Chemokine (C-C motif) ligand 3 (CCL3), also known as macrophage inflammatory protein-1α, is a cytokine involved in inflammation and activation of polymorphonuclear leukocytes. CCL3 has been detected in infiltrating cells and tumor cells. Chondrosarcoma is a highly malignant tumor that causes distant metastasis. However, the effect of CCL3 on human chondrosarcoma metastasis is still unknown. Here, we found that CCL3 increased cellular migration and expression of matrix metalloproteinase (MMP)-2 in human chondrosarcoma cells. Pre-treatment of cells with the MMP-2 inhibitor or transfection with MMP-2 specific siRNA abolished CCL3-induced cell migration. CCL3 has been reported to exert its effects through activation of its specific receptor, CC chemokine receptor 5 (CCR5). The CCR5 and AMP-activated protein kinase (AMPK) inhibitor or siRNA also attenuated CCL3-upregulated cell motility and MMP-2 expression. CCL3-induced expression of MMP-2 and migration were also inhibited by specific inhibitors, and inactive mutants of AMPK, p38 mitogen activated protein kinase (p38 or p38-MAPK), and nuclear factor κB (NF-κB) cascades. On the other hand, CCL3 treatment demonstrably activated AMPK, p38, and NF-κB signaling pathways. Furthermore, the expression levels of CCL3, CCR5, and MMP-2 were correlated in human chondrosarcoma specimens. Taken together, our results indicate that CCL3 enhances the migratory ability of human chondrosarcoma cells by increasing MMP-2 expression via the CCR5, AMPK, p38, and NF-κB pathways. Chondrosarcomas are the third most common bone tumors, after myelomas and osteosarcomas. Chondrosarcomas are rapidly progressive, pathologically diverse, and highly malignant, and to date, surgical resection remains the primary treatment for these sarcomas. They also have the potential for distant metastasis . TherefoMetastasis is a multistage process that requires cancer cells to escape from the primary tumor, survive in the circulation, seed at distant sites, and grow. Metastasis increases cell motility, induction of vascular and lymphatic angiogenesis, and migration and invasion to other organs . The invChemokines are low-molecular weight secretory proteins that can regulate the chemotaxis and the metabolic activity of specific leukocyte subsets. Their production, in general, is stimulated by pro-inflammatory cytokines, growth factors, and by pathogenic stimuli arising in inflammatory tissues. In diseased tissues, different tumor cell types trigger different complex chemokine networks that influence the quality and quantity of immune-cell infiltration and, consequently, the proliferation, survival, spread, and angiogenic response of malignant cells . CCL3, aEpidemiological studies have shown that energy availability is associated with an increased risk of several metabolic diseases such as obesity, hypertension, diabetes, and induced cancers ,16. Aber172), p38, p-p38, inhibitor of kappa B (IκB), p-IκBα, IκB kinase (IKKα/β) (Ser180/181), NF-κB p65 subunit (p65), p-p65 (Ser536), and MMP-2; control shRNA (sc-108060) and CCL3 shRNA (sc-44722-SH) plasmids were purchased from Santa Cruz Biotechnology . AMPK inhibitors (Ara A and compound C), p38 inhibitor (SB203580), IκB protease inhibitor (TPCK), NF-κB inhibitor (PDTC), and MMP-2 inhibitor were purchased from Calbiochem . CCL3 and CCR5 monoclonal antibody (mAb) were purchased from Abcam . Met-RANTES was purchased from R&D Systems . The p38 dominant-negative MAPK mutant was provided by Dr. J. Han . The IKKα (KM) (K44A) and IKKβ (KM) (K44A) mutants were gifted by Dr. H. Nakano . All other chemicals were purchased from Sigma-Aldrich .Anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase, rabbit polyclonal antibodies specific for β-actin, AMPK, phospho(p)-AMPK (Thr2.The human chondrosarcoma cell line (JJ012) was kindly provided by the laboratory of Dr. Sean P. Scully  and originated from Dr. Joel Block . JJ012 cells were cultured in a complete medium containing Dulbecco’s modified Eagle’s medium (DMEM)/α-minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS). The human chondrosarcoma cell line (SW1353) was obtained from the American Type Culture Collection. SW1353 cells were cultured in a complete medium containing DMEM supplemented with 10% FBS. All experiments with cells were maintained at 37°C in a humidified atmosphere of 5% COCCL3 shRNA or control shRNA plasmids were transfected into JJ012 cells by using the transfection reagent, Lipofectamine 2000. At 24 h after transfection, stable transfectants were selected with 10 μg/mL puromycin (Life Technologies). The selection medium was replaced every 3 days and 2 weeks after selection, and puromycin-resistant cells and their clones were isolated.4 cells in 100 μL of serum-free medium was placed in the upper chamber, and 300 μL of the same medium containing CCL3 was placed in the lower chamber. The plates were incubated for 24 h at 37°C in 5% CO2, and cells were then fixed in methanol for 15 min and stained with 0.05% crystal violet in PBS for 15 min. Cells on the upper side of the filters were removed with cotton-tipped swabs, and the filters were washed with PBS. Cells on the underside of the filters were examined and counted under a microscope. Each clone was plated in triplicate in each experiment, and each experiment was repeated at least 3 times [The migration assay was performed using the Transwell assay  in 24-well dishes. For the invasion assay, filters were precoated with 30 μL Matrigel Basement Membrane Matrix  for 30 min. The procedure for both migration and invasion assays was as follows. Before the migration assay was performed, cells were pretreated for 30 min with different concentrations of inhibitors, including the CCR5 mAb, compound C, Ara A, SB208530, TPCK, PDTC, MMP-2 inhibitor, or vehicle control (0.1% DMSO). Approximately 1 × 10 3 times .5 cells/well in culture medium. At 24 h after seeding, the confluent monolayer of culture was scratched with a fine pipette tip, and migration was visualized by microscopy. The rate of wound closure was observed at the indicated times [For wound-healing migration assays, cells were seeded on 12-well plates at a density of 1 × 10ed times .2, 1 μM ZnCl2, 0.02% thimerosal, and 1% Triton X-100). Zymographic activity was revealed by staining with 1% Coomassie Blue.Supernatants collected from JJ012 cell cultures were mixed with sample buffer without reducing agents or heating. Samples were loaded onto a 10% SDS-PAGE gel containing 1 mg/ml gelatin and electrophoresed under constant voltage. Subsequently, the gel was washed with 2.5% Triton X-100 to remove SDS, rinsed with 50 mM Tris–HCl, pH 7.5, and then incubated overnight at room temperature with a developing buffer , the threshold was set above the non-template control background and within the linear phase of target gene amplification.Quantitative real-time polymerase chain reaction (qPCR) analysis was carried out using TaqMan® one-step PCR Master Mix . Total complementary DNA (100 ng/25 μL reaction) was mixed with sequence-specific primers and TaqMan® probes according to the manufacturer’s instructions. All target gene primers and probes were purchased commercially, and GAPDH was used as the internal control (Applied Biosystems). qPCR assays were carried out in triplicate with a StepOnePlus sequence detection system. The cycling conditions were 10 min polymerase activation at 95°C followed by 40 cycles at 95°C for 15 s and 60°C for 60 s. To calculate the cycle number at which the desired transcript was detected  according to the manufacturer’s recommendations. At 24 h after transfection, the cells were pretreated with inhibitors for 30 min, and then, CCL3 or vehicle was added for 24 h. Cell extracts were then prepared, and luciferase and β-galactosidase activities were measured [Human chondrosarcoma cells were plated on 24-well culture plates with coverslips. Cells were treated with CCL3 (30 ng/ml) and washed twice with ice-cold phosphate-buffered saline. Immunofluorescence staining using a primary anti-p65 monoclonal antibody was performed as described previously .The human chondrosarcoma tissue array was purchased from Biomax . The tissues were placed on glass slides, rehydrated, and incubated in 3% hydrogen peroxide to block the endogenous peroxidase activity. After trypsinization, sections were blocked by incubation in 3% bovine serum albumin (BSA) in PBS. The slides were incubated at 4°C overnight with a 1:50 diluted primary antibody, which was either monoclonal mouse anti-human CCL3, CCR5, or MMP-2 antibody. After being washed 3 times with PBS, samples were treated with goat anti-mouse IgG biotin-labeled secondary antibodies at a dilution of 1:50. The bound antibodies were detected with an ABC kit . The slides were stained with the chromogen diaminobenzidine, washed, counterstained with Delafield’s hematoxylin, dehydrated, treated with xylene, and mounted. The intensity of staining was evaluated as 0, 1+, 2+, 3+, 4+, and 5+ for no staining, very weak staining, weak staining, moderate staining, strong staining, and very strong respectively. IHC score was determined as the sum of the intensity score.t test. Statistical comparisons of more than two groups were performed using one-way analysis of variance with Bonferroni’s post-hoc test. Analyzing patterns of staining in immunohistochemical studies statistical comparison of two tissue scores was performed using the Regression Analysis Method. In all cases, p < 0.05 was considered significant.Data are presented as mean ± standard error of the mean (SEM). Statistical comparison of two groups was performed using the Student’s Previous study suggest that CCL3 is overexpressed in many cancer types . HoweverIt has been reported that CCL3 promotes tumor metastasis . We therPrevious studies have shown significant expression levels of MMP-1, -2, -3, -9, and -13 in human chondrosarcoma cells ,27. We tA previous study showed that CCL3 affects cell migration through binding to the cell surface CCR5 receptor . Pretrea172 in a time-dependent manner  or were transfected with control siRNA, AMPKα1 siRNA, AMPKα2 siRNA, p38 mutant, IKKα mutant, or IKKβ mutant (B) before exposure to CCL3. NF-κB luciferase activity was measured, and the results were normalized to the β-galactosidase activity and expressed as the mean ± SE for three independent experiments performed in triplicate. *P < 0.05 compared with control. #P < 0.05 compared with CCL3-treated group.AMPKα1 or AMPKα2 siRNA inhibited AMPKα1 or AMPKα2 expression. JJ012 cells were transfected with AMPKα1 or AMPKα2 siRNA for 24 h, the AMPKα1 or AMPKα2 expression was examined by western blotting. Click here for file


N-hydroxyacrylamide-derived HDAC inhibitor, inhibited human colorectal cancer HCT116 cell growth in vitro and in vivo. The results of NCI-60 screening showed that MPT0E028 inhibited proliferation in both solid and hematological tumor cell lines at micromolar concentrations, and was especially potent in HCT116 cells. MPT0E028 had a stronger apoptotic activity and inhibited HDACs activity more potently than SAHA, the first therapeutic HDAC inhibitor proved by FDA. In vivo murine model, the growth of HCT116 tumor xenograft was delayed and inhibited after treatment with MPT0E028 in a dose-dependent manner. Based on in vivo study, MPT0E028 showed stronger anti-cancer efficacy than SAHA. No significant body weight difference or other adverse effects were observed in both MPT0E028-and SAHA-treated groups. Taken together, our results demonstrate that MPT0E028 has several properties and is potential as a promising anti-cancer therapeutic drug.Recently, histone deacetylase (HDAC) inhibitors have emerged as a promising class of drugs for treatment of cancers, especially subcutaneous T-cell lymphoma. In this study, we demonstrated that MPT0E028, a novel  Histone deacetylase inhibitors (HDACi) are a promising new class of anticancer agents. HDACi inhibit tumor progression through their ability to regulate gene expression by promoting acetylation of histone and non-histone proteins N-hydroxyacrylamide group play an important role in HDAC activity N-hydroxy-acrylamide (MPT0E028) has the best inhibitory activity of HDACs. In this study, we examined the antitumor activities of MPT0E028 in several cancer cell lines from the NCI-60 cancer cell panel. We investigated the effects of MPT0E028 on cell cycle progression and apoptosis and explored possible molecular mechanisms that underlie its anticancer activity. In addition, we examined the effect of MPT0E028 on the growth of human colorectal cancer HCT116 cells in vivo, using a tumor xenograft model, which confirmed the antitumor effect of MPT0E028. Our results suggest that MPT0E028 is a promising therapeutic candidate for the treatment of human cancers.A close view of these clinical trials with small molecule HDACi indicated the hydroxamic acid or MPT0E028 and SAHA was synthesized by Dr. Jing-Ping Liou’s Lab. , and the purity is more than 98% . RPMI 162/95% air.The human colorectal cancer cell line HCT116, breast cancer cell line MDAMB231 and human umbilical vein endothelial cells (HUVEC) were purchased from American Type Culture Collection . Ovarian cancer cell line NCI-ADR was obtained from the DTP Human Tumor Cell Line Screen . HCT116, MDAMB231 and NCI-ADR cells were cultured in RPMI 1640 with 10% heat-inactivated fetal bovine serum (v/v) and penicillin (100 units/mL)/streptomycin (100 µg/mL). HUVEC was cultured in M199 with 20% heat-inactivated fetal bovine serum (v/v) and penicillin (100 units/mL)/streptomycin (100 µg/mL). All cells were maintained in a humidified incubator at 37°C in 5% COhttp://www.dtp.nci.nih.gov/branches/btb/ivclsp.html).The NCI-60 cancer cell lines screening was conducted by the NCI’s Developmental Therapeutics Program . After incubation with vehicle (0.1% DMSO), MPT0E028 or SAHA for 48 h, cells were fixed with 10% trichloroacetic acid and then stained with sulforhodamine B at 0.4% (w/v) in 1% acetic acid. Excess Sulforhodamine B was washed away by 1% acetic acid and dye-containing cells were lysed with 10 mmol/L Trizma base. The absorbance was read under wavelength of 515 nm. By measuring time zero (T0), control growth (C), and cell growth in the presence of the drug (xT), the percentage growth was calculated. Percentage growth inhibition was calculated as 100-[(xT–T0)/(C-T0)]×100. Growth inhibition of 50% (GI50) is determined at the drug concentration that results in 50% reduction of total protein increase in control cells during the compound incubation.HCT116, MDAMB231 and NCI-ADR cells were seeded in 96-well plates in medium with 5% fetal bovine serum overnight. Cells were fixed with 10% trichloroacetic representing cell population at the time of drug treatment (T0). Other cells were treated with MPT0E028 or SAHA for 48 h. After 48 h incubation at 37°C, cells were stained with 0.1% crystal violet/20% methanol for 10 minutes and washed for 3 times. Then the dye was eluted by 0.1 M sodium citrate/75% ethanol, and the absorbance is measured at 550 nm.The HUVECs were seeded into 96-well culture plates (5000 cells/well) in quadruplicate. After 24 h incubation, cells were fixed with 0.1% crystal violet/20% methanol, representing cell population at the time of drug treatment ] for 20 min at room temperature. Then, the cells were centrifuged and resuspended with 0.5 mL PI solution containing Triton X-100 , RNase (100 µg/mL), and PI (80 µg/mL). DNA content was analyzed with the FACScan and CellQuest software (Becton Dickinson).After the treatment of vehicle (0.1% DMSO), MPT0E028 or SAHA for the indicated time courses, the cells were harvested by trypsinization, fixed with 75% (v/v) alcohol at 4°C overnight. After centrifugation, cells were incubated in 0.1 mol/L phosphate-citric acid buffer . Cell lysates were centrifuged at 13,000 rpm for 30 min. For Western blot analysis, the amount of protein (40 µg) was separated by electrophoresis in a 10% or 15% polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane. After 1 h incubation at room temperature in PBS/5% nonfat milk, the membrane was washed with PBS/0.1% and incubated with the indicated antibodies at 4°C overnight. After washings with PBS/0.1% Tween 20, the corresponding secondary antibodies were applied to the membranes for 1 h at room temperature. The membranes were then washed with PBS/0.1% Tween 20 and the detection of signal was done with an enhanced chemiluminescence detection kit .7 cells/mL. Each mouse was injected s.c. in the right flank with 1.0×107 cells (0.2 mL cell suspension). Tumors were monitored twice weekly and then daily as their volumes approached 1,200 mm3. Tumor size, in mm3, was calculated from: where w = width and l = length in mm of the tumor. Tumor volume = (2w×l)/2. All animal experiments followed ethical standards, and protocols have been reviewed and approved by Animal Use and Management Committee of National Taiwan University .The human HCT116 colorectal adenocarcinoma cells used for implantation were harvested during log phase growth and resuspended in phosphate-buffered saline at 5×10Female nude-athymic mice were 8 weeks old, and had a body weight (BW) range of 17.2–22.0 g, on D1 of the study. The animals were fed ad libitum water  and PicoLab Rodent Diet 20 Modified and Irradiated Lab Diet® consisting of 20.0% crude protein, 9.9% crude fat, and 4.7% crude fiber. The mice were housed on National Taiwan University Laboratory Animal Center, NTUMC, on a 12-hour light cycle at 21–23°C and 60–85% humidity.P = 0.05. Kaplan-Meier plots show the percentage of animals remaining in the study versus time. The Kaplan-Meier plots use the same data set as the logrank test.The logrank test was used to determine the statistical significance of the difference between the TTE values of two groups, except any NTR deaths. Statistical and graphical analyses were performed with Prism 3.03 (GraphPad) for Windows. The two-tailed statistical analyses were conducted at p<0.05; **p<0.01; ***p<0.001; compared with the respective control group).The tumor growth curves show the group median tumor volume, on a log scale as a function of time. When an animal exits the study due to tumor size or TR death, the final tumor volume recorded for the animal is included with the data used to calculate the median at subsequent time points. Therefore, the final median tumor volume shown by the curve may differ from the MTV, which is the median tumor volume for mice remaining in the study on the last day . If more than one TR death occurs in a group, the tumor growth curves are truncated at the time of the last measurement that precedes the second TR death. Tumor growth curves are also truncated when the tumors in more than 50% of the assessable animals in a group have grown to the endpoint volume. .Data were expressed as mean±SEM of the indicated number for separate experiments. Statistical analysis of data was performed with one-way ANOVA followed by the 1) is depicted in 2) was reacted with sodium cyanoborohydride in the presence of acetic acid afforded the methyl indoline-5-carboxylate (3) in 93% yield. The N1-sulfonylation of indoline (3) with benzenesulfonyl chloride in pyridine and then LiAlH4-mediated C5-ester reduction, followed by the PDC oxidation produced the desired 1-benzenesulfonylindoline-5-carbaldehyde (4) in 42% yield (3 steps). Wittig reaction of methyl(triphenylphosphoranylidene)acetate with the indoline-5-carboxyaldehyde 4T and treatment of lithium hydroxide (LiOH) in MeOH gave cinnamic acid (5) in 73% yield. The conversion of carboxylic acid (5) to the hydroxamic acid (1) was accomplished in 72% yield by the treatment of NH2OTHP in the presence of PYBOP and Et3N, followed by trifluoroacetic acid-mediated deprotection. The detailed experimental information for the preparation of MPT0E028 is available in the supplemental section.The synthesis of MPT0E028  . MPT0E028 also inhibited cell proliferation significantly in these two cell lines (50 = 3.57±0.45 µM), suggesting MPT0E028 specifically targets malignant tumor cells  . Two othll lines  but showor cells . To inveor cells . The refactivity  and induactivity  in a conactivity . MPT0E0250 = 11.1±2.8 nM) and in HCT116 cells (IC50 = 4.43±0.5 µM), which were approximately 9–30 times more potent than SAHA   . The potctively) . These rBecause α-tubulin and histone H3 are common downstream targets of HDACs, we examined the effects of MPT0E028 on α-tubulin and histone H3 acetylation in HCT116 cells using western blot analysis. As shown in in vivo tumor HCT116 xenografts in a nude mice model . Control mice received the vehicle (0.5% carboxymethyl cellulose +0.1% Tween 80). Tumors in all 3 groups were allowed to reach an endpoint volume of 1,200 mm3. The median time to endpoint (TTE) for control mice was 16.6 days. In mice that were orally treated with SAHA , the TTE increased to 27.0 days, which corresponds to a T-C of 10.0 days and a percent tumor growth delay (TGD) of 63%. According to the logrank analysis, SAHA produced significant antitumor activity (P = 0.0251). Oral treatment with MPT0E028  increased the median TTE to 36.9, 19.2, and 15.5 days, respectively, corresponding to a T-C of 20, 2.6, and 0 days and a percent TGD of 122%, 16%, and 0%, respectively. Based on the logrank analysis, MPT0E028 exhibits significant antitumor activity at 200 mg/kg (P = 0.0031). Notably, 3 mice in a group that were treated with 200 mg/kg MPT0E028 and 1 mouse in another group that was treated with 100 mg/kg MPT0E028 showed complete tumor regression after treatment  and then tumors were carefully removed and subjected to western blot analysis. As shown in in vitro. Taken together, MPT0E028 induced a dose-dependent delay and inhibition of tumor growth and displayed stronger anti-tumor activity in vivo than SAHA.Finally, we evaluated the efficiency of MPT0E028 and SAHA using ce model  , that has a potent HDACi activity. MPT0E028 was designed based on our prior research with microtubule destabilizing agents using indoline-1-arylsulfonamides as a core structure N-hydroxyacrylamide functional group at the 5-position of indoline ring, we afforded the desired MPT0E028.HDACs are important targets for cancer therapy because of their ability to transcriptionally regulate the expression of genes that are involved in cell proliferation, differentiation, and apoptosis n vitro and in vivo. We found that MPT0E028 induced cytotoxicity in numerous human cancer cell lines from the NCI-60 panels and performed mechanistic studies in HCT116 cells, which showed high sensitivity to MPT0E028. When compared to SAHA, treatment with MPT0E028 induced stronger inhibition of cancer cell  but not normal cell growth (GI50 = 3.57±0.45 µM) and produced a significantly higher number of sub-G1 (apoptotic) cells as determined by flow cytometry. Also, MPT0E028 induced more profoundly caspase 3 and PARP activation. Taken together, MPT0E028 inhibits cancer cells growth and induces apoptotic cell-death.Furthermore, we evaluated the anti-cancer activity of MPT0E028 iWe demonstrated that MPT0E028 inhibits the activity of HDAC1, HDAC2 and HDAC8 in class I as well as HDAC6 in class IIb, but not HDAC4 in class IIa, and consistently induces acetylation of histone H3 and α-tubulin. Class I HDACs have been shown to be overexpressed in human colorectal cancer cells Finally, we examined the efficiency of MPT0E028 against the human HCT116 colorectal adenocarcinoma cell growth in mice. Median tumor growth and Kaplan-Meier curve analysis demonstrated strong antitumor activity of MPT0E028. Daily administration of MPT0E028 resulted in significant antitumor activity. Moreover, compared to SAHA, MPT0E028 displayed stronger anti-tumor activity. Based on these results, MPT0E028 is a novel synthetic HDACi with unique pharmacologic properties that should be tested in human colorectal cancer therapy.in vitro and in vivo. The results presented here show that MPT0E028 inhibits HDACs activity and has antitumor properties that are more potent than SAHA, which is currently in clinical use in subcutaneous T-cell lymphoma. Thus, MPT0E028 is suitable for further testing as a novel anti-cancer agent.In summary, we have identified a novel inhibitor of HDAC activity, MPT0E028, with antitumor activity Data S1Chemistry. Nuclear magnetic resonance (1H NMR) spectra were obtained with Bruker DRX-500 spectrometer (operating at 500 MHz), with chemical shift in parts per million  downfield from TMS as an internal standard. High-resolution mass spectra (HRMS) were measured with a JEOL (JMS-700) electron impact (EI) mass spectrometer. Flash column chromatography was done using silica gel . All reactions were carried out under an atmosphere of dry nitrogen. 2,3-Dihydro-1H-indole-5-carboxylic acid methyl ester(3): To a stirred solution of methyl indole-5-carboxylate (2)  in AcOH (2 mL), sodium cyanoborohydride  was added to the reaction mixture at 0°C. The reaction was warmed to room temperature and stirred for 2 h. The reaction was quenched with water at 0°C, concentrated NaOH was added up to pH 10. The aqueous layer was extracted with CH2Cl2 (15 mL×3). The combined organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure to give a yellow residue, which was purified by silica gel chromatography (EtOAc: n-hexane = 1∶2) to afford 3 (0.28 g), yield 93%. 1H NMR : δ 3.06 , 3.65 , 3.84 , 6.53–6.55 , 7.75–7.76 . 1-Benzenesulfonyl-2,3-dihydro-1H-indole-5-carbaldehyde(4): To a solution of 3  in pyridine (2 mL), benzenesulfonyl chloride  was added. The reaction mixture was refluxed overnight. The mixture was then purified by silica gel chromatography (EtOAc: n-hexane = 1∶3) to afford the 1-benzenesulfonylindoline (0.40 g). 1H NMR : δ 2.99 , 3.87 , 3.97 , 7.45–7.48 , 7.56–7.59 , 7.66 , 7.75 , 7.82 , 7.90 . LAH  was added to a solution of 1-benzenesulfonylindoline  in THF (10 mL) at 0°C. The reaction mixture was warmed to room temperature and stirred for 2 h before it was quenched with water and then extracted with CH2Cl2 (15 mL×3). The combined organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure. The residue was further treated with PDC -mediated oxidation in the presence of molecular sieves (0.63 g) and CH2Cl2 (10 mL) stirring at room temperature overnight. The reaction mixture was filtered through celite and purified by silica gel chromatography (EtOAc: n-hexane = 1∶2) to afford 4 (0.19 g), 42% yield. 1H NMR : δ 3.05 , 4.01 , 7.46–7,49 , 7.58–7.62 , 7.71 , 7.75 , 7.84 , 9.85 . 3--acrylic acid(5): To a solution of 4  in CH2Cl2 (10 mL) was treated with methyl (triphenylphosphoranylidene) acetate . The reaction mixture was stirred at room temperature for 3 h, was then quenched with water, and extracted by CH2Cl2 (15 mL×3). The combined organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure to give a yellow residue, which was then purified by silica gel chromatography (EtOAc: n-hexane = 1∶3) to afford the acrylic acid methyl ester (0.20 g). 1 M LiOH aqueous solution  was added to a solution of acrylic acid methyl ester  in dioxane (15 mL). The reaction mixture was stirred at 40°C overnight before it was concentrated under reduced pressure. The residue was dissolved in water and concentrated HCl was added up to acidic pH to give the precipitation, which was dried by vacuum to afford 5 (0.16 g), 73% yield. 1H NMR : δ 2.92 , 3.96 , 6.33 , 7.38 , 7.41 , 7.50–7.53 , 7.55 , 7.58–7.64 , 7.82 . 3--N-hydroxy-acrylamide : NH2OTHP  was added to a stirred solution of 5 , PYBOP , triethylamine  in DMF (1.5 mL). The reaction mixture was stirred at room temperature for 1 h before it was quenched with water, followed by extraction with EtOAc (15 mL×3). The combined organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure. The residue was purified by silica gel chromatography (CH2Cl2: CH3OH = 30∶1 : 1%NH3(aq)) to give a white solid, which was treated with TFA  in the presence of CH3OH (25 mL) and stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure to give a white residue, which was recrystallized by EtOAc/CH3OH to afford compound 1 (0.09 g), 72% yield. mp: 162–164°C. 1H NMR : δ 2.91 , 3.96 , 6.32 , 7.32 , 7.37–7.39 , 7.46 , 7.50–7.53 , 7.58–7.64 , 7.82 . MS (EI) m/z: 170 (100%), 344 . HRMS (EI) for C17H16N2O4S (M+): calcd, 344.0831; found, 344.0829.(PDF)Click here for additional data file.Data S2The effects of MPT0E028 on cell growth in human MDAMB231, NCI-ADR and HUVEC cells. Concentration-dependent effect of MPT0E028 and SAHA on cell growth in (A) MDAMB231, (B) NCI-ADR and (C) HUVEC cells. Cells were incubated without or with the indicated concentrations of MPT0E028 or SAHA for 48 h. Cell growth was evaluated by SRB and crystal violet assay. Data were expressed as mean±S.E.M. of at least 3 independent experiments.(PDF)Click here for additional data file.Data S3Inhibition of total HDAC activity by MPT0E028 and SAHA in MDAMB231 and NCI-ADR cells. (A) MDAMB231 and (B) NCI-ADR cells were treated with the indicated concentrations of MPT0E028 and SAHA for 24 h, and the total lysate were subjected to total HDAC enzyme activity detection. Data are expressed as the mean±S.E.M. of at least 3 independent experiments.(PDF)Click here for additional data file.


Glucose turnover  was assessed under fasted, resting conditions using primed, constant infusions of  glucose. Glucose Ra and Rd (mg kg-1 min-1) were higher for MP (2.8 ± 0.1 and 2.7 ± 0.1) compared to HP  and LP  diets. Glucose levels (mmol/L) were not different (P > 0.05) between LP (4.6 ± 0.1), MP (4.8 ± 0.1), and HP (4.7 ± 0.1) diets.Using a randomized, crossover design, volunteers consumed 4 week eucaloric diets providing either a low (0.8 g kg-1 d-1. Findings suggest that consumption of protein in excess of the recommended dietary allowance but within the current acceptable macronutrient distribution range may contribute to the regulation of blood glucose when carbohydrate intake is reduced by serving as a gluconeogenic substrate in endurance-trained men.Level of protein consumption influenced resting glucose turnover in endurance athletes in a state of energy balance with a higher rate of turnover noted for a protein intake of 1.8 g kg Glucose rates of appearance (Ra) and disappearance (Rd) were calculated using a modified version of the Steele equation [Glucose turnover was assessed after 3 wks of each 4 wk diet intervention using a 120 min primed, constant infusion of [6,6-equation ,12.Plasma insulin and glucose concentrations were determined using a commercial RIA  and automated glucose oxidase-peroxidase method , respectively.P < 0.05. Data were analyzed using SPSS  and expressed as means ± SEM.Baseline participant characteristics and macronutrient data were described using common descriptive statistics. Shapiro-Wilk tests of normality confirmed that plasma glucose, insulin, and glucose turnover data were normally distributed. Repeated measures ANOVA  were used to evaluate effects of dietary protein intake on glucose turnover, insulin, and glucose. In cases in which significant main effects (diet or time) or interactions were present, post hoc analyses were conducted by using Bonferroni adjustments to reduce the type I error rate. The alpha level for significance was set at P < 0.05) were noted for glucose turnover. Ra (mg kg-1 min-1) was greater for MP (2.8 ± 0.1) compared to HP  and LP  diets . Rd (mg kg-1 min-1) was also greater for MP (2.7 ± 0.1) than for HP  and LP  diets . No difference was observed between LP and HP for Rd.Diet main effects (Ra mg kg- min-1 waP < 0.05) was observed for plasma insulin, as mean insulin concentrations (pmol/L) were greater (P < 0.01) for LP (49.4 ± 6.4) compared to MP (22.8 ± 2.7) and HP (16.2 ± 0.6) diets. Insulin levels did not change over time (P > 0.05). No main effects of time or diet were observed for plasma glucose (mmol/L), as levels remained steady over time and were not different between the LP (4.6 ± 0.1), MP (4.8 ± 0.1), and HP (4.7 ± 0.1) diets (P > 0.05). No interactive effects (P > 0.05) were observed for plasma glucose and insulin concentrations.A main effect of diet , energy restricted diet stabilized blood glucose and lowered the postprandial insulin response compared to a diet providing protein at 0.8 g kg-1 d-1 [-1 d-1 increased hepatic glucose output (Ra) compared to that observed when protein intake was 0.7 g kg-1 d-1 [Findings from other studies indicate that level of protein intake contributes to glucose homeostasis -3,13. Inkg-1 d-1 . Contrarkg-1 d-1 . Also, t-1 d-1following weight loss [Level of dietary protein can affect glucose utilization by: 1) influencing fasted and postprandial insulin secretion; and 2) providing amino acids which serve as substrates and mediators of hepatic gluconeogenesis ,15. In tght loss . Our finght loss .The contribution of amino acids to hepatic glucose production as gluconeogenic substrates and through the glucose-alanine cycle is well documented -20. In tGreater carbohydrate uptake and subsequent oxidation likely increased metabolic demand for endogenous hepatic glucose production accounting for the differences noted in glucose Ra in the MP diet. Consistent with our hypothesis, Jungas et al. reported an increase in protein oxidation concomitant with a greater contribution of amino acids to hepatic gluconeogenesis with modest increases in dietary protein . Therefo-1 d-1 did not appear to provide any additional benefit towards the regulation of blood glucose. While our findings must be interpreted cautiously due to the specific population studied , small sample size, and state of energy balance  during which the experimental diets were implemented, the concept is nonetheless intriguing. That is, when carbohydrate intake is within 55-70% of the total energy consumed and adequate to support glycogen replenishment (7.4 g carbohydrate kg-1 d-1), dietary protein at a level that exceeds the RDA but is well within the AMDR may contribute to maintenance of blood glucose by serving as gluconeogenic substrate.In summary, this investigation demonstrated that glucose turnover is influenced by level of dietary protein routinely consumed by a group of endurance-trained men. A novel aspect of this work is that chronic consumption of dietary protein above 1.8 g kgNancy R. Rodriguez has received honorarium for participation in the speaker bureau for the NCBA and serves on the Protein Advisory Board for the NCBA. Remaining author(s) declare that they have no competing interests.SMP participated in manuscript preparation, CSS, MAP, PCG, DRB, and BTB participated in data collection, statistical analysis, and manuscript preparation. NRR served as the principal investigator and contributed to study design, data collection, and manuscript preparation. All authors read and approved the final manuscript.


Although AML1 and C/EBP-ϵ mRNA peaked at F1 and F4, respectively, in healthy individuals, C/EBP-ϵ was maximized at F2/F3 in all patients, two of whom showed simultaneous peaks of AML1 at F2. Thus, this fractionation is useful to detect mistimed induction of granulopoiesis-regulating genes in myelodysplastic syndromes.We applied our new method, maturity-dependent fractionation of bone marrow-derived neutrophil progenitors, to a study of gene expression profiles during granulopoiesis in myelodysplastic syndromes. CD34 The six fractions derived from MDS patients well correlated with morphological maturation of neutrophil progenitors (Table Heparinized BM blood was divided into low (<1.065 g/mL), intermediate (between 1.065 g/mL and 1.080 g/mL), and high density (>1.080 g/mL) populations. The overall recovery rate 73.7 ± 12.4%) did not differ from the results of healthy BM [.4% did nThe BM-derived fractions and a peripheral blood-derived neutrophil fraction [F7] were subjected to quantification of mRNA of acute myeloid leukemia 1 (AML1), CCAAT-enhancer-binding protein-epsilon (C/EBP-ϵ), and ecotropic viral integration site 1 (EVI1) as previously described . As showThese aberrant gene inductions were not detectable by analyses of only a hematopoietic stem cell fraction ,5, or deThere are no relevant conflicts of interest to disclose for any of the authors.H H performed this research. Y S designed this study, analyzed the data, and wrote the paper. T S collected patients’ samples and revised the paper critically. K I and K A performed cell isolation by fluorescence-activated cell sorter. T O took part of density centrifugation and immunostaining. H K, K O, and H N chose appropriate patients and collected the specimens. Y T and J K critically discussed this study and revised the paper. All authors read and approved the final manuscript.


Rhizomucor pusilis by Microbial Type Culture Collection (MTCC), Chandigarh, India. An extracellular polygalacturonase (PGase) from R. pusilis was purified to homogeneity by two chromatographic steps using Sephadex G-200 and Sephacryl S-100. The purified enzyme was a monomer with a molecular weight of 32 kDa. The PGase was optimally active at 55°C and at pH 5.0. It was stable up to 50°C for 120 min of incubation and pH condition between 4.0 and 5.0. The stability of PGase decreases rapidly above 60°C and above pH 5.0. The apparent Km and Vmax values were 0.22 mg/mL and 4.34 U/mL, respectively. It was the first time that a polygalacturonase enzyme was purified in this species. It would be worthwhile to exploit this strain for polygalacturonase production. Polygalacturonase from this strain can be recommended for the commercial production because of its constitutive and less catabolically repressive nature, thermostability, wide range of pH, and lower Km properties. However, scale-up studies are needed for the better output for commercial production.A thermophilic fungal strain producing polygalacturonase was isolated after primary screening of 40 different isolates. The fungus was identified as  Becaused xylose , 6.Mucor and Aspergillus [Pectinolytic enzymes are of prime importance for plants as they help in cell-wall extension and softening of some plant tissues during maturation and storage , 8. Theyergillus . Preparaergillus . Due to Higher cost of the production is perhaps the major constraint in commercialization of new sources of enzymes. Though, using high yielding strains, optimal fermentation conditions, and efficient enzyme recovery procedures can reduce the cost. In addition, technical constraint includes supply of cheap and pure raw materials and difficulties in achieving high operational stabilities, particularly to temperature and pH. Literature highlighting the optimization, biochemical characterization, genetics, and strain improvement studies of pectinases from mesophilic fungi is available –18. Howe4·7H2O-0.5, MnSO4·5H2O-0.01, (NH4)2SO4-2.0, Agar-20.00 supplemented with mineral salt solution of composition g/100 mL CuSO4·5H2O-0.04, FeSO4-0.08, Na2MoO4-0.08, ZnSO4-0.8, Na2B4O7-0.004, and MnSO4-0.008. To the above medium distilled water was added to make 1 liter solution. The pH of the media was adjusted to 5.5–6.0 by using 1.0 N HCl/1.0 N NaOH. The salt solution with pectin, and agar were autoclaved separately. The sterilization was done at 121°C (10 lbs) for 15 minutes to avoid pectin degradation. After sterilization the pectin media and agar were mixed aseptically. Ampicillin (100 mg/mL) was also added to restrict bacterial growth. The inoculated plates were incubated at 50°C for 5–7 days. The cultures were further screened by subculturing on YPSS (Yeast soluble starch agar) medium having the following composition : Pectin-15, Yeast extract-0.4, K2HPO4-0.23, KH2PO4-0.2, MgSO4·7H2O-0.05, and Citric acid-0.052. The pH of the media was adjusted to 5.5 by using 1.0 N HCl/1.0 N NaOH. Pectin utilization was detected by flooding the culture plates with freshly prepared 1% Cetrimide solution and allowed to stand for 20–30 minutes. A clear zone around the colonies against a white background (of the medium) indicates the ability of an isolate to produce pectinase. Based on screening, the isolated fungi were identified up to genus level by examining the morphological characters following Dubey and Maheshwari [Pectinase producing fungi were isolated from various soil, decayed fruits, and other vegetables collected from different fruits markets of western Uttar Pradesh by using modified pectin agar medium of following composition  : Pectin-4)2SO4-0.68, KH2PO4-0.33, FeSO4-0.15, and Sugarcane bagasse-11.6). The flasks were inoculated with 2 mL spore suspension containing 106 spores mL−1, which was obtained from a five-day agar slant. Final moisture was adjusted to 70%. The pH of the media was adjusted to 5.5–6.0 by using 1.0 N HCl/1.0 N NaOH [The solid state cultivation was carried out in 250 mL Erlenmeyer flasks containing 15 g of basal medium . The crude enzyme was loaded on a Sephadex G-200 column (1 × 50 cm) preequilibrated with sodium acetate buffer . Fractions in 3 mL volume were collected at a flow rate of 24 mL/hour. The eluted fractions were monitored at 280 nm for protein and assayed for enzyme activity. Fraction with highest polygalacturonase activity was loaded on Sephacryl S-100 column (1.6 cm × 60 cm) preequilibrated with same at a flow rate of 20 mL/hour. Fractions of 1.5 mL were collected and monitored for proteins and polygalacturonase activity.2, and 0.5 mL of 1% solution of polygalacturonic acid (PGA). Blank was prepared for each sample by boiling the reaction mixture before the addition of substrate. Tubes were incubated at 37°C for one hour. The reaction was stopped by heating at 100°C for 3 minutes. 0.5 mL of the solution mixture was taken and analyzed for reducing sugars by Nelson-Somogyi method. Final volume was made up to 2 mL in both sample and standard tubes with distilled water. 1.0 mL of alkaline copper tartrate was added and kept for 10 minutes. Tubes were cooled and 1.0 mL of arsenomolybdate reagent was added to each of the tubes. Final volume was made up to 10 mL volume in each tube with distilled water. The absorbance of blue color was recorded at 620 nm after 10 minutes. The PGase activity was assayed by estimating the amount of reducing sugar released under assay conditions. Polygalacturonase activity was measured by determining the amount of reducing groups released according to the method described by Nelson  and modiμmol mL−1 min−1 galacturonic acid under standard assay conditions.The amount of galacturonic acid released per mL per minute was calculated from standard curve of galacturonic acid. One unit of enzyme activity is defined as the enzyme that releases 1 The protein content of the enzyme solution is estimated by the method developed by Lowry et al.  with BovThe purified enzyme was subjected to electrophoretic studies to determine molecular weight. SDS PAGE (12.5%) was conducted by using a slab gel apparatus with notched glass plate system . Gels ofThe substrate specificity of the purified enzyme was determined by using various substrates in the reaction mixture for enzyme assay. The various substrates used were polygalacturonic acid, pectin, xylan, galactose, and cellulose at 0.1% (w/v) .Km and Vmax were evaluated.The optimum temperature was estimated by performing the standard assay within the temperature range of 30–80°C. Incubating the enzyme for 4 hours at temperature from 30–80°C in assay buffer and then measuring the remaining activity by standard assay determined the inactivation temperature. The optimal pH for PGase activity was evaluated by varying the pH of the reaction mixture between 3.0 and 9.0 at increments of 1.0. Activity was then assessed under standard conditions. The effect of substrate concentration on PGase activity was assayed in standard assay by using PGA in various concentrations (0.1–1.0 mg) and Mucor, Aspergillus, Penicillium, Rhizopus, and Trichoderma were isolated. Similar results were also reported by different coworkers: Mucor [Aspergillus [Penicillium [Rhizopus [Tricoderma [Thermophilic fungal strains isolated from various precollected samples from different fruits markets of western Uttar Pradesh were purified and their cultural and morphological characteristics were examined according to the method described by Dubey and Maheshwari . A totals: Mucor , Aspergiergillus , Penicilicillium , RhizopuRhizopus , and Triicoderma .Rhizomucor pusillus by Microbial Type Culture Collection (MTCC), Chandigarh, India.High pectinase producing strains were further screened semiquantitatively by plate assay method. Twelve different isolates were further screened by solid state fermentation . The resR. pusillus was induced by pectin (pure) (1.5% w/v) as carbon source, urea (0.3% w/v) as nitrogen source, and MnSO4 (0.2% w/v) as mineral supplement at 45°C. The maximum reported polygalacturonase activity of Mucor genevensis is 5.0 U/mL [Mucor sp. 7 is 15.2 U/mL[Penicillium viridicatum is 18 U/mL [Mucor circinelloides is 9.15 U/mL [Rhizomucor pusillus is available in the literature. However it is evident from this work that this strain is a hyperproductive one and is suitable for further studies.The production of PGase by 5.0 U/mL , Mucor s15.2 U/mL, Penicil 18 U/mL , and MucThe crude enzyme was extracted from fermented media by a process of filtration and centrifugation to remove mycelia and other media components; the crude was concentrated to 150 mL. The crude extract contains evaluated protein and specific activity of 6.38 mg and 4.97 U/mg, respectively.The purification of the PGase activity was carried out by two chromatographic steps.A. kawachii IFO 4033. Mohamed et al. [Tricoderma harzianum. Celestino et al. [Acrophialophora nainiana. Saad et al. [Mucor rouxii NRRL 1894. Thakur et al. [Mucor circinelloides. These factors mainly depend not only on the strain but also the methods adopted for purification. Generally an increase in fold purification results in a gradual decrease in yield.First, 6.38 mg of protein from CE (150 mL) with a total activity of 31.74 U were loaded on a Sephadex G-200 column. The eluted fractions with PGase activity were identified and pooled, concentrated to 1 mL, and applied on a Sephacryl S-100 column eluted with sodium acetate buffer . PGase activity was detected in fractions eluted between 54 and 75 mL. These last were pooled and concentrated to 1 mL. d et al.  reportedo et al.  reportedd et al.  reportedGeotrichum candidum-34.5 kDa [Rhizopus sp. LKN-38.5 kDa [Verticillium albo-atrum-37 kDa [Fusarium solani-38 kDa [Fusarium moniliforme-30.6 kDa [Trichoderma harzianum-31 kDa [Acrophialophora nainiana-35.5 kDa [The purified PGase was homogenous as judged by electrophoresis gel , where o34.5 kDa , Rhizopum-37 kDa , Fusariui-38 kDa , Fusariu30.6 kDa , Trichodm-31 kDa , and AcrThe maximum PGases specificity was observed when polygalacturonic acid was used as substrate. Assuming it as 100%, almost half (57%) activity was expressed with pectin and only 37.8% activity was expressed with cellulose . PreviouKm and Vmax values of PGases of R. pusillus are 0.22 mg/mL and 4.34 U/mL, respectively, by plotting the Lineweaver Burk plot (Km of 0.071 nmol/mL and Vmax of 432 nkat/mg of endo-PG for Fusarium moniliforme. Saad et al. [Km of 1.88 mg/mL and Vmax of 0.045 mole/mL/min for Mucor rouxii. Thakur et al. [Km of 2.2 mM and Vmax of 4.81 IU/mL for Mucor circinelloides. From these studies it is evident that the kinetic properties of PG vary with the source of the enzyme. But in most cases the Km value is low, which agrees with the result of the present study.The urk plot . Bonnin d et al.  reportedr et al.  reportedR. pusillus was 55°C (Thermoascus aurantiacus at 60°C; the enzyme was stable at 60°C for 1 hour. Kaur et al. [Sporotrichum thermophile apinis at 55°C and the enzyme was stable up to 4th hour at 65°C. Thakur et al. [Mucor circinelloides at 42°C and the enzyme was stable up to 4th hour at 42°C. Andrade et al. [The optimum temperature of the purified polygalacturonase from was 55°C . The enzwas 55°C . This agr et al.  reportedr et al.  reportede et al.  reportedR. pusillus was 5.0 (Aspergillussojae-5.0 [Mucor rouxii-4.5 [Mucor circinelloides-5.5 [Cylindrocarpon destructans-5.0 [The pH optima of the purified polygalacturonase from  was 5.0 . The enz was 5.0 . Previouojae-5.0 , Mucor ruxii-4.5 , Mucor cides-5.5 , and Cyltans-5.0 . This istans-5.0 . In the


Background. Although the benchmark in the treatment of rheumatoid arthritis remains methotrexate, only 70% of patients respond. Thus, there is a need for predictive biomarkers. This study planned to evaluate serum levels of myeloid progenitor inhibitory factor-1 (MPIF-1) and monocyte chemoattractant protein 2 (MCP-2)—as biomarkers. Methods. Patients with rheumatoid arthritis (RA) having high disease activity (DAS28-3v ≥ 5.1) were treated with oral methotrexate (MTX) for 12 weeks. Disease activity was measured by DAS28-3v (Modified Disease Activity Score 3 variables). Serum samples were stored at baseline and 12 weeks. Results. This study included 46 patients (F : M = 35 : 11) having mean (±SD) age of 42.6 ± 11.3 yrs, disease duration of 4.7 ± 4.5 yrs, and DAS28-3v of 6.1 ± 0.8. Serum MPIF1 was elevated in patients compared to controls , but there was no difference in MCP2 levels . Baseline MPIF-1 level was lower in good responders  compared to poor responders  . On ROC analysis, baseline MPIF1 performed reasonably to predict good response; that is, ΔDAS28-3v ≥ 1.2 . Conclusions. Lower baseline MPIF1 level predicted a good response to methotrexate at 12 weeks. Rheumatoid arthritis is an autoimmune chronic inflammatory disease predominantly affecting the joints . There iChemokines are small proteins involved in chemoattraction of a variety of cells to site of inflammation, being important in the pathogenesis of RA and may serve as biomarkers . MCP-2 . In this study only those patients who started with 15 mg methotrexate were included after consent.Inclusion criteria were (a) fulfillment of the 1987 American College of Rheumatology (ACR) criteria for rheumatoid arthritis , (b) 18 Patients were started on 15 mg per week dose of methotrexate, which was escalated by 2.5 mg biweekly (maximum 25 mg) till 12 weeks. In addition, folate supplementation (5 mg twice a week = 10 mg/week) was given. Patients were monitored 4 weekly with complete blood counts and liver function tests, and methotrexate was withheld/not increased in case of cytopenias or transaminitis.√ (tender joints) + 0.28*√ (swollen joints) + 0.70* Ln (ESR)] *1.08 + 0.16, where joint count was done on 28 joints and ESR was measured using Westegren method (first hour). Change in disease activity (∆DAS28-3) was calculated as the difference in DAS28-3 at baseline (0 week) “minus” final (12 weeks). Patients were stratified into 3 categories of response based on change in disease activity (∆DAS28-3). The three categories were good, moderate, and poor responsers, defined as ∆DAS28-3 ≥ 1.2, 0.6–1.2, and <0.6, respectively. In addition, the functional status of the patients was measured using Indian health assessment questionnaire at baseline and 12 weeks [The modified Disease Activity Score using 3 variables (DAS28-3v) was calculated to measure the disease activity . This wa12 weeks .Blood sample was obtained at baseline and 12 weeks; serum was separated and stored at −80°C. In addition, serum of controls was also stored. ELISA for MPIF-1 and MCP-2 (RayBio) was done on the stored samples after completion of original study. Minimum detectable limits of MPIF-1 and MCP-2 were 7 pg/mL and 1.5 pg/mL, respectively.t test was used to compare the chemokine levels between patients and controls. Pearson's correlation was used to look at the correlation between chemokine levels and disease activity. In addition, multivariable linear regression and receiver operator curve (ROC) analysis were done. Analysis was done using SPSS v15 and GraphPad Prism (version 5).Student's This study included 46 patients (F : M = 35 : 11) with rheumatoid arthritis. Their mean age was 42.6 ± 11.3 yrs and duration of disease was 4.7 ± 4.5 years. Baseline disease activity (DAS28-3v) was 6.1 ± 0.8, HAQ score was 1.3 ± 0.7, and 30 (65%) were rheumatoid factor positive. At 12 weeks, the mean dose of methotrexate reached was 24.3 ± 2.0 mg/week and mean (±SD) change in DAS28-3v and HAQ was 0.5 ± 0.6 and 0.3 ± 0.5, respectively.P < 0.001). However, there was no difference in the MCP-2 level  (P = 0.04) (r2 = 0.28). On ROC analysis, baseline MPIF-1 had area under curve of 0.68 (95% CI 0.50–0.87) and a level of <946 pg/mL was found to have the best predictive value with a sensitivity and specificity of 55.6 and 81% to predict good response to MTX.Baseline level of MPIF-1 was elevated in patients compared to controls  . On stra P = ns) . Also, b = 0.04) . On multP = ns). However, MCP-2 level increased at 12 weeks . There was a lack of correlation between change in DAS28 (∆DAS28-3v) and change in MPIF-1 levels (∆MPIF-1)  or MCP-2 levels (∆MCP-2) .Although baseline MPIF-1 was elevated and predicted response, it did not significantly change after 12 weeks  levels to be raised 4-fold in serum of rheumatoid arthritis (RA) patients compared to controls. This is similar to a previous study that found a 1.3-fold higher level in plasma of patients . Higher Lower MPIF-1 levels at baseline were associated with better response to methotrexate at 12 weeks. Indeed, MPIF-1 was modestly accurate on ROC analysis (AUC = 0.68) in identifying patients with good response (∆DAS28 ≥ 1.2). Although it had modest predictive ability, as a comparison, a recent prediction model to predict methotrexate response in JIA, using SNPs in 4 genes and ESR, could only have an AUC of 0.65 . This isThis study did not find any difference in MCP-2 levels between patients and controls, similar to a previous study . HoweverThe strength of our study is the longitudinal design. Important drawbacks are the limited number of patients and the short followup of only 12 weeks. This short duration was chosen, as 12 weeks is fast emerging as a decision point as per both guidelines and major trials, when other drugs  are added in the face of inadequate response to MTX –23. We pTo conclude, this study found that MPIF-1 may be a useful biomarker for predicting response to methotrexate in rheumatoid arthritis, with lower levels baseline predicting higher change in DAS28-3v (better response).


Erythrina verna, popularly known as “mulungu”, is a Brazilian medicinal plant used to treat anxiety. Erythrina alkaloids have been described in several species of Erythrina, which have biological and therapeutic properties well known that include anxiolytic and sedative effects.in vitro metabolism of erythraline (1), the major spirocyclic alkaloid of Erythrina verna, was studied in the pig cecum model and by biomimetic phase I reactions. The biomimetic reactions were performed with Jacobsen catalyst to produce oxidative metabolites and one metabolite was isolated and evaluated against cancer cells, as HL-60 (promyelocytic leukemia), SF-295 (Glioblastoma) and OVCAR-8 (ovarian carcinoma).In this work, Erythraline exhibited no metabolization by the pig microbiota and a main putative metabolite was formed in a biomimetic model using Jacobsen catalyst. This metabolite was isolated and identified as 8-oxo-erythraline (2). Finally, erythraline and the putative metabolite were tested in MTT model and both compounds showed no important cytotoxic activity against tumor cells.The alkaloid erythraline was not metabolized by intestinal microbiota, but it was possible to identify its oxidative metabolite from biomimetic reactions. So these data are interesting and stimulate other studies involving this alkaloid, since it is present in phytomedicine products and there are not reported data about the metabolism of erythrina alkaloids. Erythrina (Fabaceae) comprises 115 species distributed in tropical and subtropical Forest . E. . E. Eryt anxiety . Systema anxiety –11] and andEryth anxiety . Take to anxiety . In addi anxiety , which sin vitro biomimetic models were developed for phase I metabolism investigations. Our group recently applied Jacobsen catalyst ]) to produce similar metabolites of CYP450 enzymes yielding the product in good scale . Th. Th17]. od scale ,22. Compl trials ,23,24. IE. verna were acquired commercially of a registered supplier of plant matter. 5.4 kg of powered stem bark was extracted by percolation with ethanol and after solvent elimination afforded the ethanolic extract (488.72 g).2Cl2:MeOH (8:2) and extracted with 5% HCl (pH 2); the resulting acid fraction was adjusted to pH 10 with NH4OH and exhaustively extracted with CH2Cl2. This extract was concentrated under reduced pressure to yield the alkaloid extract (4.51 g). This extract (4.34 g) was fractionated by column chromatography on silica gel and eluted with hexane:ethyl acetate gradient system  to yield 155 fractions. The fractions 9–25 (875.0 mg) were purified by HPLC to obtain the alkaloid erythraline (367.5 mg), which was submitted to Mn  oxidation procedure.The ethanol extract (469.76 g) was solubilized in CHAll the reactions were performed in a 10 mL glass vessel equipped with a magnetic stirring bar at room temperature. The reactions were performed in dichloromethane using Mn , erythraline (1) and oxidant (PhIO) in the proportion 1:30:30 (μmol). After 24 hours (reaction time), the samples were analyzed by GC-MS. Control reactions were carried out in the absence of catalyst, under the same conditions as the catalytic runs. No products were detected.2O (A) both with TFA 0.02% (v/v) as solvents. The elution profile was 0–25 min – 15-65% B, 25–30 min – 25-80% B, 30–32 min – 80-100% B and 32–35 min – 100% B. The compound 8-oxo-erythraline (2) was isolated from this procedure and characterized by MS and NMR.The Mn  oxidation reaction was submitted to semipreparative LC using the following conditions: C18 column , flow rate 9 mL/min and acetonitrile (B) and H8-oxo-erythraline (2): brown amorphous solid;1H NMR  δH 6.79 , 6.65 , 6.64 , 6.26 , 6.01 , 5.86 , 5.83 , 3.81 , 3.69 , 3.58 , 3.27 , 3.07 , 2.91 , 2.73 , 1.62 ; 13C NMR  δC 171.6 (C-8), 157.7 (C-6), 147.2 (C-15), 146.1 (C-16), 136.8 (C-2), 129.5 (C-13), 127.7 (C-12), 123.8 (C-1), 119.7 (C-7), 109.5 (C-17), 105.0 (C-14), 101.2 (O-CH2-O), 74.8 (C-3), 67.3 (C-5), 56.5 (OCH3), 41.1 (C-10),38.0 (C-4), 27.3 (C-11). ESI MS (pos. ion mode) m/z 312.1232 [M + H]+ .Two animals  used to carry out the pig cecum model experiments were bred without antibiotics on the diet, also called biodynamic conditions. To ensure that the matrix (feces) were not exposed to the air and kept under anaerobic conditions after the slaughtering of the pig, the ceca were firstly ligated in both orifices and stored in an anaerobic jar with Anaerocult A® (Merck) to be transported to the laboratory. The ceca were obtained from fresh slaughtered pigs, which are bred in a biodynamic farm  and afterwards delivered to the market (as food). Thus, it was not mandatory to be approved by ethical committees, since the ceca are a waste from the farm production.2 atmosphere inside a hermetic chamber. All the vessels and solutions used were previously flushed with a mixture of the gases N2 and CO2 . The cecum of both animals were opened and the inside content was split in 20 g (±1.0 g) portions in BD Falcon™ tubes (n = 6). Half of these tubes (n = 3) were sterilized  to be used as a negative control (DA). The active inoculum (not autoclaved) was denominated as CA for each animal separated. To each of these tubes (n = 6) were added 20 mL of 0.15 M PBS (pH 6.4) containing a trace element solution 0.0125% v/v  and a Na2S-solution 11.1% (575.9 mg/100 mL of 0.037 M NaOH solution). The suspensions obtained were afterwards filtered through a net lace in order to avoid large particles at the inoculum suspension. All the procedure were done in duplicate for each single animal.All the steps of the inoculum preparation were done under COIn a 2 mL Eppendorf® tube, 100 μL of 1.0 mM erythraline solution in MeOH was mixed with 900 μL of the inoculum suspension. The samples were prepared in duplicate for each time point  and incubated at 37°C. The same procedure was applied to DA samples (negative control) and using 1.0 mM quercetin solution as positive control incubated with active inoculum. On this study two animals  were used in order to consider the possible differences between the animals microbiota.g for 10 minutes. The supernatant was collected and used for chromatographic analysis. For this analysis were used an High Performance Liquid Chromatograph (HPLC) equipped with Diode Array Detector (DAD) Jasco XLC™, a reversed phase column Nucleodur C18 ISIS, 5 μm, 150 × 2 mm (Macherey-Nagel) and water (A):methanol (B) (both 0.1% formic acid) at the flow 0.4 mL min-1. The elution gradient applied was the following: 0 – 2 min (10% B), 2 – 12 min (10 → 100% B), 12 – 14 min (100% B), 14 – 17 min (100 → 10% B) always keeping an equilibration time between each injection of three minutes. The wavelength used for the quantification was 254 nm and the calibration curve showed r = 0.9957.Reached the respective incubation time the samples were immediately stored at -80°C in order to stop the possible reactions. Prior to the sample preparation the inoculum was thawed in a water bath at 37°C and 1.0 mL of hydrochloric acid in methanol (1% v/v) was added. The samples were sonicated for 15 minutes and afterwards centrifuged at 20°C and 12000 × H-tetrazolium bromide (MTT) assay + as base peak in the mass spectrum obtained by HRESI in positive ionization mode and its molecular formula was determined to be C18H17NO4. The 1H NMR spectrum showed signals at δH 5.83 and 5.86  that characterized the methylenedioxy group  and five olefinic hydrogens, which belongs to the aromatic ring [δH 6.65 (H-14) and 6.64 (H-17)] and conjugated diene protons . The carbonyl presence at C-8 was confirmed mainly by downfield shift of H-7 (+0.23 ppm) compared with the observed for erythraline and the characteristic chemical shift of the carbonyl . Eleven carbon resonances were observed in DEPT 135° spectra, including six methynes, four methylenes and a methoxyl group. Therefore, the absence of one methylene carbon suggested the oxidation in one of them, as well as the absence of two aliphatic protons  in the 1H NMR when compared with the spectrum of erythraline. Thus, the structure of 2 was determined as 8-oxo-erythraline , respectively, and 3.9 and 18.5 μg/mL for 8-oxo-erythraline (2). In the present study, the highest evaluated concentration was 5 μg/mL using a colon adenocarcinoma cell HCT-116, a metastatic melanoma cell MALME-3 M and a metastatic prostate cell PC-3 M, and no significant change on cell viability was observed for both compounds regardless of the cell type used. These data reinforce that those alkaloids could not be considered highly toxic to cancer cell lines, as previously observed for other alkaloids [The final step was to define if erythraline and the putative metabolite showed cytotoxic activity in MTT model. For both compounds 1 and 2, a weak cytotoxicity was observed in the hepatocyte carcinoma Hep-G2 and cervix carcinoma HEP-2 cell lines with IClkaloids ,27.The results indicated that the alkaloid erythraline is not metabolized by the pig cecum microbiota and one putative metabolite was formed in a biomimetic model using Jacobsen catalyst, which was isolated and identified as 8-oxo-erythraline. Erythraline and its metabolite showed no cytotoxic activity against tumor cells. Our findings are extremely relevant, since erythrina alkaloids are present from phytomedicines used in the therapeutic and there is no information on their metabolism and they could be used for pre-clinical trials.The authors declare that they have no competing interest.TG, DBS, ACB conducted extraction and isolation of the metabolites, biomimetic reactions and the interpretation of all these data. LRS conducted the study in the pig cecum model and H-UH supervised this study design and interpretation data. LVCL conducted the evaluation of cytotoxicity activity against tumor cell. TG, DBS, NPL and JLCL wrote and revised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/14/61/prepubIn vitro metabolism studies of erythraline, the major spiroalkaloid from Erythrina verna.Click here for file


Polypedates teraiensis to establish reference ranges for its hematological and serum biochemical parameters. The peripheral blood cells were differentiated as erythrocytes, lymphocytes, eosinophils, neutrophils, monocytes, basophils, and thrombocytes, with similar morphology to other anurans. Morphology of blood cells did not vary according to sex. The hematological investigations included morphology and morphometry of erythrocytes, morphometry of leucocytes, packed cell volume (PCV), hemoglobin content (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), erythrocyte or red blood cell (RBC) count, leukocyte or white blood cell (WBC) count, differential leukocyte count, and neutrophil to lymphocyte (N/L) ratio. Besides, protein, cholesterol, glucose, urea, uric acid, and creatinine content of blood serum were assayed. Hematological parameters that differed significantly between sexes were RBC count, length and breadth of RBC, neutrophil %, N/L ratio, area occupied by basophils, and diameter of large lymphocyte and eosinophils. The level of glucose, urea, and creatinine in blood serum also significantly differed between sexes.Blood was analyzed from eighty  adult individuals of  Polypedates maculatus, have been studied by Mahapatra et al. [P. maculatus is P. teraiensis which is arboreal and mostly found in bushes, plantations, and gardens and rarely enters human habitation [P. teraiensis was described by us earlier [Hematology is the most straightforward and less invasive technique to access the health status of natural population of vertebrates . These pa et al. . Anotherbitation . Blood c earlier  and herePolypedates teraiensis . Different types of blood cells present in the smear were identified following Turner , and HeaChemicals used for the serum biochemical studies were of analytical grade. Bovine serum albumin (BSA) was obtained from SIGMA Chemicals Co., USA. Folin-phenol reagent was obtained from Sisco Research Laboratory, Mumbai, India. Eco-pak glucose kit, autozyme urea enzymatic kit, infinite liquid uric acid kit, and autozyme creatinine kit were procured from Accurex Biomedical Private Limited, Mumbai, India. All other chemicals were of the highest purified grade available. For the estimation of total protein and total cholesterol, regular laboratory methods, that is, those of Lowry et al.  and RoseP > 0.05; averages were compared with student's t-test. The significance level was P ≤ 0.05. Statistical analyses were performed by SPSS (version 10.00).Statistical analysis was done following Kolmogorov-Smirnov test, In the present study, SVL (snout to vent length) in males ranged from 6.0 to 7.0 cm with a mean of 6.42 ± 0.43 cm and in females it ranged from 6.0 to 9.2 cm with a mean of 7.47 ± 1.36 cm. In case of males the average body weight was 16.87 ± 0.62 g (ranged from 16.0 to 17.5 g) and in females average body weight was 19.37 ± 1.93 g (ranged from 17.5 to 21.5 g). Hematological and biochemical parameters investigated in the present study are represented in Tables Polypedates maculatus [The characteristics shape of amphibian erythrocytes is elliptical form . Apart faculatus . Besidesaculatus . DividinThe lymphocytes observed were of two distinct forms, that is, large lymphocytes  and smalμm and 8.95 ± 0.12 μm in males and 19.77 ± 1.53 μm and 8.62 ± 0.34 μm in females, respectively. The mean length of erythrocytes in females was more than the males and the difference was statistically significant . Arserim and Mermer [μm, 14.59 μm) than males  in Rana macrocnemis. The mean length and breadth of erythrocytes in both males and females were found to be less when compared to other Rhacophorid anurans, that is, Buergeria buergeri , Rhacophorus annamensis , and Rhacophorus schlegelii  as reported by Kuramoto [t = 2.65, df = 6, P = 0.03). The surface area occupied by the erythrocytes was more in females than in males (μm2) in comparison to females (210.58 ± 38.279 μm2) has been reported in adult frogs of Polypedates maculatus [μm and 5.0 ± 0.08 μm in males and 7.5 ± 1.73 μm and 5.5 ± 0.90 μm in females, respectively. The surface area of nuclei ranged from 23.01 to 25.85 μm2 with a mean of 23.98 ± 1.27 μm2 in case of males. In females it ranged from 20.7 to 49.64 μm2 with a mean of 33.0 ± 12.80 μm2. However, there was no significant difference between sexes for the parameters studied on nuclei of RBCs. Length and breadth of erythrocytes remained 16.87 ± 1.37 d Mermer  have repin males , but thiThe erythrocytes were larger in size in tadpoles of this species  in compaKnowledge on erythrocytes in vertebrates provides us with much valuable information. Snyder and Sheafor  have dest = 2.26, df = 18, P = 0.036). The diameter of eosinophils was more in females (12.0 ± 0.31) than in males (11.5 ± 0.45) and the difference was significant . There was no difference in mean size of neutrophils in both sexes. The area occupied by the large lymphocytes, small lymphocytes, basophils, and monocytes was more in males than in females. The area occupied by eosinophils was more in females than in males. There was no difference in mean area of neutrophils in both sexes , while for other leucocytes the difference remained insignificant. The diameter of neutrophils (16.98 μm), basophils (13.69 μm), eosinophils (16.30 μm), and monocytes (14.30 μm) was more in Rana macracnemis [μm, lymphocytes = 13.6 μm, eosinophils = 16.2 μm, monocytes = 15.2 μm, and basophils = 16.9 μm) have been reported in R. catesbeiana by Coppo et al. [Amongst the leucocytes, mean diameter of large lymphocytes, small lymphocytes, monocytes, and basophils remained more in males than in females . The sizth sexes . The difonocytes .30 μm wa3 of blood but in females it was 0.62 ± 0.017 millions/mm3 of blood. The number of RBCs remained more in females than in males. This difference was statistically significant . The gram percentage of hemoglobin per 100 mL of blood varied from 5.8 to 6.1 with a mean of 5.95 ± 0.12 among males. In females it ranged from 5.5 to 6.2/100 mL with a mean of 5.82 ± 0.29/100 mL. The mean packed cell volume (PCV) was more in females (51.55 ± 0.95) than in males (50.625 ± 1.10)  . The meanificant . In Poly3 in males and 12.13 × 10³±0.69/mm3 in females, the difference was not significant .More WBCs were observed in females than in males . As meannificant . Howevernificant . The dift = 3.328, df = 6, P = 0.058). According to Davis [The neutrophil to lymphocyte (N/L) ratio in females (0.48 ± 0.02) was more than in males (0.42 ± 0.03)  and the to Davis , the aveto Davis . For ecoto Davis .t = 3.70, df = 6, P = 0.010), urea , and creatinine . No significant differences were observed for the level of protein, cholesterol, and uric acid in the blood within the sexes.The levels of blood serum protein, cholesterol, glucose, urea, and uric acid were found to be higher in females than in males. However, the serum creatinine value was more in males than in females . The difPolypedates teraiensis, the values can serve as general reference values for future investigations involving this species and other anuran species. This study would be helpful to understand the relevance of the data described earlier for the tadpoles of this species.As the study represents the first hematological serum biochemical investigation of the Rhacophorid frog,


Due to relative stability of the initial oligomers of stefin B, electrospray ionization mass spectrometry (ESI MS) could be applied in addition to size exclusion chromatography (SEC). These two techniques enabled us to separate and detect distinguished oligomers from the monomers: dimers, trimers, tetramers, up to decamers. The amyloid fibril formation process was followed at different pH and temperatures, including such conditions where the process was slow enough to detect the initial oligomeric species at the very beginning of the lag phase and those at the end of the lag phase. Taking into account the results of the lower-order oligomers transformations early in the process, we were able to propose an improved model for the stefin B fibril formation.Oligomers are commonly observed intermediates at the initial stages of amyloid fibril formation. They are toxic to neurons and cause decrease in neural transmission and long-term potentiation. We describe an  Stefin B, together with stefin A and some cathepsins have been identified in the core of amyloid plaques of various origins z+), which depends on the conformational state of the protein. Conformational changes are evident from the shifts in charge-state distribution of protein ions in ESI MS spectra, and rely on the well-confirmed assumption that in the electrospray ionization process, proteins with compact conformations produce lower charge-state ions than proteins with more open conformations 2/[T]) for the tetramer formation is 325 ± 180 μM. Transitions between dimers and tetramers are fitted to square function according to a second order reaction.Two equilibrium constants were calculated from these data . At pH 7et al. [Škerget et al.  proposedThe dissociation of part of a dimer to a monomer at pH 7.5 and 50 °C  indicateBased on the NMR protection pattern , it was cis-trans proline isomerization (stefin B has five proline residues), which is also responsible for the conformation of the loop between strands four and five [Lowering the pH to 4.7 and adding 10% TFE affects the structure of both stefin B monomers and dimers, which was shown by NMR . Shown cand five . Those detc.) [Stefin B cytotoxicity was extensively studied earlier –33. Monoetc.) . Previouetc.) . Wild-tyetc.) . It alsoetc.) . There aetc.) ,33. The In conclusion, in previous studies based on the NMR determined model of the structure for the stefin B mature fibrils  and toxiw/v) polyethyleneimine. Stefin B was further purified using affinity chromatography  and size exclusion chromatography . Proteins were then stored at −20 °C in 0.01 M phosphate buffer, pH 6.1, 0.06 M NaCl. The recombinant proteins have Ser at position 3 instead of Cys to prevent covalent disulfide bond formation, which anyway does not occur in vivo.Recombinant human stefin B (S3E31) was used for all studies. Protein inserted in pET11a vector was expressed in BL21 pLysS (DE3) cells. Expression was induced with IPTG . After induction, cells grew for 3 h. Cells were lysed and resuspended in lysis buffer . Sample was homogenized with ultraturax; DNA and some other proteins were precipitated with 4% (On the Superdex™ 75 column (10 × 300 mm) , connected to FPLC  stefin B elutes as a set of well-defined oligomers, allowing isolation of monomers, dimers, tetramers and higher-order oligomers. SEC was performed at room temperature. Flow was 0.5 mL/min. 500 μL of protein (~3 mg/mL) were applied to the column. 0.01 M phosphate buffer, pH 7.0, 0.15 M NaCl was standard buffer, only when preparing samples for ESI MS 0.02 M ammonium acetate, pH 7.5 was used. We isolated different oligomers by loading them to Superdex™ 75 column and concentrate them with Amicons ultra 3 kDa  before usage.Samples for ESI MS were prepared using Superdex™ 200 GL column (10 × 300 mm)  connected to GE AKTA Purifier system . The flow rate was 0.75 mL/min. Sample volume was 100 μL and 0.2 mL fractions were collected.m/z using following instrument parameters: ion spray voltage 5500 V; source gas 45 L/min; curtain gas 20 L/min; declustering potential 60 V; focusing potential 320 V; detector voltage 2350 V. The obtained spectra were deconvoluted by program Bioanalyst 2.0 . The resolution of QStar ELITE  for a 6000 Da protein is 11,000.Selected fractions from SEC  were injected directly into the ESI-Q-TOF mass spectrometer  by a syringe pump at the flow rate of 8 μL/min (sample volume was 50 μL). ESI MS spectra were recorded in the positive mode in region 500–7000 v/v) TFE, 25 °C, 300 rpm) and pH 7.5  TFE, 50 °C, 300 rpm). At certain time points, 50 μL aliquots were taken, 570 μL of ThT with absorbance 0.66 at 416 nm were added and fluorescence was measured .Thirty four micromolar protein was incubated at pH 4.8  TFE, 25 °C, 300 rpm) and pH 7.5  TFE, 50 °C, 300 rpm)) were taken at the plateau of the process (~95 h for both samples). Protein concentration was 34 μM. 20 μL of solution, diluted 10–50 times as appropriate, were applied to a formvar and carbon coated grid. After 5 min the samples were soaked away and the grid was stained with 1% uranyl acetate.Two different samples .v/v) TFE, 25 °C, 300 rpm) and pH 7.5  TFE, 50 °C, 300 rpm) were taken at the plateau of the reaction (~38 h (pH 7.5), ~200 h (pH 4.8)). Protein concentration was 34 μM. 15 μL of each sample were applied onto common mica which was cleaved earlier with sellotape to have an ultraflat surface. After 10 min, samples were washed away with 1 mL of water and mica was dried with nitrogen flow.Two different samples at pH 4.8  and tapping mode  in ambient air.Images were recorded with Nanoscope IIIa , scanner E in contact mode  , connected to FPLC  was used with the constant flow 0.5 mL/min at room temperature. Column was equilibrated with 0.01 M phosphate buffer, pH 7.0, 0.15 M NaCl. Fifty microliters of sample were applied on the column. Afterwards, the peaks were integrated and ratio between different oligomers was calculated. As control we used isolated oligomers which were >95% pure.v/v) TFE, 25 °C) and 7.5  TFE, 50 °C, 300 rpm). Every hour an aliquot of 50 μL was analyzed by SEC.Oligomers were incubated in two different buffers at pH 5.8  TFE; 0.1 M phosphate buffer, pH 5.8, 0.15 M NaCl, 9% (v/v) TFE and 0.1 M sodium acetate, pH 4.8, 0.15 M NaCl, 9% (v/v) TFE. Protein concentration was 34 μM. Samples were incubated 2 h at 23 °C. Results obtained at 40 °C and with no TFE added were very similar and are not shown.For pH dependent oligomer transformations, three different buffers were used: 0.1 M phosphate buffer, pH 7.0, 0.15 M NaCl, 9% (v/v) TFE, 50 °C, 300 rpm and 0.1 M phosphate buffer, pH 5.8, 0.15 M NaCl, 9% (v/v) TFE, 25 °C, 300 rpm. Samples were incubated at defined conditions for five hours. Every hour an aliquot was taken and analyzed by SEC. Protein concentrations were 4, 9, 34, 75 and 100 μM at pH 5.8 and 34, 75 and 100 μM at pH 7.5. Also, the control samples (before changing conditions) were analyzed by SEC. Reactions were started from the oligomer mixture (wild type protein after purification). Differences in the certain oligomers content in the control are due to sample concentration .For concentration dependent oligomer transformations, two different buffers were used: 0.02 M ammonium acetate, pH 7.5, 0.15 M NaCl, 9% (v/v) TFE, 23 °C) and pH 7.5 . Protein concentration was 30 μM. At different time points, aliquots were taken, filtered and analyzed by ESI MS. Average charge state (qav) was computed as follows:SEC-purified oligomers were incubated at pH 5.8 .Different conditions like pH, temperature, and ionic strength in the case of stefin B all lead to the same result at the end of the process—In all cases fibrils do not differ significantly.The new results  towards the end of the lag phase) are consistent with previous studies ,70.Taking into account the already published (described) and the present results, we were able to improve the model for stefin B amyloid fibril formation  further We believe that stefin B is a good model protein as it has certain advantages over other amyloidogenic proteins . Thus, this research provides valuable information for a deeper understanding of the mechanism of amyloid fibril formation and some generalization to other proteins can be made.


Leishmania mexicana cysteine protease CPB2.8 suggested that it differs significantly from bovine cathepsin B and thus could be a good drug target. High throughput screening of a compound library against this enzyme and bovine cathepsin B in a counter assay identified four novel inhibitors, containing the warhead-types semicarbazone, thiosemicarbazone and triazine nitrile, that can be used as leads for antiparasite drug design. Covalent docking experiments confirmed the SARs of these lead compounds in an effort to understand the structural elements required for specific inhibition of CPB2.8. This study has provided starting points for the design of selective and highly potent inhibitors of L. mexicana cysteine protease CPB that may also have useful efficacy against other important cysteine proteases.Cysteine proteases of the papain superfamily are present in nearly all eukaryotes. They play pivotal roles in the biology of parasites and inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas’ disease and leishmaniasis. Homology modeling of the mature  T. cruzi (cruzain) . In. InCP229 assumed  [38]. BeL. mexicana (CPB2.8∆CTE). In a separate assay, the compounds were evaluated for their ability to inhibit cathepsin B from bovine spleen. BtCatB was chosen over the higly similar HsCatB because of easy accessibility. Two unexpected results emerge from this study. First, molecules with novel cysteine protease warheads were not identified in this study. This is startling, given the effort devoted to identify inhibitors bearing covalent reversible warheads in screening libraries amino]-4-guanidinobutane) were from Sigma-Aldrich Inc., St. Louis, MO, USA, unless stated otherwise. The Z-F-R-AMC substrate was from Bachem. All chemicals and the irreversible cysteine protease inhibitor E-64 (1--1,3,5-triazine-2-carbonitrile (purity of >95%). CP229988 was ordered from Chemical Diversity Labs, Inc., San Diego, CA; the IUPAC name is 3-[(1H-benzimidazol-2-yl)-2H-1-benzopyran-2-ylidene]-2-carbaldehyde semicarbazone (purity of >95%). 2O to lower the DMSO concentration in the assay. Final concentrations of the test compounds from 30 µM to 0.3 nM were used. For the L. mexicana CPB2.8∆CTE assay, 5 µl test compound or 5 µl DMSO in acetate buffer (10% DMSO) was added to each well of the microtiter plate. Subsequently, 25 µl of a premix containing sodium acetate buffer , titriplex II (f.c. 1 mM) , DTT (f.c. 10 mM) , triton (f.c. 0.01%) , BSA (f.c. 0.1 mg/ml)  and enzyme was added to each well, except the wells for the blanks, which include the premix without enzyme. After an incubation time of 10 min, the reaction was started by addition of substrate . The reaction was stopped after 35 min incubation at room temperature by addition of 10 µl inhibitor E64 (f.c. 16.7 µM) and enzyme activity was measured with a SpectraFluorPlus  plate reader using excitation at 360 nm with emission at 465 nm. The assay procedure for BtCatB was the same as for the CPB2.8∆CTE assay with the exceptions of the final concentration of the substrate Z-F-R-AMC (100 µM) and the stop of the assay using a stop buffer containing sodium acetate and sodium chlorine acetate (f.c. 41.7 mM each). The activities of CPB2.8∆CTE and BtCatB were measured in a homogeneous fluorescence endpoint assay using Z-F-R-AMC as substrate, modified from published methods ,54. The The percentage enzyme activity was calculated using the formula (V-B)/(C-B) x 100%, with V being the absorbance of the assay containing the test compound, B being the absorbance of the negative control (‘blank’) and C being the absorbance of the positive control. In primary screening, all compounds of the screening library were tested at 10 µM in double point measurements. Z´ factors, which describe the quality of an assay, were in the range of 0.85-0.99. The Z´ factor is defined in terms of four parameters: the means and standard deviations of both the positive (p) and negative (n) controls: Z´= 1-((3 x (STDEVp + STDEVn))/(MEANp - MEANn)) .50 determination from liquid compound stock) using CPB2.8∆CTE and BtCatB to get the activity profile of the compounds. Hit confirmation (IC50 determination from freshly dissolved solid compound stock) was performed for all compounds with an IC50 ≤ 30 µM on CPB2.8∆CTE. Compound concentrations used for IC50 determinations were as follows: 30 µM, 10 µM, 3 µM, 1 µM, 0.3 µM, 0.1 µM, 0.03 µM, 0.01 µM, 0.003 µM, 0.001 µM, and 0.0003 µM. Enzyme activities were expressed as percentages of residual activity compared with an uninhibited control and were plotted versus increasing inhibitor concentrations. IC50 values were calculated using the four-parameter equation model 205 and the option “unlock” from the XLfit add-in  in Excel . All values are mean values from at least three independent assays to ensure statistically significant results. The theoretical hit limit for a HTS primary screen can be calculated as MEAN + 3 x STDEV of inhibitions from diverse compounds. If a normal distribution for inhibition is expected, 99.73% of inactive compounds are within this limit. In our case, based on a preliminary screen of >3000 diverse compounds, the hit limit was determined between 12.8% and 22.9% inhibition. For practical reasons, screening hit limits are normally set above this limit, aiming at approximately 0.2-0.4% hits from a diverse library. Therefore, 50% inhibition at 10 µM compound concentration compared to the positive controls was considered a hit compound and were subjected to hit verification  with [S] = 20 μM and Km = 21.3 μM for CPB2.8∆CTE[PB2.8∆CTE. It is aA 2-fold serial dilution of the compounds was performed (0.5 to 500 µM) in DMSO  and added in duplicate to 96-well microtiter plates. Phosphate-buffered saline  was added to give a total volume of 200 µl and a final DMSO concentration of 5% (vol/vol) in all wells. The plates were incubated at room temperature for 22 min, and the relative solubilities of the compounds were determined by measuring forward-scattered light using a NEPHELOstar laser-based microplate nephelometer . Wells containing only buffer and 5% (vol/vol) DMSO were used as controls. Data analysis was carried out using Excel . 2O to CH3CN supplemented with 0.1% formic acid, and eluent B was CH3CN), and a column temperature of 313 K. MS detection was performed using an MSD 1100 electrospray ionization (ESI) and atmospheric pressure ionization (API)  single quadrupole mass spectrometer. Nuclear magnetic resonance (NMR) spectroscopy was carried out on a Bruker Avance DRX 400 MHz NMR spectrometer . Compound purity and molecular mass were confirmed by liquid chromatography-mass spectrometry (LCMS) experiments, which were performed on an Agilent LC7 MSD (Mass Selective Detector) 1100 LC-MS . The liquid chromatography conditions were as follows: a Zorbax SB (stable bound) C18 column, 1.8-µm particle size, column dimensions of 4.6 by 30 mm, 0.1 µl/min flow rate, gradient of 10 to 100% eluent B over 3 min (eluent A was 95:5 HL. mexicana CPB2.8∆CTE homology model and the sulfur atom of the C29 residue in the BtCatB X-ray structure (PDB ID 1QDQ) were defined as the link atoms for the covalent bond. The three-dimensional (3D) illustrations of the covalent docking results depicted in Covalent docking was performed as previously described . Version


Rickettsia gravesii is a new Rickettsia species closely related to the human pathogen Rickettsia massiliae. Here, we describe the genome sequence of R. gravesii strain BWI-1, isolated from Amblyomma triguttatum triguttatum ticks collected from humans on Barrow Island, Western Australia. Rickettsia gravesii is a novel species isolated from Amblyomma triguttatum triguttatum ticks removed from humans on Barrow Island, Western Australia, after there was anecdotal evidence of a disease possibly of rickettsial origin in the region  was grown in XTC and L929 cells, and its genomic DNA was extracted using a phenol-chloroform protocol. Sequencing was performed using the MiSeq platform  with a 2 × 250-bp paired-end run after library preparation with the Nextera XT sample preparation kit (Illumina). De novo genome assembly was done using the CLC Genomics Workbench 4.9 . The resulting contigs were reordered in Mauve 2.3.1 (R. massiliae strain MTU5 (GenBank accession no. CP000683)  inflation parameter of 1.5.P000683) , tRNAscaP000683) , and RNAP000683) , respectOrthoMCL , with a R. gravesii BWI-1T is made up of 28 chromosomal contigs exhibiting an average length and coverage of 47,415 bp and 185×, respectively, arranged in a single scaffold, for a chromosome size of 1,327,625 bp . We also detected a 19,874-bp plasmid (pRgr) with a G+C content of 31.8% and 91% sequence identity  to Rickettsia monacensis strain IrR/Munich plasmid pRM (accession no. EF564599). The chromosome contains 1,675 protein-encoding genes and, like other Rickettsia species, 3 noncontiguous rRNAs , 33 tRNAs, and 3 other RNAs. In addition, the pRgr plasmid contains 24 protein-encoding genes, including a split sca12 gene and a proline-betaine transporter gene, but no RNAs.The draft genome sequence of R. massiliae MTU5, the R. gravesii chromosome exhibits a high level of synteny with the exception of four inversions of 37,978 bp, 4,791 bp, 2,782 bp, and 1,339 bp. Moreover, several genes are lacking in the R. gravesii genome, including paaJ (acetyl-coenzyme A [CoA] acetyltransferase), def3 (polypeptide deformylase), and genes for several transposases and inactivated derivatives.Compared to that of AWXL00000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no.