observed every day. Clinical signs such as significant-reduced locomotion, signs of severe diarrhea, severe piloerection or a sudden weight loss (>20%) were recorded. If animals reached these endpoints they were euthanized by exsanguination under 2,2,2-tribromoethanol anesthesia . All injections were performed under the 2,2,2-tribromoethanol anesthesia method. Animal studies have been approved by the Nanjing Medical University’s Institutional Animal Care and Use Committee .The male severe combined immuno-deficient (SCID) mice were implanted subcutaneously  followed by Turkey's test. All data was expressed as mean ± standard deviation (SD). A value of First, we tested the potential effect of liposomal C8 (ceramide) on HCC cell proliferation, and compared its activity with free C8 ceramide. MTT assay results in At the meantime, as shown in Next, we studied the potential role of liposomal C8 on HCC cell apoptosis. HepG2 cells were treated indicated concentrations of liposomal C8, and following cell apoptosis was tested by three independent assays: TUNEL staining assay , histoneTo study the role of apoptosis in liposomal C8-indcued HepG2 cytotoxicity, caspase inhibitors were applied. As demonstrated, the caspase 3 specific inhibitor z-VAD-fmk and the caspase-9 specific inhibitor Z-LEHD-fmk significantly attenuated liposomal C8-indcued HepG2 cell growth inhibition  . As demoin vitro anti-HCC activity.Further studies were also performed to investigate the role of ASK1 in liposomal C8-induced actions. ShRNA strategy was applied. As demonstrated, targeted-shRNA significantly downregulated ASK1 expression in the stable HepG2 cells . ImportaOver-activation of AKT-mTOR  signaling plays an important role in regulating several key cancerous behaviors of HCC . ExistinTo study the role of AKT-mTOR inactivation in liposomal C8’s actions, we introduced a constitutively-active AKT1 (“ca-AKT1”) into HepG2 cells, and stale cell line was selected . Westernin vivo anti-tumor activity of liposomal C8 [i.v. injection of liposomal C8 at 5 and 15 mg/kg (body weight) dramatically inhibited HepG2 xenograft growth in SCID mice . Further, ca-AKT1 introduction had no detectable effect on JNK activation by liposomal C8 (Data not shown). It should be noted that ASK1-JNK inhibition or ca-AKT1 introduction only inhibited, but not reversed, liposomal C8-induced cytotoxicity against HCC cells, indicating that other signalings could also play a role in mediating liposomal C8’s actions in HCC cells. For example, a recent study by Chen et al., showed that C6 ceramide activated AMP-activated protein kinase (AMPK)-dependent p53 signaling to promote cancer cell apoptosis . It was In vivo, liposomal C8 injection also failed to induce apparent toxicities to tested mice. Our results here are consistent with other studies using other liposomal systems [Intriguingly, we showed that the cytotoxicity by liposome C8 ceramide was only observed in HCC cells, but not in non-cancerous HL7702 hepatocytes, suggesting a neoplasm-selective mechanism.  systems ,30. For  systems . One exp systems , contribin vitro findings along with in vivo results support further development of liposomal C8 as a novel and efficient anti-HCC agent.In summary, we demonstrate that liposomal C8 ceramide exerts potent anti-tumor activity in pre-clinical HCC models. Molecularly, ASK1-JNK activation and AKT-mTOR inactivation mediate, at least in part, liposomal C8-induced actions in HCC cells. Our S1 FigData represent the means of three independent experiments ± SD.(EPS)Click here for additional data file.S2 FigBar = 25 μm. Data represent three independent experiments.(EPS)Click here for additional data file.S3 Fig# indicates statistically significant differences compared to liposomal C8 only group.Data represent the means of three independent experiments ± SD. The asterisks (*) indicate statistically significant differences compared to “C” group. (EPS)Click here for additional data file.


Durvalumab monotherapy has shown durable responses in a NSCLC cohort from a Phase I/II study in heavily pre-treated patients with solid tumors[Platinum-based doublets are standard of care (SoC) first-line treatment for advanced NSCLC, but durable benefit is observed infrequently, with resistance to chemotherapy invariably developing. The blockade of immune checkpoints is a promising novel approach in cancer treatment. Blocking co-inhibitory molecules, such as programmed cell death-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), from binding with their ligands can restore T-cell responses against tumors. As these pathways are non-redundant, dual targeting may have additive or synergistic antitumor activity. Durvalumab (MEDI4736) is a selective, high affinity human IgG1 mAb that blocks programmed cell death ligand-1 (PD-L1) binding to PD-1  NSCLC who are wild-type for epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) will be randomized 1:1:1) to receive durvalumab (20 mg/kg IV every 4 weeks for up to 12 months) plus tremelimumab (1 mg/kg IV every 4 weeks for up to 4 doses); durvalumab monotherapy (20 mg/kg IV every 4 weeks for up to 12 months); or SoC chemotherapy (Figure :1:1 to rClinicalTrials.gov identifier NCT02453282.


The major sources of scar-forming myofibroblasts during liver fibrosis are activated hepatic stellate cells (HSC) and portal fibroblasts (PF). In contrast to well-characterized HSC, PF remain understudied and poorly defined. This is largely due to the facts that isolation of rodent PF for functional studies is technically challenging and that PF cell lines had not been established. To address this, we have generated two polyclonal portal myofibroblast cell lines, RGF and RGF-N2. RGF and RGF-N2 were established from primary PF isolated from adult rat livers that underwent culture activation and subsequent SV40-mediated immortalization. Specifically, Ntpdase2/Cd39l1-sorted primary PF were used to generate the RGF-N2 cell line. Both cell lines were functionally characterized by RT-PCR, immunofluorescence, immunoblot and bromodeoxyuridine-based proliferation assay. First, immortalized RGF and RGF-N2 cells are positive for phenotypic myofibroblast markers alpha smooth muscle actin, type I collagen alpha-1, tissue inhibitor of metalloproteinases-1, PF-specific markers elastin, type XV collagen alpha-1 and Ntpdase2/Cd39l1, and mesenchymal cell marker ecto-5’-nucleotidase/Cd73, while negative for HSC-specific markers desmin and lecithin retinol acyltransferase. Second, both RGF and RGF-N2 cell lines are readily transfectable using standard methods. Finally, RGF and RGF-N2 cells attenuate the growth of Mz-ChA-1 cholangiocarcinoma cells in co-culture, as previously demonstrated for primary PF. Immortalized rat portal myofibroblast RGF and RGF-N2 cell lines express typical markers of activated PF-derived myofibroblasts, are suitable for DNA transfection, and can effectively inhibit cholangiocyte proliferation. Both RGF and RGF-N2 cell lines represent novel in vitro cellular models for the functional studies of portal (myo)fibroblasts and their contribution to the progression of liver fibrosis. Portal fibroblasts (PF) are defined as resident spindle-shaped fibroblasts found in the portal mesenchyme, with a peribiliary distribution. During Cell culture reagents and media were obtained from Life Technologies . Molecular biology reagents and kits were obtained from Life Technologies and Qiagen . All other reagents and chemicals were of the highest quality available.All rat experiments were performed in accordance with regulations approved by the University of Arkansas for Medical Sciences Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (12 weeks/400 g) were purchased from Charles River Laboratories  and used for two-step collagenase liver perfusion protocol,16.2+/Mg2+ buffer (Life Technologies) supplemented with heparin  for blanching. Upon inferior vena cava transection to allow blood and fluid drainage, the liver was further perfused with HBSS buffer minus Ca2+/Mg2+ alone, then with a collagenase (type 2 blend) solution  in HBSS plus Ca2+/Mg2 buffer. The liver was then removed from the rat and triturated in cold Leibovitz's media (Life Technologies), to tease away parenchymal cells from the biliary tree. The recovered hilar remnants were further digested in a solution of pronase  in Dulbecco's modified Eagle's medium/F-12 media  supplemented with type 2 collagenase and DNAse (Sigma-Aldrich), followed by mesh filtration . The remaining pronase-resistant hilar remnants were recovered and further digested in a solution of hyaluronidase  in DMEM/F-12 media supplemented with type 2 collagenase and DNAse, again followed by mesh filtration. The liberated cells from both pronase and hyaluronidase digestion steps were combined and washed with a solution of DNAse in RPMI 1640 media (Life Technologies). Finally, the resulting cell suspensions of non-parenchymal cells  were plated in medium containing Dulbecco's modified Eagle's medium/F-12 supplemented with 10% fetal bovine serum, 2% penicillin-streptomycin, 0.3% gentamycin, and 0.1% fungizone (Life Technologies).Primary PF were isolated from freshly perfused livers of healthy rats, as previously described,16. The PRIMARY PF)[RGF and RGF-N2). In the case of RGF-N2 cell line, primary isolated cells were sorted before plating and immortalization, using a rabbit polyclonal antibody raised against PF marker rat Ntpdase2 [Primary isolated rat PF were grown on plastic for 2 days before media change, and then passaged every 4 days twice, at which time cell purity approaches 100% and myofibroblastic differentiation is observed (IMARY PF). To immo, Canada) pre-labe, Canada) coupled Acta2 , Eln , Col1α1 , Entpd2 , Nt5e , and housekeeping B2m  and Hprt1  genes in rat species. The following PCR protocol was used: initial denaturation step at 95°C for 3 minutes, followed 40 repetitions of a 2-step cycling program consisting of 10 seconds at 95°C, 30 seconds at 55 or 57°C.Total RNA extractions were performed from rat liver tissue, primary isolated PFs, and SV40-immortalized RGF and RGF-N2 PFs, using a RNeasy Plus Kit (Qiagen). Quantification of total RNA concentration was performed using the Qubit RNA Assay Kit with a Qubit 2.0 Fluorometer (Life Technologies). Treatment of isolated total RNA samples with RNAse-free Ambion DNAse1 enzyme (Life Technologies) was performed to remove any genomic DNA contamination. Complementary DNA (cDNA) synthesis reaction was performed using the iScript Reverse Transcription Supermix . Primary and immortalized cell cDNA reaction products were further used as templates for polymerase chain reaction (PCR) amplification with the TopTaq Master Mix Kit (Qiagen). Target-specific PCR primers sets  were designed using NCBI’s Primer-BLAST program and are g for 15 min, and protein concentration was determined with the BCA Protein Assay kit (Thermo Scientific) using bovine serum albumin (BSA) as standard. Proteins (40 g per well) were resolved by SDS-PAGE under reducing conditions, and transferred onto a polyvinylidene difluoride membrane . Membranes were blocked with 5% Carnation skimmed milk in 1X Phosphate-Buffered Saline (PBS) or 1X Tris-Buffered Saline (TBS), incubated with the following primary antibodies (diluted in 5% BSA in 1X PBS or 1X TBS): mouse monoclonal anti-pan cytokeratin  and anti-glial fibrillary acidic protein  antibodies or rabbit polyclonal: anti-αSMA , anti-elastin antibody , anti-platelet derived growth factor receptor, beta polypeptide , anti-epidermal growth factor receptor , and anti-glyceraldehyde-3-phosphate dehydrogenase  antibodies, followed by appropriate Molecular Probes goat Alexa Fluor-conjugated anti-mouse IgG and Fluor-conjugated anti-rabbit IgG antibodies (diluted in 0.5% Milk in 1X PBS or 1X TBS), and bands were visualized by use of a Typhoon imaging system   .L, Ectonucleotidases-Ab) and anti-rat elastin , overnight at 4°C. Slides were washed 3 times with 1X PBS, then incubated with appropriate Molecular Probes goat Alexa Fluor-conjugated anti-mouse IgG and anti-rabbit IgG antibodies (Life Technologies) for 1 h at room temperature. Slides were washed 3 times with 1X PBS, 1 time with deionized water, and subsequently mounted in ProLong Gold Anti-fade reagent with 4,6-diamidino-2-phenylindole (DAPI) nuclear stain medium (Life Technologies). Slides incubated with secondary antibody alone were used as a control for specificity of fluorescence detection. Confocal fluorescence microscopy images were acquired using Zeiss LSM 510 Meta imaging system   solution  for 15 min at room temperature and washed in 1X PBS. Fixed cells were then incubated with the following mouse monoclonal antibodies: anti-β-actin , anti-rat Cd73 , anti-αSMA , anti-simian virus SV40 T antigen , and rabbit polyclonal antibodies: anti-rat Ntpdase2  .4) were transfected immediately after trypsinization with a monomeric GFP (mGFP) mammalian expression vector  using Fugene6 transfection reagent (Promega) at 1:3 (microgram(s) of DNA/microliter(s) of reagent) ratio or Lipofectamine2000 at 1:6 ratio, and grown for 48 h. Transfected cells were then fixed in neutral 4% PFA solution, washed in 1X PBS, and mounted in ProLong Gold Anti-fade reagent with DAPI, prior to confocal fluorescence microscopy imaging  were plated in individual wells of 6-well plates and overlaid with culture media containing bromodeoxyuridine-labeling reagent from a commercial cell proliferation kit  for a 24-hour period, to allow incorporation. On day 2, RGF and RGF-N2 cells were added (20 x 103 cells count per well) at an estimated 2:1 ratio to RGF and RGF-N2 cells, as previously described. Both Mz-ChA-1 and RGF or RGF-N2 cells were maintained in co-culture for an additional 24-hour period, in DMEM-high glucose supplemented with 10% fetal calf serum, 2% penicillin-streptomycin. On day 3, proliferation rate of Mz-ChA-1 cells was assessed by ELISA, according to the manufacturer’s instructions (Roche Biosciences). Primary isolated rat PFs were used as experimental control cells (not shown). Labeled Mz-ChA-1 cells grown alone were included as baseline controls , as previously described. All celcontrols .P-value was considered significant, when P < 0.05.Statistical analysis was performed using one-way ANOVA followed by Bonferroni's post-hoc multiple comparisons test. Entpd2/Cd39l1) and Thy1.1+ (Cd90.1/Thy1a) markers were used for cell sorting and enrichment of PF from primary isolated cell preparation pools[NTPDase2) and, an Alexa 647-conjugated mouse monoclonal anti-rat Thy1.1+, which selectively stained approximately less than 4% of primary isolated cell preparation (THY1.1+). Both non-sorted and Ntpdase2-sorted primary isolated PF cell preparations were cultured on plastic for 10 days, before transfection with SV40 large T antigen expression vector was performed. Polyclonal RGF and RGF-N2 cell lines were obtained by serial dilutions  and, then, constant passaging steps. Data presented here are from RGF and RGF-N2 cells after 45 passages.To generate the immortalized rat PF cell lines, we first isolated primary rat PF from livers of healthy rats. Primary isolated cells were either immediately plated in the case of RGF cell line, or used for cell staining/sorting before plating in the case of RGF-N2 cell line. Based on published literature, ectonucleoside triphosphate diphosphohydrolase 2/Ntpdase2 [αSMA isoform (Acta2)[Timp1)[Eln)[Fbln2)[Col15α 1)[Lox), and lysyl oxidase-like 1–4 (Loxl1-4)[Cygb)[Vim)[Des) and Lecithin-retinol acyltransferase (Lrat)[(Nt5e) and Tissue non-specific alkaline phosphatase (Alpl) enzymes mRNAs was also detected. Both RGF and RGF-N2 also express several key receptors for signal transduction in myofibroblasts[Pdgfrβ/Cd140b), Transforming growth factor, beta receptor 1 (Tgfβr1/Alk5), Epidermal growth factor receptor (Egfr), Insulin growth factor 1 receptor (Igf1r/Cd221), and Tumor necrosis factor receptor superfamily 1a (Tnfrs1a/Cd120a) and 1b (Tnfrs1b/Cd120b) members. Moreover, Interleukin-4 receptor alpha (Il-4ra/Cd124), Interleukin-13 receptor, alpha 1 (Il-13ra/Cd213A1), Cd200 (Mox2), and Cd9 gene products were also detected. Interestingly, expression of Vascular endothelium growth factor receptor-2 (Vegfr2/Cd309) appears to be weakly detected or absent in RGF-N2 cells, when compared to RGF cells and primary isolated aPF cells. Expression of G protein-coupled bile acid receptor 1 (Gpbar1/Tgr5) mRNA was observed in primary isolated aPF cells but not in RGF and RGF-N2 cells. Separately, expression of markers for other parenchymal and non-parenchymal liver cell types was studied, and showed that RGF and RGF-N2 cells express neither Albumin  nor F4/80  markers mRNAs, although Keratin 19  mRNA expression was detected  relatively to primary isolated aPF, nonetheless with still detectable expression. Second, the cellular origin or identity of RGF and RGF-N2 cell lines was further established, following analysis of total protein extract samples by immunoblot for expression of several proteins including simple epithelium keratins, Gfap, αSMA, Elastin, Pdgfrβ and Egfr fibroblast-specific marker Elastin  for mRNA expression of typical myofibroblastic markers . Like prCol1α 1), and micm (Acta2),23, tiss2)[Timp1) mRNAs (Fmp1)[Eln),25, fibun)[Fbln2), type XVCol15α 1), lysyl o(Loxl1-4) markers.-4)[Cygb), along wygb)[Vim), Glial fygb)[Vim) mRNAs wase (Lrat) markers.broblasts,32 includetected . Expressdetected . In addie RT-PCR . We founand Egfr . Immunoband Egfr . Additioand Egfr . Protein Elastin . Furthermembrane . As expemembrane . Taken tmembrane . In contIgf1r/Cd2, and TumIgf1r/Cd2, and Tumefficacy , comparaαSMA (mRNA and protein), Pdgf receptor-β(mRNA and protein), type I Collagen α1 (mRNA), and PF-specific Elastin (mRNA and protein), type XV Collagen α1 (mRNA) and Fibulin-2 (mRNA), an expression pattern also observed in cultured primary isolated aPF (mRNA). Importantly, RGF and RGF-N2 cell lack expression of HSC-specific Desmin and Lrat marker mRNAs. Both cell lines do express cholangiocyte Keratin 19 mRNA, although no corresponding protein expression was noted by immunoblot analysis, using a polyclonal pan cytoKeratin antibody . We sought to test the achievability of DNA transfer in RGF and RGF-N2 cell lines, which is an important feature to perform functional studies. Using a commercially-available transfection reagent, both RGF and RGF-N2 were found to be transfectable with DNA . Lastly, we demonstrated Ntpdase2-expressing RGF and RGF-N2 cell lines have the ability to block proliferation of Mz-ChA-1 cholangiocarcinoma cells, as was previously demonstrated for rat cultured primary isolated aPF. Importantly, we obviously cannot claim that the described polyclonal cell lines are functionally representative of all types of activated liver myofibroblasts originating from portal areas of the fibrosing liver. Specificity and reliability of cell markers, currently used to define cellular sources of liver myofibroblasts including portal fibroblasts, are still debated[in vitro study of liver portal (myo)fibroblasts functions.We report, in the present paper, the generation and functional characterization of two novel rat SV40-immortalized PF cell lines: RGF and RGF-N2. The RGF cell line was established by immortalization of cultured primary isolated aPF, while the RGF-N2 cell line was established by immortalization of cultured Ntpdase2-sorted primary isolated aPF. We performed preliminary studies to screen RGF and RGF-N2 cell lines for expression of common liver myofibroblast-associated genes. RGF and RGF-N2 express classical pan-myofibroblast antibody  and a mol debated,34. It il debated–38, as wS1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.


The identification of acetylation-mediated auto-proteolysis provides a new biochemical mechanism for tau self-regulation and warrants further investigation into whether auto-catalytic functions of tau are implicated in AD and other tauopathies.Tau proteins are abnormally aggregated in a range of neurodegenerative tauopathies including Alzheimer’s disease (AD). Recently, tau has emerged as an extensively post-translationally modified protein, among which lysine acetylation is critical for normal tau function and its pathological aggregation. Here, we demonstrate that tau isoforms have different propensities to undergo lysine acetylation, with auto-acetylation occurring more prominently within the lysine-rich microtubule-binding repeats. Unexpectedly, we identified a unique intrinsic property of tau in which auto-acetylation induces proteolytic tau cleavage, thereby generating distinct N- and C-terminal tau fragments. Supporting a catalytic reaction-based mechanism, mapping and mutagenesis studies showed that tau cysteines, which are required for acetyl group transfer, are also essential for auto-proteolytic tau processing. Further mass spectrometry analysis identified the C-terminal 2 Tau proteins are expressed primarily in the nervous system and are comprised of six isoforms containing up to two N-terminal repeats 0N, 1N, or 2N) and either three 3R-tau) or four 4R-tau) repeat domains that contribute to tau-microtubule (MT) binding, thereby regulating MT stability  and homogenized. The cell lysates were heated to 100°C for 10 min, rapidly cooled on ice for 20 min and centrifuged at 70,000xg for 30 min. Supernatants were dialyzed into FPLC buffer A , applied onto a HiTrap Sepharose HP IEX cation-exchange column , and eluted with a 0–0.4 M NaCl gradient using an AKTA-Pure FPLC system . The fractions were checked for the presence of the tau proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) followed by Coomassie Blue R-250 staining. The fractions containing enriched purified tau were pooled together and dialyzed against 100 mM sodium acetate buffer, pH 7.0. Importantly, all purified tau proteins were subject to a critical heat-denaturation step for 30 min at 100° to inactivate any endogenous bacterial enzymes and simultaneously enrich for heat-stable tau proteins. Proteins were concentrated using Amicon Ultra centrifugal filter units , and protein concentrations were determined using the bicinchoninic acid protein assay  with bovine serum albumin as the protein standard.All full-length , tau-K18 (4R), and tau-K19 (3R) proteins were purified using an AKTA-Pure FPLC chromatography system (GE). The proteins were expressed in Escherichia coli BL21(DE3) RIL strain. Bacteria were grown in LB media containing ampicillin at 37°C and induced with isopropyl-D -thiogalactopyranoside at a final concentration of 0.8 mM when the OD reached 0.6. After agitation for 2 h, cells were harvested by centrifugation. The pellet was re-suspended in high-salt RAB buffer [0.1 M MES, 1 mM EGTA, 0.5 mM MgSOin vitro using acetyl-CoA, butyryl-CoA, or propionyl-CoA (Sigma) in acetylation reaction buffer  at 37°C for the indicated timepoints from 1 day—7 days. Purified recombinant CBP protein (Enzo Life Sciences) (0.5 ug) was incubated in the acetylation reactions, where indicated, leading to maximal tau acetylation. Reaction products were analyzed by resolving proteins on 15% SDS-PAGE gels followed by Coomassie staining or subsequent immunoblotting with the indicated tau antibodies, where indicated. For tau pharmacological inhibition studies, tau proteins were pre-incubated on ice with 100 μM chymostatin, 1 mM N-ethylmaleimide (NEM), 4mM iodoacetamide (IA), 10 μM ALLN, and 10 μM Antipain (Sigma) followed by the above described acetylation reactions. All reactions were performed with a minimum of N = 3 independent experimental replicates. For tau fragmentation studies, error bars represent standard deviation (SD) among replicates and statistical calculations were performed using student t-test with significant p-values ≤ 0.01.Tau K18 or full-length tau proteins were either mock acetylated with coenzyme A alone or modified 2) followed by 5-min heat denaturing at 95° in SDS sample buffer and analysis of N- and C-terminal tau epitopes by immunoblotting.Calpain protease digestions were performed using 20 μM recombinant tau (2N4R) and increasing concentrations of 0–0.5 Units recombinant calpain-2 (Millipore) and incubated at 30° for 10-min in calpain reaction buffer  and 0.4 mM butyryl or propionyl-CoA were incubated in 30 μl of reaction buffer  for 1 hr at 37°C. Acylated tau products were analyzed by SDS-PAGE and Coomassie staining followed by gel excision for mass spectrometry using LTQ XL* Linear Ion Trap Mass Spectrometer (Thermo Scientific). Data was acquired using Xcalibur software (Thermo Scientific) and analyzed using Mascot, Scaffold, and PEAKS softwares.SEC was performed using an Acquity UPLC system equipped with a photodiode array detector . Injections of 15 μl were separated with an Acquity BEH200 SEC 1.7 μm (4.6 × 300 mm including a 4.6 × 30 guard column) using 100 mm sodium acetate, pH 5, with 300 mm NaCl at 0.3 ml/min over 30 min. Sample peaks were detected and analyzed using absorbance at 220 nm.Polyclonal anti-acetyl tau K280 antibodies were previously described . Anti-ac4, 0.02 M NaF, 2 mM DTT, pH 7.4). All buffers were supplemented with protease inhibitor cocktail, phosphatase inhibitor, and HDAC inhibitors (2 uM Trichostatin A and 10 mM nicotinamide). The high-salt RAB homogenates were incubated at 4° C for 20 min to depolymerize MTs, then centrifuged at 100,000 xg for 30 min at 4° C to yield the high-salt (HS) supernatant fraction. Pellets were re-homogenized and centrifuged in 3 vol/g of cold High Salt RAB buffer. Resultant pellets were homogenized in 3 vol/g RIPA buffer  and centrifuged at 100,000g for 30min followed by another RIPA extraction containing 20% sucrose, thus allowing myelin floatation. Finally, the resultant insoluble pellets were extracted in 1 vol/g tissue in urea buffer . Tau proteins present in soluble high-salt (HS) and insoluble urea fractions were analyzed by SDS-PAGE electrophoresis and western blotting using tau-5, T46, and K9JA antibodies to detect total tau protein fragmentation patterns.All brain tissue was acquired from the Center for Neurodegenerative Disease Research (CNDR) at the University of Pennsylvania brain bank. Frontal cortex from control and Braak stage VI Alzheimer’s disease cases were used to prepare gray matter, which was homogenized in 3 vol/g of cold High Salt RAB buffer  was strongly auto-acetylated in the presence of radiolabeled [etyl-CoA , lane 1,us study . Thus, Cin vitro tau auto-acetylation on tau fragmentation. Previous studies of bacterial acetyltransferases have hinted that auto-acetylation could be coupled to downstream post-translational processing including proteolysis [in vitro. Surprisingly, we noticed that acetyl-CoA, but not CoA, led to progressive fragmentation of tau-K18 into low molecular weight (LMW) species that were < 10 kDa  was employed to detect more slowly eluting low molecular weight tau fragments . SEC anain vitro [in vitro acetylation reaction, a cysteine-less tau-K18 protein containing C→A substitutions at both C291 and C322 (2CA) was sufficient to abrogate acetylation-induced fragmentation  and N-ethylmaleimide (NEM) were sufficient to prevent fragmentation of tau-K18  and 0N4Rlabeling .282LDLSNVQSK290 (2nd MTBR) at the start of the 17 kDa fragment and 341SEKLDFKDR349 (4th MTBR) at the start of the 12 kDa fragment  or +70 (butyrylation) mass additions were identified  to yield slightly larger molecular mass fragments. Alternatively, smaller C-terminal tau fragments could be targeted for degradation in vivo. In this regard, we note that mass spectrometry analysis of the ~12 kDa tau fragment identified an extreme N-terminal sequence 341SEKLDFKDR349 that is immediately adjacent to a putative recognition motif for chaperone-mediated autophagy (CMA), 336QVEVK340 [Based on our results, we hypothesized that acetylation-induced tau C-terminal fragments containing portions of the MTBR might accumulate in AD brain and potentially seed tau aggregation or alter synaptic signaling, for example by modulating the recently described KIBRA protein leading to memory loss . Our anaQVEVK340 . Thus, iIn summary, we provide evidence for a novel acetylation-induced auto-proteolytic cascade that generates N- and C-terminal tau fragments with potential implications for normal tau physiology and its pathological aggregation. Future efforts to characterize the disease relevance of tau auto-regulation could provide unique opportunities to directly modulate tau catalytic functions as a new therapeutic avenue for AD and other tauopathies. Tau auto-proteolytic fragmentation could also provide opportunities for biomarker discovery in tauopathies characterized by the accumulation of tau fragments in CSF and other patient biofluids.S1 FigFull-length 2N4R tau proteins were incubated with increasing concentrations of recombinant calpain-2 (0-.5U enzyme/reaction). Reactions containing calpain-induced tau fragments were analyzed by immunoblotting using a panel of tau antibodies detecting N-terminal (T14 and tau-5 antibodies) or C-terminal (K9JA and T46) tau epitopes. The calpain-mediated fragmentation patterns defined by the 15 kDa N-terminal fragment and 25 kDa C-terminal fragment are distinct from tau auto-acetylation induced fragmentation .(TIF)Click here for additional data file.S2 FigCBP-catalyzed tau acetylation was performed in co-transfected QBI-293 cells. Cell lysates were analyzed by tau immunoblotting using T46, K9JA, and tau-5 antibodies. Proteolytic tau low molecular weight smears were observed upon full tau acetylation in the presence of wild-type CBP, but were reduced with an enzymatically inactive CBP mutant (CBP-LD).(TIF)Click here for additional data file.S1 Tablein vitro acylation reactions were performed for 1hr at 37° using propionyl-CoA and butyryl-CoA, slightly longer acyl-coA derivatives compared to acetyl-CoA, and 1.0 μg reaction products were resolved by gel elecrophoresis and analyzed by mass spectrometry (see Tau etry see . Unambig(XLS)Click here for additional data file.


NT5E gene, is the dominant enzyme in the generation of adenosine from degradation of AMP in the extracellular environment. Nonsense  and missense  NT5E gene mutations in three distinct families have been shown recently to cause premature arterial calcification disease in human patients. However, the underlying mechanisms by which loss-of-function NT5E mutations cause human disease are unknown. We hypothesized that human NT5E gene mutations cause mistrafficking of the defective proteins within cells, ultimately blocking NT5E catalytic function. To test this hypothesis, plasmids encoding cDNAs of wild type and mutant human NT5E tagged with the fluorescent probe DsRed were generated and used for transfection and heterologous expression in immortalized monkey COS-7 kidney cells that lack native NT5E protein. Enzyme histochemistry and Malachite green assays were performed to assess the biochemical activities of wild type and mutant fusion NT5E proteins. Subcellular trafficking of fusion NT5E proteins was monitored by confocal microscopy and western blot analysis of fractionated cell constituents. All 3 F1, F2, and F3 mutations result in a protein with significantly reduced trafficking to the plasma membrane and reduced ER retention as compared to wild type protein. Confocal immunofluorescence demonstrates vesicles containing DsRed-tagged NT5E proteins  in the cell synthetic apparatus. All 3 mutations resulted in absent NT5E enzymatic activity at the cell surface. In conclusion, three familial NT5E mutations  result in novel trafficking defects associated with human disease. These novel genetic causes of human disease suggest that the syndrome of premature arterial calcification due to NT5E mutations may also involve a novel “trafficking-opathy”.Ecto-5′-nucleotidase/CD73/NT5E, the product of the  NT5E gene encodes the membrane-associated protein ecto-5′-nucleotidase/CD73/NT5E [EC 3.1.3.5], a 70 kDa, glycosylphosphatidylinositol (GPI)-anchored ectoenzyme and a key component of purinergic signaling pathways NT5E gene mutations: nonsense mutation  in exon 3, missense mutation  in exon 5, and nonsense  in exon 9 NT5E mutations were found to be associated with loss of functional enzymatic activity, and cause an imbalance in the metabolic pathway of pyrophosphate in circulation, eventually leading to abnormal vascular calcification. However, the precise molecular mechanisms by which NT5E mutations induce cellular defects have not been identified. Of note, in silico analysis of the potential effects of each mutation on NT5E protein structure indicated that, although all three actually result in NT5E proteins lacking catalytic activity, alterations would affect distinct features of newly-synthesized NT5E proteins such as conformation and/or membrane anchoring  proteins tagged with red DsRed monomer fluorescent probe see “; 2 NT5E anti-DsRed, green) and monoclonal anti-hNT5E clone 2B6  clearly co-localized with intrinsic DsRed fluorescence . We next extracted total proteins from the same transfected cells and analyzed their contents by immunoblot in wild type DsRed-hNT5E fusion proteins. In these experiments, we used COS-7 cells transiently transfected with DsRed monomer expression vector, and mock-transfected COS-7 cells as controls for antibody specificity. Immunoblot analysis with anti-DsRed antibody . The same anti-DsRed antibody also detected in the sample corresponding to COS-7 cells overexpressing DsRed-hNT5E a single band, with a molecular weight around 90 kDa . That molecular weight value would closely correspond to the predicted molecular weight of DsRed-hNT5E wild type fusion protein after maturation . In contrast, the same anti-hNT5E clone 2B6 antibody detected a major band in the sample corresponding to DsRed-hNT5E-overexpressing COS-7 cells with a molecular weight around 90 kDa , as it was observed with anti-DsRed immunoblot . As expected, again, no band was detected in mock-transfected COS-7 cells. We finally tested the biochemical AMPase activity at the cell surface of intact COS-7 cells overexpressing DsRed-hNT5E fusion proteins by enzyme immunohistochemistry, using adenosine monophosphate (AMP) substrate (COS-7 DsRed hNT5E WT + AMP). Such activity could be blocked in these cells when specific NT5E inhibitor α/β-methylene-ADP was added to the incubation medium. Similar results were obtained with the expression vector for wild type hNT5E protein (data not shown). As expected, mock-transfected COS-7 cells as well as COS-7 cells transfected with expression vectors for mutant DsRed-hNT5E F1-3 fusion proteins did not exhibit any AMPase activity (COS-7 DsRed hNT5E F1-3 + AMP). Taken together, these results show that addition of red fluorescent DsRed monomer probe does not affect the expression, distribution and AMPase activity of wild type hNT5E mature protein, therefore validating use of DsRed-hNT5E-encoding expression vectors for tracking studies.In COS-7 cells transiently transfected with wild type DsRed-hNT5E vector, double immunofluorescence staining  with polantibody  detectedturation  and is gturation  did not ubstrate . Brown pWILD TYPE-MERGED, arrows), and to less extent within the cytoplasm, exhibiting a distinctive punctate distribution pattern. In contrast, DsRed-hNT5E F1–3 fusion proteins were primarily observed within the cytoplasm, decorating vesicular-like structures (Family1-3-MERGED). This localization of mutant DsRed-hNT5E proteins differed from DsRed monomer distribution, which was also observed in the cytoplasm but was more evenly scattered (DsRed monomer-MERGED). Most importantly, DsRed-hNT5E F1-3 fusion proteins and DsRed monomer did not significantly overlap with the one of GFP-PM marker at the level of plasma membrane. In transfected cells labeled with cytoplasm-targeting GFP fusion protein (GFP-CYTO), DsRed-hNT5E wild type fusion protein was again observed lining the plasma membrane and following a clear punctate distribution pattern within the cytoplasm . This localization was clearly distinct from the one of GFP-CYTO marker. A similar conclusion was drawn after analysis of DsRed-hNT5E F2 fluorescence signal (Family2-MERGED). DsRed-hNT5E F1 and F3 fluorescence signals did partially co-localize with GFP-CYTO marker . DsRed monomer fluorescence signal was not observed at the plasma membrane level, but was uniformly distributed within cytoplasm, overlapping significantly with the GFP-CYTO marker (DsRed monomer-MERGED). In transfected cells labeled with endoplasmic reticulum-targeting GFP fusion protein (GFP-ER), DsRed-hNT5E wild type fusion protein was mainly detected at the plasma membrane level and only partially co-localized with the GFP-ER marker . While absent from the plasma membrane, DsRed-hNT5E F2 fluorescence signal also partially overlapped with the one of GFP-ER marker (Family2-MERGED). In contrast, DsRed-hNT5E F1 and F3 proteins significantly co-localized with the one of GFP-ER marker and were detected only within the cytoplasm . Finally, DsRed monomer fluorescence signal was again absent from the plasma membrane, and detected within the cytoplasm partially co-localizing with the one of GFP-ER marker (DsRed monomer-MERGED). Taken together, these results suggest that mutant DsRed-hNT5E F1-3 fusion proteins, in addition to be enzymatically inactive, assume intracellular trafficking pathways clearly distinct from the one of DsRed-hNT5E wild type fusion protein. While the DsRed-hNT5E wild type fusion protein is observed within ER structures and cytoplasm to some extent, and mainly at the level of plasma membrane, mutant DsRed-hNT5E fusion proteins are mainly detected within cytoplasm (F1 and F3) and ER vesicles (F1–3) compartments. Notably, subcellular distribution of DsRed-hNT5E F2 fusion protein is comparable to the one of DsRed-hNT5E wild type fusion protein, except for its absence from the plasma membrane.COS-7 cells transiently expressing wild type and mutant human DsRed-hNT5E  were labeled with GFP fusion proteins  specifically targeted to plasma membrane, cytoplasm and endoplasmic reticulum compartments. In transfected cells labeled with plasma membrane-targeting GFP fusion protein (GFP-PM), only fluorescence signal for DsRed-hNT5E wild type fusion protein was observed at the level of the plasma membrane, predominantly co-localizing  with the one for GFP-PM . As expected, examination of hydrophobic (plasma membrane-enriched) protein fraction shows that only DsRed-hNT5E wild type protein is abundantly present at the plasma membrane of all DsRed-hNT5E fusion proteins . Finally, analysis of microsomes-enriched protein fraction shows that DsRed-hNT5E F1–3 fusion proteins are significantly less abundant than their wild type counterpart, with DsRed-hNT5E F1 fusion protein being almost absent . Taken together, these results indicate that, although all DsRed-hNT5E fusion proteins are produced roughly to the same extent, only DsRed-hNT5E wild type protein is able to efficiently reach the plasma membrane compartment. These data also suggest that mutant protein trafficking mechanisms are deficient within and beyond the ER compartment as DsRed-hNT5E F1-3 fusion protein contents are greatly reduced in the microsome-enriched fraction, when compared to DsRed-hNT5E wild type protein.Total, hydrophobic, and microsome-enriched protein fractions were extracted from transiently-transfected COS-7 cells and analyzed for their contents in DsRed-hNT5E fusion proteins. Analysis of the total protein fraction reveals that DsRed-hNT5E wild type and mutant fusion proteins are produced to similar levels in COS-7 cells upon transient transfection (WT). Mock-transfected COS-7 cells or COS-7 cells transiently transfected with expression vectors for DsRed-hNT5E F1–3 fusion proteins had no detectable activity (MOCK and F1-3). Transfected COS-7 cells were also incubated in presence of sodium butyrate (NaB), or 4-phenyl sodium butyrate (4PBA) molecules for 24 hours (prior to ending 72-hour transfection reaction) and cell lysates extracted and tested for AMP hydrolysis activity. This experiment was designed to test whether both NaB and 4PBA molecules could be used as molecular chaperones and induce the recovery of biological AMPase activity from mutant DsRed-hNT5E proteins, as it had been previously done for other mutant proteins such as CFTR The ability to degrade AMP nucleotide substrate of DsRed-hNT5E fusion proteins was tested using the Malachite Green assay for detection of released inorganic phosphate. AMPase activity assays were performed on intact cells or using lysates from transiently-transfected COS-7 cells. In both assays, only samples from COS-7 cells transfected with expression vector for DsRed-hNT5E wild type fusion protein exhibited AMP hydrolysis , WT. MocNT5E genetic mutations in the context of the newly-described hereditary vascular calcification disorder due to these mutations, now shown to cause disease in three distinct family cohorts In the present report, we report here novel trafficking defects caused by Importantly, caution is required when interpreting data presented here and making general assumptions with regard to CD73 cellular dynamics and contribution to cell functions. Because of the chosen heterologous over-expression system, all DsRed-NT5E fusion proteins were produced at similar mRNA (not shown) and protein  levels. NT5E mutations? On one hand, besides the impact on pyrophosphate balance in the circulation described in the original article NT5E mutations. Future work should certainly explore the potential impact of NT5E mutations in other diseases relevant to NT5E (non-)catalytic activity functions such as, organ injury/fibrosis What could be the functional consequences of th and 27th amino acids (aa) of hNT5E protein sequence (NP_002517.1th and 79th base pairs (bp) of human NT5E consensus coding DNA sequence (NCBI Nucleotide ID: NM_002526.3[78–79]). In a first step, the pDsRed-Monomer-C vector was modified by directionally ligating a synthetic DNA polylinker containing a single Spe1 restriction (enzyme cutting) site into Sal1/HindIII restriction sites. In a second step, the signal peptide sequence of hNT5E protein  were used to generate mammalian expression vectors encoding a fusion protein consisting of the DsRed fluorescent probe attached to the N-terminal end of the mature form of human NT5E (hNT5E) protein (NCBI Protein ID: NP_002517.1[27–549]), after peptide signal .CTGGTGCCCAGGGGCAGC-, an internal EcoR1 restriction site (5′end) and a single Spe1 restriction site (3′end) were added to the corresponding 1647 bp-long coding hNT5E sequence (NM_002526.3[79–1725]) by high fidelity PCR using hNT5E cDNA as template and the following primersThe obtained PCR product was ligated into pCRII TOPO vector (Invitrogen) and ligation products were used for transformation bacteria following manufacturer's instructions. Plasmid DNA isolated from at least 3 individual colonies was analyzed by automated sequencing (UAMS DNA Sequencing Core Facility). The plasmid DNA from one selected clone was then Nhe1-digested, purified and ligated into the single Nhe1 restriction site of pDsRed-Monomer-C vector (located 16 bp 5′ upstream of in-frame DsRed-Monomer sequence). The ligation products were used for bacteria transformation, and plasmid DNA isolated from at least 3 individual colonies was analyzed by automated sequencing. This step was carried out only once as nucleotide sequence analysis  shows full (100%) homology between the wild type and mutant sequences. In a third and final step, a consensus thrombin recognition motif was added to the 5′end of remaining portion of the hNT5E protein sequence (NP_002517.1[27–574]). A single Sal1 restriction site, a thrombin recognition sequence -Sal1In: forward, -CAGGGGCAGCGAATTCTGGGAGCTTACGATTTTGC-;Spe1: reverse, -ATATTACTAGTCTATTGGTATAAAACAAAGATCAC- .An aliquot (1/10) of the obtained PCR product was then used as template for a nested high fidelity PCR reaction using the following primersSal1Out: -TTAAGGCCTCTGTCGACCTGGTGCCCAGGGGCAGC- ;Spe1: reverse, -ATATTACTAGTCTATTGGTATAAAACAAAGATCAC- . The obtained nested PCR product was ligated into pCRII TOPO vector (Invitrogen). The ligation products were used for bacteria transformation and plasmid DNA isolated from at least 3 individual colonies analyzed by automated sequencing. The plasmid DNA from one selected clone was then was Sal1- and Spe1-digested, purified and directionally ligated into Sal1/Spe1restriction sites of pDsRed-Monomer-C vector. This step was carried out for each wild type and 3 mutant cDNAs as nucleotide sequence analysis shows expected variations between the wild type and mutant sequences 5/coverslip) 24 hrs post-transfection and cultured for additional 48 hrs. For each assay, experiments were performed 3 times, except for enzymatic assays with sodium butyrate and 4-phenyl sodium compounds 2 times. The COS-7 cell line was used in this study because it exhibits negligible basal levels of nucleotide-metabolizing enzyme activities African green monkey COS-7 cells (ATCC CRL-1651) were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum and 1% penicillin-streptomycin (Invitrogen). Transient transfection of COS-7 cells using plasmids encoding cDNAs for DsRed-hNT5E wild type and mutant forms was carried out with Lipofectamine 2000 reagent (Invitrogen) for 72 hrs, according to the manufacturer's instructions. Where appropriate, protein concentration was estimated using the Microplate BCA Protein Assay Kit  with reference to bovine serum albumin (BSA) as a protein standard. For enzymatic activity assays on protein extracts, cells were incubated in absence or presence of 5 mM sodium butyrate  or 5 mM 4-phenyl sodium butyrate (Sigma) 48 hrs post-transfection for additional 24 hrs. For enzymatic activity assays on intact cells, cells were split 24 hrs post-transfection, plated in 24-plate wells and cultured for additional 48 hrs. For fluorescence microscopy, cells were split and plated on sterile coverslips (10Transfected COS-7 cells were fixed in freshly prepared 4% (wt/vol) paraformaldehyde in neutral PBS (pH 7.2) for 20 min at room temperature. For enzyme histochemistry experiments, hNT5E AMPase activity was localized using a modified Wachstein/Meisel lead phosphate method, as previously described Protein extracts from transfected COS-7 cells were prepared, as previously described g for 5 min) in cold 1X PBS supplemented with protease inhibitors cocktail  before preparation of various protein fractions. Protein samples were prepared from the following cell compartments: total proteins using a mild-strength lysis buffer , hydrophobic (plasma membrane-associated) proteins using the MEM-PER Membrane Protein Extraction kit (Pierce Biotechnology), and crude microsomal fraction proteins using the Endoplasmic Reticulum Isolation Kit (Sigma). Protein fractions were separated by SDS-PAGE under reducing conditions, and transferred onto a polyvinylidene difluoride membrane . Membranes were blocked with 1X Tris-Buffer Saline, 5% milk, 0.1% Tween-20 and incubated with rabbit polyclonal anti-DsRed antibody (Clontech) followed by fluorescent-labeled goat anti-rabbit IgG antibody, or mouse monoclonal anti-hNT5E antibody (clone 2B6) followed by fluorescent-labeled goat anti-mouse IgG antibody, and bands were visualized by use of a Typhoon 9400 imaging system . Membranes were subsequently stripped and incubated with anti-calreticulin , and anti-EGFR  antibodies, and mouse monoclonal anti-beta actin clone AC-15 , followed by fluorescent-labeled goat anti-rabbit IgG or anti-mouse IgG antibodies, and band intensity signals were visualized and quantified by use of a GE Typhoon imaging system. Band densitometry analysis was performed using GE Image Quant TL software, version 7.0.Following transfection (72 hrs), cells were scraped and pelleted (500×www.graphpad.com.One-way ANOVA with Bonferroni's post-test was performed using GraphPad Prism version 5.0d for Mac, GraphPad Software, San Diego California USA,


VMHD becomes significant during systole when rapidly ejected blood from the left ventricle into the aortic arch interacts with the strong magnetic field  Phase Contrast Cine MRI slices were obtained in three healthy volunteer subjects n=3) along the aortic arch Fig.  to quant along thmpatible . For eacAfter MLR was performed for each subject Table , aortic Relatively accurate beat-to-beat stroke volume and blood flow velocity estimates can be obtained from MHD voltages extracted from 12-lead ECG, providing a means for enhanced patient monitoring inside the MRI bore. A relatively short Phase-Contrast Cine measurement is required to provide the required patient-specific parameters.NIH U41-RR019703, NIH R03 EB013873-01A1, SBIR-1 R43 HL110427-01.


Pseudomonas aeruginosa produces N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule for bacterial communication. C12 has also been reported to induce apoptosis in various types of tumor cells. However, the detailed molecular mechanism of C12-triggerred tumor cell apoptosis is still unclear. In addition, it is completely unknown whether C12 possesses any potential therapeutic effects in vivo. Our data indicate that, unlike most apoptotic inducers, C12 evokes a novel form of apoptosis in tumor cells through inducing mitochondrial membrane permeabilization independent of both pro- and anti-apoptotic Bcl-2 proteins. Importantly, C12 inhibits tumor growth in animals regardless of either pro- or anti-apoptotic Bcl-2 proteins. Furthermore, opposite to conventional chemotherapeutics, C12 requires paraoxonase 2 (PON2) to exert its cytotoxicity on tumor cells in vitro and its inhibitory effects on tumor growth in vivo. Overall, our results demonstrate that C12 inhibits tumor growth independent of both pro- and anti-apoptotic Bcl-2 proteins, and through inducing unique apoptotic signaling mediated by PON2 in tumor cells. Pseudomonas aeruginosa uses N-(3-oxododecanoyl)-L-homoserine lactone (C12) as a quorum sensing signal [Quorum sensing is a bacterial intercellular communication system that constitutively produces, releases, and detects small diffusible autoinducers that are similar to mammalian hormones . The graactone C1 as a quoin vitro is relevant to tumor growth in vivo has never been studied.Recent studies have shown that C12 is not only important in regulating bacterial virulence genes but also interacts with eukaryotic cells and modulates cell physiology, such as triggering cell death . C12 hasResistance toward apoptosis is a hallmark of most, perhaps all, types of human cancer , 10. Bcl2+-dependent lactonase and arylesterase activities [Pseudomonas aeruginosa quorum sensing by inactivating C12 [As a lactone, C12 is known to be hydrolyzed into a carboxylic acid by the lactonase paraoxonase 2 (PON2), which belongs to a gene family  with Cativities , 20. In ting C12 . PON2 anting C12 –24. The ting C12 , 24. In ting C12 .in vitro and the inhibitory effects of C12 on tumor growth in vivo. The role of pro- and anti-apoptotic Bcl-2 and PON2 proteins in C12-induced apoptosis was systematically examined. C12 inhibited the tumor growth through inducing apoptosis in a PON2-dependent but not Bcl-2 protein-dependent manner. Our study reveals a unique anti-tumor function of C12, which may lead to the development of new therapeutic agents for cancer.To gain insights into the mechanism of C12-evoked tumor cell apoptosis, we evaluated the cytotoxic effects of C12 on tumor cells The cytotoxic effects of C12 on tumor cells have been reported previously –8, 26, bin vivo.To investigate the relevance of C12 cytotoxicity on transformed cells to tumor growth in animals, we examined the effects of C12 on the growth of established Lewis Lung Carcinoma (LLC) tumors. As shown in Figure L ABT-737 at a constant dose ratio were measured, and the combination index (CI) values were determined  and cytochrome c release. Depolarization of Δψmito was evaluated by determining the changes in fluorescence of the voltage-dependent dye JC1 released from mitochondria into the cytosol and nucleus. Within minutes of C12 exposure, mitochondria in A549-vector cells and A549-Bcl-2 overexpressing cells were depolarized to the same degree  in C12-treated athymic nude mice  [in vitro as well as blocking tumor growth in vivo independent of both pro- and anti-apoptotic Bcl-2 proteins.Many neoplastic cells show an increased ratio of anti-apoptotic to pro-apoptotic Bcl-2 proteins, which enables them to survive even under the conditions that would normally initiate apoptotic signaling , 44. An 2) . The pol2) . Further2) , 17. ComThe apoptosis cascade induced by C12 in tumor cells is unique, evident by its rapid pro-apoptotic effects, such as depolarizing mitochondrial membrane potential within minutes Figures , releasiThis study also shows that endogenous PON2 is essential for C12‘s cytotoxicity in human lung tumor cells and inhibitory effects on tumor growth Figures –9, consiin vitro and block tumor growth in vivo is mediated through PON2 -homoserine lactone (C12), polybrene and actinomycin D were purchased from Sigma . Propidium iodide (PI), CM-H2DCFDA (5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester), TMRE  and TOTO-3 were obtained from Invitrogen . Unless otherwise stated, all reagents were dissolved in dimethyl sulfoxide (DMSO). Dulbecco's Modified Eagle's Medium (DMEM), penicillin/streptomycin, trypsin, and L-glutamine were obtained from Mediatech , and fetal bovine serum was purchased from Gemini . Caspase-Glo assay 3/7 kit was purchased from Promega . Antibodies (Abs) for western blot were anti-β-actin mAb (Sigma), anti-Bcl-2 mAb; anti-Bax pAb , anti-Bak pAb , anti-PON2 pAb; anti-PON3 pAb; anti-Mcl-1 pAb , anti-Noxa pAb , anti-Puma pAb  peroxidase-conjugated goat anti-rabbit IgG  and peroxidase-conjugated goat anti-mouse IgG (Thermo Fisher).L-IRES-EGFP was a gift from Dr. Levi Beverly (University of Louisville). The plasmid identities were validated by sequencing. Lentiviral human PON2 shRNA plasmid was purchased from Santa Cruz.The retroviral expression vector pBABE-IRES-mKate2 was generated by replacing EGFP cDNA in pBABE-IRES-EGFP with mKate2 cDNA. Human PON3 cDNA  or murine PON3 cDNA  was cloned into pBABE-IRES-mKate2 to generate pBABE-hPON3-IRES-mKate2 or pBABE-mPON3-IRES-mKate2. pBABE-hBcl-2-IRES-EGFP was generated by cloning human Bcl-2 cDNA into pBABE-IRES-EGFP. pMIG-hMcl-1-IRES-EGFP and pMIG-hBcl-xL or Mcl-1 were produced by retroviral infection as described previously [2 humidified incubator at 37°C.Retrovirus and lentivirus were generated as described previously . HCT116 eviously . To geneeviously . NHBE aneviously . Cells w®-3 Iodide using flow cytometry  as described previously [50 of C12 and ABT-737 on NCI-H1299 or A549 was first determined. Various combinations of C12 and ABT-737 at a constant ratio above or below their IC50 values were added to cells. Following incubation for the indicated time, cell viability was evaluated and Fa  was determined as the ratio between the cell death levels of drug-treated cells and those of untreated control cells. Combination index (CI) was calculated by the CompuSyn software .Cell viability of NCI-H1299 or A549 re-expressing PON3 cells was measured by TOTOeviously . Cell vieviously . The synCaspase-3/7 activities were measured using a Caspase-Glo assay kit  as described previously . The promito probe JC1 in growth media for 10 minutes at room temperature followed by three washes with Ringer's solution. Dye-loaded cells were mounted onto a chamber on the stage of a Nikon Diaphot inverted microscope with a Fluor 20x objective (0.75 NA) at room temperature. Treatments were made by diluting stock solutions into Ringer's solution at the indicated concentrations. Fluorescence imaging measurements of Δψmito were performed as reported previously [Cells were incubated with 10 μM Δψeviously , 34. BriTo image mitochondrial morphology and membrane potential using confocal microscopy, cells were transfected with mitochondrial matrix-targeted GFP plasmid  using an Amaxa nucleofector device , seeded onto 35 mm glass coverslips and placed in a tissue culture incubator. After 16–24 hours coverslips were mounted in a recording chamber positioned on the stage of an inverted microscope . Cells were visualized using a PlanApo 60x, 1.42 NA oil immersion objective and confocal time series images acquired using a VT-Infinity 3 . The chamber was perfused with Hank's Balanced Salt Solution containing 20 nM mitochondrial membrane potential probe tetramethylrhodamine, ethyl ester (TMRE) at room temperature. After a 15 minute equilibration period, GFP and TMRE were alternately excited using the 488 and 568 nm lines respectively of a Krypon-Argon laser. The emitted fluorescence was filtered using a dual bandpass filter set  and collected and using HCImage software . Mitochondrial morphology was analyzed using ImageJ to determine the mean area/perimeter ratio and inverse circularity as the measurement of interconnectivity and elongation respectively, as described previously .For immunofluorescence staining of A549 and HCT116 cells, cells plated 24 hours earlier were incubated for 3 hours with either vehicle (DMSO) or 50 μM C12 in Ringer's solution. The immunofluorescence staining of cytochrome c was carried out as described previously . Images For immunofluorescence staining of tumor sections, tumor sections (5 μm) were treated with antigen retrieval procedure by boiling in 10% Triton x-100, then slowly cooled down at room temperature. After incubating with the blocking buffer , the slides were incubated with antibodies against activated caspase-3  overnight at 4°C. Following three 10-minute washes, slides were incubated with goat anti-rabbit IgG  for 1 hour. The fluorescence was visualized by confocal microscopy using a 40x CFI Plan Fluor objective (NA 0.6) and was analyzed using ImageJ software (NIH) as described previously .For C12 toxicity studies, DMSO or C12 (25 mg/kg/day) was administered intraperitoneally each day in 8 week-old C57BL/6 mice or in 6 week-old athymic nude mice. All organs were collected and weighted at sacrifice.6 Lewis Lung Carcinoma cells on the right flank. Tumors were measured daily with dull edged Vernier calipers (V = L × W2/2). After tumor size reached around 100 mm3, animals with size-matched tumors were divided into control group and C12 group. DMSO or C12 was administered intraperitoneally each day. At the end of the experiments, tumors were excised for apoptosis evaluation. For the study of transplanted human tumors, female athymic nude mice (5–7 weeks) were purchased from Harlan Laboratories . Wild-type HCT116 cells and Bak/Bax DKO HCT116 cells (3.0 × 107/ml), wild-type A549 cells and Bcl-2-overexpressing A549 cells (3.0 × 107/ml), A549-control-shRNA cells and A549-PON2-shRNA cells (3.0 × 107/ml) were resuspended and mixed 1:1 (vol/vol) with Matrigel (BD Biosciences), then 0.1 ml mixture of cells and Matrigel was injected s.c. on the right flank of mice. All animals with size-matched tumors were divided into two groups (control and C12) when the tumor size reached around 100 mm3. For HCT116 and Bak/Bax DKO HCT116 tumors, DMSO or C12 was administered intraperitoneally each day. DMSO or etoposide (40 mg/kg/day) was administered intraperitoneally once every other day. For wild-type A549 tumors, animals were treated with C12 for 6 times/week. For Bcl-2 over-expressing A549 cells tumors, DMSO or C12 was administered with C12 each day. For A549-control-shRNA and A549-PON2-shRNA tumors, DMSO or C12 was administered twice every three days. At the end of the experiments, tumors were excised for apoptosis evaluation.For transplanted tumors in C57BL/6 mice, eight-week old C57BL/6 female mice  were inoculated subcutaneously (s.c.) with 1×10Tumor tissues along with corresponding adjacent normal tissues from non small cell lung carcinoma (NSCLC) patients were acquired from the James Graham Brown Cancer Center Bio-Repository at University of Louisville following an approved IRB protocol. Frozen tissue samples were resuspended in tissue protein extraction reagent (Thermo Fisher) supplemented with protease inhibitors  and phosphatase inhibitors . Following homogenization and centrifugation, protein concentration was determined by BCA assay (Thermo Fisher). Thirty micrograms of total protein was electrophoresed in 4–12% Bis/Tris gels . PON2 was detected by western blot as described previously .3 per well) for 24 hours. Cells were subsequently treated with DMSO or 100 μM CM-H2DCFDA in growth medium for 1 hour at 37°C. Growth medium was then removed and cells were washed twice with Ringer's Buffer. DMSO or 100 μM C12 in Ringer's Buffer was added to cells. The fluorescence signal (Ex/Em = 492 nm/520 nm) was measured kinetically at 37°C using the Gemini EM microplate spectrofluormeter . Data was plotted as relative fluorescence units (RFUs) versus time and the slope was determined (RFU/min) as an indicator of intracellular ROS level.A549 or NCI-H1299 cells were cultured in 96-well white-walled plates (7.5 × 10t-test. A p value < 0.05 was considered significant.Results are presented as mean ± standard deviation of at least three independent experiments. Statistical analysis was performed using Student's two tail


Interleukin-6 (IL-6) and soluble IL-6 receptor (sIL-6R) have been demonstrated to play a significant role as inflammatory mediators in systemic juvenile idiopathic arthritis (SJIA). Tocilizumab, a humanized anti-IL-6 receptor antibody, becomes a new biologic treatment for SJIA nowadays but the correlation of serum IL-6 levels and serum sIL-6R levels with disease activity in SJIA with and without tocilizumab treatment are still unclear.To determine the correlations of serum IL-6 levels and serum sIL-6R levels with disease activity in SJIA patients with and without tocilizumab treatmentSJIA patients in pediatric rheumatology clinic, Ramathibodi hospital between September 2011 and June 2013 were enrolled in this study. Patients were followed up three times in 2-3 months interval. Fifteen healthy children were included as normal controls. Demographic data was collected. During the visit, patients were evaluated according to Juvenile Arthritis Disease Activity Score-71 (JADAS-71) and blood samplings were collected for complete blood count, erythrocyte sedimentation rate, IL-6 levels, and sIL-6R levels, then patients were categorized into 4 groups; 1) active disease with systemic features and arthritis 2) active disease with only arthritis 3) remission on medication 4) remission off medicationForty-two SJIA patients, 131 blood samplings were included in this study. Seventeen patients (40%) were treated with tocilizumab during the study. Serum IL-6 levels in patients without tocilizumab treatment significantly elevated in active disease with systemic features and arthritis [median (IQR) = 101.8 (303.2) pg/mL] when compared to active disease with only arthritis [median (IQR) = 4.5 (23) pg/mL], and remission on medication [median (IQR) = 1.5 (0.55) pg/mL], whereas serum IL-6 levels in patients with tocilizumab treatment were not different between groups but there were significantly different when compared to healthy children (p < 0.05). In addition, the correlation between serum IL-6 levels and JADAS-71 in patients without tocilizumab treatment  was stronger than patients with tocilizumab treatment . Serum sIL-6R levels in SJIA patients with and without tocilizumab treatment were significantly higher when compared to healthy children (p < 0.05). Interestingly, in patients with tocilizumab treatment, serum sIL-6R levels were extremely higher  than patients without tocilizumab treatment [median (IQR) = 94.2 (82.7) ng/mL].The correlation between serum IL-6 levels and disease activity in patients without tocilizumab treatment was stronger than patients with tocilizumab treatment. In addition, serum sIL-6R levels in patients with tocilizumab treatment were extremely higher than patients without tocilizumab treatment.None declared.


Overall our data indicate that this interaction promotes anti-tumor effects in breast cancer cells. To assess the in vivo relevance of this interaction, we induced tumors in nude mice using MCF-7 cells expressing both ADAMTS-12 and fibulin-2 that showed a remarkable growth deficiency. Additionally, we also found that ADAMTS-12 may elicit pro-tumor effects in the absence of fibulin-2. Immunohistochemical staining of breast cancer samples allowed the detection of both ADAMTS-12 and fibulin-2 in the connective tissue surrounding tumor area in less aggressive carcinomas. However, both proteins are hardly detected in more aggressive tumors. These data and survival analysis plots of breast cancer patients suggest that concomitant detection of ADAMTS-12 and fibulin-2 could be a good prognosis marker in breast cancer diagnosis.Balance between pro-tumor and anti-tumor effects may be affected by molecular interactions within tumor microenvironment. On this basis we searched for molecular partners of ADAMTS-12, a secreted metalloprotease that shows both oncogenic and tumor-suppressive effects. Using its spacer region as a bait in a yeast two-hybrid screen, we identified fibulin-2 as a potential ADAMTS-12-interacting protein. Fibulins are components of basement membranes and elastic matrix fibers in connective tissue. Besides this structural function, fibulins also play crucial roles in different biological events, including tumorigenesis. To examine the functional consequences of the ADAMTS-12/fibulin-2 interaction, we performed different  Extracellular matrix (ECM) components and their interactions are essential for homeostasis of tissues and provide structural support to animal cells -2. TheseFBLN2, the human gene coding for fibulin-2, is found epigenetically silenced in B cell acute lymphoblastic leukemia [Fibulin-2 illustrates an example for the contribution of fibulins to those processes. In fact, fibulin-2 is recognized as a multifunctional binding protein due to its ability to bridge diverse ECM components , 16. Forleukemia ; and losleukemia . These fADAMTS12 gene promoter is hypermethylated in colorectal carcinomas [Adamts12-deficient mouse has confirmed the tumor-protective role of this enzyme [vβ3 integrin [ADAMTS-12 is a secreted metalloprotease  and diffrcinomas . Phenotys enzyme . By contintegrin . ExploriADAMTS12 [Here we show that ADAMTS-12 and fibulin-2 are two interacting proteins. Functional consequences of this interaction were evaluated using different cell-based assays. Two breast cancer cell lines, MCF-7 and MDA-MB-231, were employed attending to the anti-tumor role of fibulin-2  and exprADAMTS12  in this www.ncbi.nlm.nih.gov) matched with extracellular matrix proteins such as NELL2, connective tissue growth factor or agrin. Interestingly, one of these clones, with a 630-bp insert, corresponded to nucleotides 3360 to 3990 of human fibulin-2 cDNA (GenBank™ accession number X82494.1). Conceptual translation of this region showed that the sequence matched with the carboxyl-end region of fibulin-2, including the final 88 amino acids of the fibulin module , ADAMTS-12 (ts) or both proteins simultaneously (fb/ts) was analyzed by Western blot  showed a smaller number and mammospheres size than MCF-7-(fb), MCF-7-(ts) or MCF-7 control cells , MCF-7-(ts) or MCF-7-(fb/ts) cells were subcutaneously injected in the flank of nude mice. Control MCF-7 cells were injected in the opposite flank and the evolution of tumor growth was followed by 6 weeks. MCF-7-(fb/ts) cells displayed a significantly reduced tumor growth rate when comparing to MCF-7-(fb), MCF-7-(ts) or MCF-7 control cells . This analysis also showed that simultaneous high ADAMTS12 and low FBLN2 expressions (Hts+Lfb) associated to better outcome. However, high expression levels of FBLN2 combined with a low expression of ADAMTS12 (Lts+Hfb) showed a slight but not significant increase of survival rate  containing a sequence enconding amino acids 768-881 of the GAL4 protein (AD) were purchased from Clontech. Screening process was performed following manufacturer´s recommendations. Briefly, a cDNA fragment encoding amino acids 702-813 of human ADAMTS-12 (GenBank™ accession number AAI31734) was PCR amplified using the primers 5´-AAACATATGCAGACTGTGAGAAAGAT GTT-3´ and 5´-TATGTCGACTGGATTGTGTACTCATA CTT-3´and inserted between the NdeI and SalI sites of the pGBKT7 vector to make a bait vector encoding a GAL4-BD-ADAMTS-12ADAMTS12 (ts), FBLN2 (fb) or both simultaneously (fb/ts), or transfected with an empty vector were selected in the presence of 1.5 μg/mL puromycin (Sigma-Aldrich).MCF-7 and MDA-MB-231 breast cancer cells, obtained from ATCC, and 293-EBNA cells producing exogenous ADAMTS-12 containing a FLAG epitope  were emp2, 0.5 mM MgCl2, 0.02 % NaN3, 1mM AEBSF and 1 % Triton X-100), containing one complete protease inhibitor cocktail tablet  per 50 mL buffer. Protein concentration was quantified using the BCA Protein Assay Kit from Pierce Technology. To immunoprecipite ADAMTS-12, cell extracts were incubated with anti-FLAG M2 affinity gel (Sigma-Aldrich) following manufacturer´s instructions. To immunoprecipitate fibulin-2, we used H-250 antibody and the Protein G Immunoprecipitation kit (Sigma-Aldrich), following manufacturer´s instructions. MCF-7 cells extract producing fibulin-2 was used as a negative control. In the case of EBNA-293 cells producing ADAMTS-12, cell extracts were incubated with the indicated amounts of fibulin-2 prior to the immunoprecipitation step. EBNA cells transfected with an empty vector (EBNAc) were used as negative control.For western blot analysis, proteins were resolved by 8 or 12 % polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane and subsequently probed with the indicated antibodies. Primary antibodies used for detection of ADAMTS-12 were H-142 (Santa Cruz Biotech) or anti-FLAG-M2 (Sigma-Aldrich), and for detection of fibulin-2 was H-250 (Santa Cruz Biotech). Anti-actin antibody was from Sigma-Aldrich. Immunoreactive proteins were visualized using HRP-peroxidase labeled anti-rabbit or anti-mouse secondary antibody and the ECL detection system (Pierce). Relative expression levels were quantified and normalized to actin levels within the same blot using the Image J software. For immunoprecipitation, MCF-7-(fb/ts) were resuspended in lysis buffer  domain of aggrecan as substrate at 0.016 μg/μl . As indicated, different amounts of fibulin-2 were added to the reaction. Reactions were performed in 20 μL for 4 hours in the buffer previously described . Degrada3 cells were seeded in 96-well plates and six replicates per condition and time point were assessed. Cell proliferation rates were determined on three (MCF-7) or four (MDA-MB-231) consecutive days using an automated microtiter plate reader Power Wave WS (BioTek).Cell proliferation was measured using the CellTiter 96 Non-radiactive Cell Proliferation Assay kit purchased from Promega. 10In vitro invasion potential was evaluated using 24-well Matrigel-coated invasion chambers with a 8 μm pore size (BD Biosciences). For MCF-7 cells, 2.5 x 104 cells were allowed to migrate for 96 h using 10 % foetal bovine serum as a chemoattractant. Cells that reached the lower surface were stained with crystal violet. At least three independent experiments were performed with triplicates for each condition. Cells were counted in eight randomly selected microscopic fields. In the case of MDA-MB-231 cells, invasion was evaluated after 24 h.4 cells were seeded per well and migration was monitored by time-lapse microscopy using a Zeiss Axio Observer Microscopy. Experiments were performed with triplicates and covered area was quantified at different times using Image J. Results were obtained after 24 h migration for MCF-7 cells and after 6 h for MDA-MB-231 cells.Migratory capacity of cells on ECM components fibronectin, laminin I, and type-I collagen was examined using the Radius™ 24-Well Cell Migration Assay kit (Cell Biolabs) following manufacturer's instructions. Briefly, 1,25 x 104 MCF-7 or 5 x 103 MDA-MB-231 cells were plated in 6-well ultralow attachment plates (Costar) and grown in MammoCult Basal Medium (Stem Cell Research) supplemented with 10 % Mammocult Proliferation Supplement, 4 μg/mL heparine and 0.5 μg/mL hydrocortisone. After 7 days, mammospheres were collected and enzymatically dissociated as previously described [4 x 10escribed . Individnu/nu Swiss mice from Charles River Laboratories) were used for in vivo studies. To induce subcutaneous tumors, three groups of five mice were injected at one flank with 7 x 106 cells of MCF-7-(fb), MCF-7-(ts) or MCF-7-(fb/ts) cells in 200 μL PBS. As a control, the opposite flank in each animal was injected with identical number of control MCF-7 cells transfected with an empty vector. Tumor growth was monitored weekly as previously described [Four to five-week-old female athymic nude mice  containing 1 % tween-20. For detection of fibulin-2 (H-250 antibody) and ADAMTS-12 (H-142 antibody), sections were heated in high pH Envision FLEX target retrieval solution at 80 °C for 20 min and then incubated for 20 min at room temperature in the same solution. Endogenous peroxidase activity (3 % H2O2) and non-specific binding  were blocked and the sections were incubated overnight at 4 °C with primary antibodies described above using a 1:100 dilution for both antibodies. As secondary antibodies we used labelled polymer-HRP ready to use from DAKO. 3-3' diaminobenzidine was employed as a chromogen. Selected slides were counterstained with haematoxylin. The intensity of immunostaining was evaluated by two independent observers directly upon microscope and was scored as absent, slight, moderate and strong. Study methodologies were conformed to the standards set by the Declaration of Helsinki and were approved by the Ethics Committee of the Universidad de Oviedo-Principado de Asturias (Spain).A breast cancer tissue array containing 16 tumors samples from different tumor stages (Acris Antibodies) was employed to evaluate www.kmplot.com [ADAMTS12, FBLN2 and both genes simultaneously. Results were represented as Kaplan-Meier long-rank test survival plots.To assess the effect of ADAMTS-12 and fibulin-2 on breast cancer prognosis, survival probability was determined using the data available at plot.com . 1592 pat test. p values under 0.05 were considered statistically significant .Statistical analysis were carried out using the GraphPad Prism 5.0 Software. Data were represented as means +/− S.E. The occurrence of significant differences was determined with the Student-Welch