Momordica charantia was determined with different subtypes of influenza A. The protein was purified from the seed of M. charantia using an anion exchanger and a Fast Protein Liquid Chromatography (FPLC) system. At the concentration of 1.401 mg/mL, the protein did not exhibit cytotoxicity in Madin-Darby canine kidney cells (MDCK) but inhibited 1 × 105 FFU influenza A/PR/8/34 H1N1 virus at 56.50%, 65.72%, and 100% inhibition by the protein treated before the virus (pretreated), the protein treated alongside with the virus (simultaneously treated), and the protein treated after the virus (posttreated) during incubation, respectively. Using 5, 25, and 100 TCID50 of influenza A/New Caledonia/20/99 H1N1, A/Fujian/411/01 H3N2 and A/Thailand/1(KAN-1)/2004 H5N1, the IC50 was calculated to be 100, 150, and 200; 75, 175, and 300; and 40, 75, and 200 μg/mL, respectively. Our present finding indicated that the plant protein inhibited not only H1N1 and H3N2 but also H5N1 subtype. As a result of the broad spectrum of its antiviral activity, this edible plant can be developed as an effective therapeutic agent against various and even new emerging subtypes of influenza A.The new antiviral activity of the protein extracted from  Influenza A viruses have continued to be a significant public health concern with epidemics responsible for serious morbidity and mortality. Epidemics of influenza occur almost every year according to an antigenic drift of the two viral surface glycoproteins, hemagglutinin (HA), and neuraminidase (NA). Currently, sixteen hemagglutinin subtypes H1–H16) and nine neuraminidase subtypes (N1–N9) have been recognized –H16 and , 3. PandMomordica charantia L. that has been reported to contain many potent antiviral activities that might be a good candidate. For instance, the proteins from this plant strongly inhibit several viruses including hepatitis B virus, dengue virus, herpes simplex virus, and Human Immunodeficiency Virus [M. charantia, an edible vegetable belonging to the Cucurbitaceae family, is commonly cultivated in Africa and Asia and is known as a bitter melon or bitter ground. It has been traditionally used as folk medicines for several ailments such as antidiabetic, antitumor, anthelmintic, antimalarial, and antiviral agents [M. charantia with various influenza A subtypes including influenza A/New Caledonia/20/99 H1N1, A/Fujian/411/01 H3N2, and A/Thailand/1 (KAN-1)/2004 H5N1. Our discovery has clearly indicated that the protein of M. charantia has an effective, broad antiviral activity against different subtypes of influenza A.Presently, the two inhibitors of the M2 protein, amantadine, and rimantadine and the two neuraminidase inhibitors, zanamivir and oseltamivir are the available anti-influenza A agents . Howevercy Virus –21. M. cl agents . For thiM. charantia were purchased from a local market in Bangkok during January-February 2006. Briefly, the seeds were cleaned and dried by removing red tissue covers. Then, they were added with 10 mM sodium phosphate buffer pH 6.3 (PBS) containing 0.15 M NaCl with a ratio of 2 mL buffer for a gram of seeds and blended with a homogenizer for 5 minutes to form fine emulsion at 4°C. The emulsion was stirred at 4°C for 1 hour, filtrated through double cheesecloth, and centrifuged at 12,000 ×g for 10 minutes at 4°C. The supernatant was called the crude protein fraction.The ripe fruits of μm) and applied to the FPLC at 4–10°C in a cool cabinet. The column was washed with PBS containing 1 N NaCl, and the protein was eluted by varying the pH and ionic strength. The proteins migrated under an electric field with the pH gradient corresponding to their pI. The UV detector was set at 280 nm and the fraction size of the proteins was collected for each 1 mL. The desirable peak of the protein fraction recorded by the UV detector was collected, and the protein sample was divided into small 0.5 mL tubes and kept at 4°C for further investigation.The crude protein was purified using a Hitrap Q anion exchanger equipped with a Fast Protein Liquid Chromatography (FPLC) system . Briefly, the crude protein fraction was filtrated with a syringe filter  using a spectrophotometer . SDS-PAGE was performed according to the method described by Laemmli using the Bio-Rad Mini Protein apparatus . Normally, 15% separating and 5% stacking gel with 100 constant voltage were used.MDCK cells was obtained from Virology Department, Microbial Research Institution, Osaka University (Riken cell bank), and maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum, 1% sodium pyruvate, 1% MEM nonessential amino acids, 1% penicillin-streptomycin solution, and 1% glutamine (Invitrogen). The influenza virus A/PR/8/34 H1N1 (PR 8) was also from Virology Department, Research Institution Microbial Disease, Osaka University. Influenza A/New Caledonia/20/99 H1N1, A/Fujian/411/01 H3N2, and A/Thailand/1(KAN-1)/2004 H5N1 subtypes were provided from Virology Department, Siriraj Medicine Faculty, Mahidol University.μL (1 × 105 cells) of MDCK cells were added in each well of a 96-well plate. After incubation at 37°C, 5% CO2 for 24 hours, twofold serial dilutions of the protein were added and incubated at 37°C, 5% CO2 for 24 hours. Ten microliters of WST-1/ECS solution were added in each well and then incubated at 37°C, 5% CO2 for 1 hour. The number of viable cells was measured using a colorimetric assay system that measured the reduction of tetrazolium salt to formazan at an absorbance of 450 nm.The cell proliferation assay kit  was used for fast and sensitivity quantification of cell proliferation and viability. The assay was based on the cleavage of tetrazolium salt WST-1 to formazan by cellular mitochondrial hydrogenases. One hundred μL/well (1 × 105 cells) of MDCK cells and incubated at 37°C for 24 hr. The cells were then added with 0.175 to 1.401 mg/mL of the protein sample and incubated at 37°C for 1 hour. The plate was washed with PBS buffer twice. Then, 1 × 105 focus-forming units/mL (FFU/mL) of the influenza virus A/PR/8/34 was inoculated in the wells, and the plates were further incubated at 37°C for 1 hr. After incubation, the cells were fixed with 500 μL of 4% paraformaldehyde. The infected cells were identified with the nucleoprotein antibody (150 μL/well) of influenza A virus (anti-NP : PBS = 1 : 1000) and incubated at room temperature for 30 min. Finally, 150 μL of fluorescein isothiocyanate conjugated goat anti-mouse (anti-FITC : PBS = 1 : 500) were added in each well. Influenza A-infected cells were observed under a fluorescence microscope.The infection of influenza virus A/PR/8/34 in MDCH cells was determined by an immunofluorescence assay. A 24-well plate was added with 1000 μL of the first antibody (Mouse IgG to influenza NP : PBS = 1 : 1000) was added in each well, and the plates were incubated at 37°C for 30 minutes. After incubation, the plates were washed with PBS buffer twice. Fifty μL of the second antibody (Rabbit IgG to Mouse IgG : PBS = 1 : 1000) was added in each well, and the plates were incubated at 37°C for 30 minutes. The plates were washed twice with PBS buffer. Finally, 50 μL of the third antibody (Goat IgG to rabbit IgG : PBS = 1 : 500) was added in each well, and the plates were incubated at 37°C for 30 minutes and washed twice with PBS buffer. After washing, 50 μL of PAP solution (diluted with PBS to 1 : 1000) was added in each well, and the plates were incubated at 37°C for 30 minutes. Simultaneously, DAB substrate solution was prepared by adding approximately 2 mg of DAB and 2 μL of H2O2 in 10 mL of PBS. After washing, 50 μL DAB substrate solution was added in each well. The plates were incubated at room temperature for 20 minutes until the solution was changed to an umber color. After the plates were washed with tap water and dried at room temperature, the number of infected cells (foci) was counted under a light microscope.After inoculation with influenza virus A/PR/8/34, MDCK cells were incubated at 37°C for 24 hours. The infected cells were fixed with methanol for 10 minutes and dried at room temperature. Fifty 5 MDCK cells were plated in each well of a 96-well plate. For the pretreated assay, the cells were added with twofold serial dilutions of different protein  concentrations ranging from 0.17 to 1.40 mg/mL and further incubated at 37°C 5% CO2 for 1 hour. After washing with PBS twice, the cells were inoculated with 1 × 105 FFU/mL (1 MOI) of influenza virus A/PR/8/34 and incubated at 37°C 5% CO2 for 24 hours. After incubation, the number of infected cells was determined by PAP staining assay as mentioned above.In each experiment, 1 × 10For the simultaneously treated assay, the protein  was added immediately after the cells were inoculated with the virus. For the posttreated assay, the protein  was added after the cells were preinoculated with the virus for one hour. The following steps were the same as described for the pretreated assay.50 values of the protein on different subtypes of influenza A were determined using 100, 25, and 5 tissue culture infectious dose50 (TCID50) of influenza A/New Caledonia/20/99 H1N1, A/Fujian/411/01 H3N2, and A/Thailand/1(KAN-1)/2004 H5N1. Briefly, various protein concentrations ranging from 12.5 to 400 μg/mL were added to each well containing 1 × 105 cells of MDCK cells. After incubation at 37°C, 5% CO2 for 1 hour, the cells were individually inoculated with 1 × 105 FFU/mL of different subtypes of influenza A and further incubation overnight. The infected cells were individually identified with nucleoprotein antibody against different influenza A viruses using an immunofluorescence assay as mentioned previously. The IC50 was determined by graphically plotting the inhibition of viral growth as a function of the protein concentrations.The ICt-tests or one-way ANOVA were used for statistical analysis. In all cases, P values <0.05 were considered statistically significant.Student's After being purified with a Hitrap Q anion exchanger and a Fast Protein Liquid Chromatography (FPLC) system, the majority of the protein band appeared on the SDS-PAGE was of 30 kDa . This paTo evaluate the cytotoxicity effect of extracted proteins, MDCK cells were grown in different dilutions of the protein for 24 hours. The protein concentration ranging from 0.002 to 1.40 mg/mL did not exhibit any cytotoxicity in MDCK cells .5 MDCK cells /2004 H5N1, the IC50 values were calculated to be 100, 150, and 200; 75, 175, and 300; and 40, 75, and 200 μg/mL, respectively . This discovery confirmed the high antiflu activity of the protein of M. charantia.activity . For the50, the protein still exhibited effective anti-influenza activity. Taken together with the former finding of its anti-HIV property, we believe that the protein of M. charantia possess the broad antiviral activity against various types of viruses in addition to HIV and influenza. According to previous publications, the antiviral and anticancer activities of the proteins belong to the action of the protein called ribosomal inactivating proteins (RIPs). So far, several RIPs have been identified from the plants in the Cucurbitaceae family such as MAP30 from M. charantia and GAP31 from Gelonium multiflorum [Momordica cochinchinensis, contained a potent antitumor activity [M. charantia is associated with the action of RIP or not, further investigation is needed.To further investigate the antiviral activity of the plant protein, the MDCK cells were prior infected with Caledonia/20/99 H1N1, A/Fujian/411/01 H3N2, and A/Thailand/1 (KAN-1)/2004 H5N1 and later treated individually with the postprotein. As expected, the protein strongly inhibited not only H1N1 but also H3N2 and H5N1 subtypes. Its inhibitory effect was almost equally potent against the three viral subtypes. At the high dose of 100 TCIDtiflorum . Additioactivity . NeverthM. charantia possessed effective antiviral activity to a broad range of influenza A subtypes including H1N1, H3N2, and H5N1. Thus, this plant protein holds a great promise to be developed as an effective therapeutic agent against various and even new emerging subtypes of influenza A such as H7N9, which is now pandemic in China.We have found that the protein purified from


The aim of this study was non-invasively to study effect of APO866 treatment on [18F]FLT and 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) uptake.APO866 is a new anti-tumor compound inhibiting nicotinamide phosphoribosyltransferase (NAMPT). APO866 has an anti-tumor effect in several pre-clinical tumor models and is currently in several clinical phase II studies. 3′-deoxy-3′-[18F]fluorothymidine ([18F]FLT) is a tracer used to assess cell proliferation In vivo uptake of [18F]FLT and [18F]FDG in human ovary cancer xenografts in mice (A2780) was studied at various time points after APO866 treatment. Baseline [18F]FLT or [18F]FDG scans were made before treatment and repeated after 24 hours, 48 hours and 7 days. Tumor volume was followed with computed tomography (CT). Tracer uptake was quantified using small animal PET/CT. One hour after iv injection of tracer, static PET scans were performed. Imaging results were compared with Ki67 immunohistochemistry.Tumors treated with APO866 had volumes that were 114% (24 h), 128% (48 h) and 130% (Day 7) relative to baseline volumes at Day 0. In the control group tumor volumes were 118% (24 h), 145% (48 h) and 339% (Day 7) relative to baseline volumes Day 0. Tumor volume between the treatment and control group was significantly different at Day 7 (P = 0.001). Compared to baseline, [18F]FLT SUVmax was significantly different at 24 h (P<0.001), 48 h (P<0.001) and Day 7 (P<0.001) in the APO866 group. Compared to baseline, [18F]FDG SUVmax was significantly different at Day 7 (P = 0.005) in the APO866 group.APO866 treatment caused a significant decrease in [18F]FLT uptake 24 and 48 hours after treatment initiation. The early reductions in tumor cell proliferation preceded decrease in tumor volume. The results show the possibility to use [18F]FLT and [18F]FDG to image treatment effect early following treatment with APO866 in future clinical studies. The use of positron emission tomography (PET) to discriminate between responders and non-responders early following treatment initiation is desirable. The PET technique allows for non-invasive characterization of biological functionality in tumor tissue. 3′-deoxy-3′-[18F]fluorothymidine ([18F]FLT) is used to assess cell proliferation in vitro and several pre-clinical human xenograft tumor models APO866 is a new anti-tumor compound which inhibits nicotinamide phosphoribosyltransferase (NAMPT), an enzyme involved in the biosynthesis of NAD. APO866 inhibits biosynthesis of cellular NAD from niacinamide by inhibiting NAMPT. Reduction in cellular NAD levels leads to ATP depletion and apoptosis APO866 treatment decreases cellular ATP content 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) has in several pre-clinical and clinical studies been tested for its ability to assess early effect of chemotherapeutics; however, with variable results. When new anti-cancer drugs are brought into clinical phase II and III studies, effective methods to discriminate between responders and non-responders are of great value. The aim of this study was therefore non-invasively to study the effect of APO866 treatment on [18F]FLT and [18F]FDG uptake and to evaluate if [18F]FLT or [18F]FDG PET could be used to monitor early effects of APO866 treatment.7 cells in 100 µL medium mixed with 100 µL Matrixgel™ Basement Membrane Matrix  were injected subcutaneously into the left and right flank respectively during anesthesia with 1∶1 v/v mixture of Hypnorm®  and Dormicum® . The cell line has been tested free of mycoplasma. Cells were cultured in RPMI  medium 1640+ GlutaMAX  supplemented with 10% fetal calf serum  and 1% penicillin-streptomycin (Invitrogen) in 5% CO2 at 37°C.Animal care and all experimental procedures were performed under the approval of the Danish Animal Welfare Council (2006/561–1124). Female NMRI  nude mice (6–8 weeks old) were acquired from Taconic Europe  and allowed to acclimatize for one week in the animal facility before any intervention was initiated. The human ovarian carcinoma cell line A2780 3. Mice in the treatment groups received APO866 (2).½ receiving either APO866 or vehicle treatment and [18F]FLT or [18F]FDG scans. Treatment was started at day 14–16 after implantation of tumor cells, where tumor volumes were on average 265 mmd APO866  in salin[18F]FLT was synthesized using 3-N-Boc-1-[5-O--3-O-nosyl-2-deoxy-b-D-lyxofuranosyl]thymine as precursor on a GE TracerLab MX Synthesizer as described earlier 2 in N2 and fixed on a bed in presence of three fiducial markers allowing fusion of PET and CT pictures. A 10 min PET scan was acquired using a MicroPET Focus 120 . After data acquisition, PET data were arranged into sinograms and subsequently reconstructed with the maximum a posteriori (MAP) reconstruction algorithm. The pixel size was 0.866×0.866×0.796 mm and in the center field of view the resolution was 1.4 mm full-width-at-half-maximum. The images were not corrected for attenuation or scatter.Mice were injected i.v. with 10.3±1.4 (mean±SD) MBq [18F]FDG or 9.5±0.9 (mean±SD) MBq [18F]FLT. Mice were fasted overnight before each [18F]FDG scan Following the microPET scan, a microCT scan was acquired with a MicroCAT® II system . A 7 minute and 10 seconds CT scan was performed with parameter settings: 360 rotation steps, tube voltage 60 kV, tube current 500 µA, binning 4 and exposure time 310 ms. The pixel size was 0.091×0.091×0.091 mm.T*W)/Dinj, where CT is radioactivity in the tissue, W is weight of the animal and Dinj is injected dose. SUVmean gives information of the mean concentration of tracer and SUVmax is a measure of the voxel within the ROI which have the highest tracer concentration. For tumor volume measurements ROIs were drawn manually by delineation of the tumor boundary by qualitative assessment in several of the coronal planes on the microCT images. Summation of voxels from all the planes generated the tumor volume.PET and microCT images were fused in the Inveon software . Before fusion region of interests (ROIs) were drawn on the CT pictures manually by qualitative assessment covering the whole tumors and subsequently tumor volume and tracer uptake, assessed by standard uptake values (SUV) mean and maximum, were generated by summation of voxels within the tomographic planes. SUV was defined as  was used. The reactions were detected using standard polymer technique with few modifications. Negative controls were performed by omission of primary antibody. Sections were dewaxed in xylene, rehydrated and pre-treated before immunostaining. The slides were incubated with MIB-1 1:100 diluted in Power Block solution  for 30 min at room temperature. The sections were rinsed in TBST (3×5 minutes) and incubated with rabbit anti-FITC  for 20 min at RT. The reactions were detected with Super Sensitive polymer-HRP reagent  and visualised with DAB ImmPACT Substrate chromogen solution (Vector laboratories cat. co. SK-4105) following the manufactures instructions. Finally, slides were counterstained with Mayers haematoxylin.The level of Ki67 present in the samples was measured as mean of three hot spots on one slide from each tumor. Hotspots were defined as areas with the highest Ki67 intensity and were identified by scanning the entire tumor slide in 10×magnification. Ki67 positive cells were defined as cells with positive nucleus staining. The Ki67 staining index was defined as the number of Ki67 positive cells divided by the total number of tumor cells in an area on the 60×magnification. Positive and negative controls from human tissue were placed on each slide for validation of the immunohistochemical procedure.Comparison of tumor volume between the treatment and control group were calculated using an unpaired student’s t-test. For intra-group comparisons of tracer uptake ANOVA with Bonferroni post-hoc test was used. All data were tested to be normal distributed by means of Kolmogorov-Smirnov test. Correlations between SUVmax ratios and tumor growth were calculated using linear regression. Calculations were made in PASW 17.0 . Data are reported as mean±SEM (unless stated otherwise) and P<0.05 was considered statistically significant.2 = 0.98) and caliper   . APO866  = 0.87) .Baseline levels of [18F]FLT uptake measured as SUVmean were 1.26±0.16 (mean±SD) and SUVmax were 2.18±0.20 (mean±SD). Baseline levels of [18F]FDG uptake measured as SUVmean were 0.67±0.14 (mean±SD) and SUVmax were 1.23±0.29 (mean±SD).Uptake of [18F]FLT measured as SUVmean in the APO866 group was 1.28±0.03 at baseline, 0.98±0.03 at 24 h, 0.90±0.03 at 48 h and 0.96±0.07 at Day 7. Compared to baseline [18F]FLT SUVmean was significantly decreased at 24 h (P<0.001), 48 h (P<0.001) and Day 7 (P<0.001) in the APO866 group. No significant changes in [18F]FLT SUVmean were observed in the control group. [18F]FLT SUVmax uptake in the APO866 group was 2.20±0.03 at baseline, 1.64±0.04 at 24 h, 1.49±0.03 at 48 h and 1.54±0.11 at Day 7. Compared to baseline [18F]FLT SUVmax was significantly different at 24 h (P<0.001), 48 h (P<0.001) and Day 7 (P<0.001) in the APO866 group  and 5. NUptake of [18F]FDG measured as SUVmean in the APO866 group was 0.70±0.04 at baseline, 0.62±0.03 at 24 h, 0.59±0.04 at 48 h and 0.40±0.08 at Day 7. Compared to baseline [18F]FDG SUVmean was significantly decreased at Day 7 (P<0.001) in the APO866 group. No significant changes in [18F]FDG SUVmean were observed in the control group. [18F]FDG SUVmax uptake in the APO866 group was 1.29±0.09 at baseline, 1.11±0.07 at 24 h, 1.08±0.09 at 48 h and 0.77±0.15 at Day 7. Compared to baseline [18F]FDG SUVmax was significantly decreased at Day 7 (P = 0.005) in the APO866 group. In the control group [18F]FDG SUVmax was 1.16±0.07 at baseline, 0.91±0.03 at 24 h (P = 0.007 vs. baseline), 1.12±0.04 at 48 h and 1.47±0.05 at Day 7 (P<0.001 vs. baseline)  and 5.2 = 0.44) and d7/d0  correlated significantly with final tumor growth and [18F]FDG SUVmax ratio 48 h/0 h  and d7/d0  correlated significantly with final tumor growth . Differences in expression of Ki67 protein between the treatment (28.3%) and control group (52.5%) were significant (P = 0.022) (Expression of Ki67 protein measured by immunohistochemistry showed a significant correlation with [18F]FLT SUVmean (r= 0.022) . Some ofIn this study we investigated the effect of APO866 treatment on tumor uptake of [18F]FLT and [18F]FDG by PET in the A2780 ovarian cancer tumor model. By groups, uptake of [18F]FLT decreased after initiation of treatment and was significantly different from baseline already at 24 hours after treatment initiation. By groups, uptake of [18F]FDG did not change on the first 1–2 days following treatment start, however at Day 7 uptake was significantly decreased to 50% of baseline uptake. At Day 7 we observed decreases in both [18F]FLT and [18F]FDG tracer uptake. On the PET images  it was mA measure of the maximal tracer uptake is often used when assessing treatment responses rather than the mean tracer uptake Lower uptake of tracer at Day 7 compared to baseline for both [18F]FLT and [18F]FDG in the treatment groups was most likely not due to partial volume effect because of a decrease in tumor volume as the tumors did not shrink during the treatment. The changes in tracer uptake were therefore assumed to be due to physiologic changes in the tumor tissue which were further confirmed by the difference in the quantity of Ki67 positive cells between the treatment and control groups. Positive correlation between [18F]FLT uptake and the amount of Ki67 positive cells have earlier been reported by others APO866 is a relatively slow acting anti-cancer drug without any immediate cytotoxicity leading to ATP depletion and apoptosis Anti-cancer drugs being cytostatic or having low cytotoxicity often require long treatment time before exerting their effect on tumor volume. The advantage of measuring physiological changes as we do in this study with [18F]FLT and [18F]FDG PET compared to the tumor volume criteria is that we are able to measure a treatment effect earlier than we can measure a change in tumor volume.When the tumors were studied individually, we found a significant positive correlation between the changes in SUVmax for both [18F]FLT and [18F]FDG between baseline and 48 hours and the tumor growth. Accordingly, those tumors which had the greatest decrease in SUVmax values for both [18F]FDG and [18F]FLT 48 hours after treatment initiation were those which had the lowest increase in tumor volume at the end of the experiment. It indicates that we, in addition to finding a decrease in the whole treatment groups, on the individual tumor level already at 48 hours after treatment initiation were able to identify the tumors that responded best to the treatment as it were those that had the greatest change in SUVmax values.In conclusion, we found a decrease in tumor [18F]FLT uptake following treatment with the nicotinamide phosphoribosyltransferase inhibitor APO866. After 7 days of treatment tumor uptake of [18F]FDG was half of baseline uptake. Additionally, we found that the tumors which responded best to the treatment were those that had the greatest change in both [18F]FLT and [18F]FDG at 48 hours after treatment initiation. These results show the possibility to use [18F]FLT and [18F]FDG to image treatment effect early following treatment with APO866 in future clinical studies.


This taurine-induced increase of PSC frequency was abolished by 0.2 μM tetrodotoxin (TTX), 1 μM strychnine or 3 μM gabazine, but was unaffected by the glutamatergic antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and (±) R(-)-3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP), suggesting that taurine specifically activates GABAergic network activity projecting to pyramidal neurons. Cell-attached recordings revealed that taurine enhanced the frequency of action potentials (APs) in pyramidal neurons, indicating an excitatory action of the GABAergic PSCs. In order to identify the presynaptic targets of taurine we demonstrate that bath application of taurine induced in GAD67-GFP labeled interneurons an inward current that is mainly mediated by glycine receptors and can generate APs in these cells. We conclude from these results that taurine can enhance network excitability in the immature neocortex by selectively activating GABAergic interneurons via interactions with glycine receptors.Although it has been suggested that taurine is the main endogenous neurotransmitter acting on glycine receptors, the implications of glycine receptor-mediated taurine actions on immature neocortical networks have not been addressed yet. To investigate the influence of taurine on the excitability of neuronal networks in the immature neocortex, we performed whole-cell patch-clamp recordings from visually identified pyramidal neurons and interneurons in coronal slices from C57Bl/6 and GAD67-green fluorescent protein (GFP) transgenic mice . In 46% of the pyramidal neurons bath-application of taurine at concentrations ≥ 300 μM significantly enhanced the frequency of postsynaptic currents (PSCs) by 744.3 ± 93.8% ( A receptors mediate depolarizing responses in immature hippocampal  are functionally expressed during pre- and early post-natal development  recorded in pyramidal neurons of the mouse neocortex at postnatal days (P) 2–4. These experiments revealed that taurine induced a massive increase in the frequency of GABAergic PSCs, which is suppressed by the glycinergic agonist strychnine and mediated an excitatory response in these cells. Recordings from genetically labeled GABAergic interneurons showed that taurine induced an inward current, mainly mediated by glycine receptors, and thereby generate action potentials (APs) in these cells. These findings suggest that in the early postnatal neocortex taurine can activate GABAergic networks via an interaction with glycine receptors on GABAergic interneurons and may thereby influence the maturation of neocortical network.Animal handling was performed in accordance with EU directive 86/609/EEC for the use of animals in research and approved by the local ethical committee . All efforts were made to minimize the number of animals and their suffering. For electrophysiological experiments coronal slices of the neocortex were prepared from pups of C57Bl/6 or GAD67-GFP transgenic mice  with infrared differential interference contrast (IR-DIC) optics  of −69.1 mV (except where noted), using the program MiniAnalysis 4.3.3 . Frequencies of APs and PSCs were determined during at least 3 min of the recording immediately before agonist application for control conditions, and during the complete period of agonist application for the agonist condition. Holding currents were determined during a ca. 1 min interval directly before agonist application for control conditions and during a similar interval in the maximal phase of the agonist response. Cells were considered as responsive to agonists, if the frequency of PSCs in the presence of agonists  was 4x larger than the standard deviation of the frequency during the control interval. For taurine concentrations ≤ 100 μM cells were only considered as non-responsive, if the cells showed a taurine-induced increase in PSC frequency at higher taurine doses. The reversal potential (ERev) was calculated from the Goldman equation using Cl- concentrations, estimated Resting membrane potential (RMP) was measured directly after establishing whole-cell configuration. All potentials were corrected for a liquid junction potential of 9.1 mV. Input resistance and capacitance were determined from amplitude and rise kinetics of voltage deflections to 300 ms hyperpolarizing current pulses. AP amplitude was defined as voltage difference between firing threshold and peak depolarization and AP width was measured at half-maximal amplitude. PSCs were detected and analyzed in continuous voltage-clamp recordings lasting at least 180 s at a holding potential , osmolarity 333 mOsm . Standard pipette solution for whole-cell recordings contained (in mM) 44 KCl, 80 K-gluconate, 1 CaCl2, 2 MgCl2, 11 ethylene glycol tetraacetic acid (EGTA), 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2 Na2-ATP, 0.5 Na-GTP, pH adjusted to 7.4 with KOH and osmolarity to 306 mOsm with sucrose. For low-Cl- pipette solution, the KCl concentration was reduced to 2 mM and the K-Gluconate concentration raised to 124 mM. For cell-attached recordings the pipette solution contained (in mM) 126 K-Gluconate, 4 KCl, 1 CaCl2, 2 MgCl2, 11 EGTA, 10 HEPES, 2 Na2-ATP, 0.5 Na-GTP, pH adjusted to 7.4 with KOH and osmolarity to 306 mOsm with sucrose.ACSF contained (in mM) 124 NaCl, 26 NaHCOTaurine was dissolved directly in ACSF, gabazine (SR-95531), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or strychnine were used from a stock solution in dimethylsulfoxide (DMSO), tetrodotoxin (TTX) from a stock solution in citrate buffer and glycine or (±) R(-)-3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP) from an aqueous stock solution. TTX was purchased from RBI , glycine and taurine from Tocris , and all other substances from Sigma. The final DMSO concentration never exceeded 0.5%.In all whole-cell experiments 0.5 % biocytin  was added to the pipette solutions to label the recorded neurons. Slices were fixed in a 4% parafolmaldehyde solution immediately after recording for more than 24 h. After washing in phosphate buffer, slices were incubated in blocking solution ) for 2 h at RT. Subsequently, biocytin-labeled neurons were stained with Cy-3 conjugated streptavidin  as described in detail before . Results were designated significant at a level of p < 0.05.All values are expressed as mean ± standard error of the mean (SEM). For statistical analysis Student n = 530), an input resistance of 1.7 ± 0.04 GΩ and an input capacity of 72.8 ± 2.5 pF. In 96% of all 450 neurons investigated for this aspect APs with an average amplitude of 54.8 ± 0.4 mV (n = 428) and duration of 4.4 ± 0.08 ms could be evoked upon a depolarization that crosses the AP-threshold of −41.7 ± 0.3 mV , an input resistance of 3.3 ± 0.3 GΩ and an input capacity of 37.2 ± 5.0 pF. Injection of a depolarizing current evoked in these neurons APs with an amplitude of 37 ± 1.8 mV (n = 35), a duration of 2.9 ± 0.3 ms and an AP-threshold of −45.1 ± 0.9 mV , with a considerable heterogeneity.In addition, 38 GFPs Figure . These GV Figure . Upon pen = 12), which reversed at −22.3 ± 1.5 mV  reduced the amplitude of taurine-induced currents by 80 ± 5.2%  was completely blocked by 1 μM strychnine (n = 9), while 3 μM gabazine (n = 8) had no effect  induced in pyramidal neurons an inward current of −260.4 ± 31.9 pA  that was completely blocked by 1 μM strychnine (n = 6), while 3 μM gabazine (n = 7) had no effect  that was completely blocked by 3 μM gabazine (n = 6), while 1 μM strychnine had no effect  that was completely blocked by 1 μM strychnine (n = 7), whereas 3 μM gabazine had no effect 106.2 ± 5.3%  that was completely blocked by 3 μM gabazine (n = 7), whereas 1 μM strychnine had no effect , 46.9 ± 7.4 (n = 20) and 52 ± 8.8 pA (n = 15) were induced by bath application of 300 μM, 1 mM and 3 mM taurine, respectively  to 0.59 ± 0.05 Hz  with the PSC frequency during the control interval. In addition, the amplitude of PSCs increased from 19.3 ± 1 pA (n = 137) to 24.6 ± 1.3 pA (n = 145) in the presence of 300 μM taurine. Furthermore, increasing the taurine concentration to 1 mM or 3 mM induced comparable effects on PSCs. Bath application of 1 mM taurine increased the frequency of PSCs in 10 out of 21 investigated neurons by 694 ± 210%, while 3 mM taurine increased the frequency in 6 out of 15 neurons and 518 ± 198%  and amplitude (97.4 ± 5%) of PSCs. In three additional TTX-treated slices that did not show spontaneous PSCs under control conditions, 300 μM taurine failed to evoke PSCs. The taurine-induced inward current was, however, not significantly affected under this condition . These observations suggest that the taurine-induced increase in PSC frequency requires the activation of presynaptic networks.Next we analyzed the pharmacological properties of the PSCs evoked by the bath application of 300 μM taurine. Bath-application of TTX inhibited the taurine-induced PSCs Figure . In the p = 0.5 and p = 1, respectively, n = 9) different from the control application in ACSF . Similar results were obtained if these glutamate receptor antagonists were applied alone .Inhibition of ionotropic glutamate receptors by bath application of 10 μM CNQX or 20 μM CPP had no significant effect on the taurine-induced PSCs. In the presence of both glutamate antagonists neither amplitude (27.13 ± 3 vs. 31.6 ± 6.3 pA) nor the frequency (0.55 ± 0.09 vs. 0.55 ± 0.11 Hz) of taurine-induced PSCs was significantly  completely blocked sPSCs in 19 out of 26 pyramidal cells investigated and massively reduced the frequency of sPSCs to 0.023 ± 0.007 Hz in the remaining seven neurons. This result indicates that the majority of sPSCs is mediated by GABAA receptors. Bath application of 300 μM taurine in the presence of 3 μM gabazine did not induce a significant (p = 0.34) increase in frequency of PSCs  reduced by 79.6 ± 8.3% (n = 8) from 21 ± 2.6 to 5.6 ± 2.5 pA in the presence of gabazine.On the other hand, bath application of the GABAn = 12) and amplitude (92 ± 11%) of sPSCs, supporting previous reports that glycine receptors are not directly involved in synaptic transmission in the developing neocortex  and amplitude  of PSCs. In addition, the taurine-induced inward current was significantly (p < 0.001) reduced by 47.7 ± 8.3% (n = 12) from 22.2 ± 2.8 to 9.7 ± 1.1 pA. In the presence of both, 1 μM strychnine and 3 μM gabazine the taurine-induced inward current was massively reduced by 95.5 ± 1.2% to 1.1 ± 0.4 pA (n = 12). Under this condition bath application of 300 μM taurine did also not increase the frequency of PSCs . In summary, these results suggest that glycine receptors are essential for the presynaptic taurine effect and also contribute to the postsynaptic taurinergic currents.Bath application of the glycine receptor antagonist strychnine (0.3–1 μM) had no significant effect on frequency . In addition, it increased the frequency of PSCs by 566 ± 219% from 0.1 ± 0.04 to 0.24 ± 0.05 Hz  and increased the frequency of PSCs in 4 out of 6 investigated pyramidal neurons by 1045 ± 831% . To prove that these glycine-induced PSCs were indeed mediated by GABAA receptors in the postsynaptic membrane, we next applied 100 μM glycine in the continuous presence of 3 μM gabazine, 10 μM CNQX and 20 μM CPP. Under this condition spontaneous PSCs were absent and bath application of 100 μM glycine did not induce PSCs  of these PSCs. For this purpose we performed voltage-clamp experiments using a pipette-solution with a Cl- concentration of 8 mM, which corresponds to an ERev of −62.5 mV. In these experiments the cell was clamped exactly to ERev, which was determined experimentally for each cell by a series of focal GABA pulses at different holding potentials. These experiments revealed that the frequency of sPSCs was reduced to from 0.042 ± 0.013 Hz (n = 7) to 0.021 ± 0.006 Hz (n = 7) if the membrane was clamped from −85 mV to ERev, indicating that a substantial fraction of sPSCs is mediated by ligand-gated Cl- channels. At this holding potential bath application of 300 μM taurine had no significant (p = 0.35) effect on the frequency of PSCs  and 630 ± 149% (n = 11), respectively, indicating that taurine initiates PSCs that are mediated via ligand-gated Cl- channels. Accordingly the taurine-induced PSCs reversed at −56.7 mV, approximately at the estimated ERev  remained undisturbed. In 28 out of 35 investigated neurons spontaneous APs at a frequency of 0.29 ± 0.9 Hz (n = 28) were observed, the remaining eight cells showed less that three APs during the control interval. Bath application of 300 μM taurine increased the AP frequency in 20 (corresponding to 57%) of these neurons by more than 50% to 0.68 ± 0.16 Hz , while the tonic current was not significantly affected . Cell-attached recordings revealed that in the presence of 0.3 μM gabazine bath application of 300 μM taurine had no effect on AP frequency in 12 investigated cells, which all revealed a taurine-induced increase in AP frequency under control conditions . This inward current was significantly attenuated by 49 ± 8%  in the presence of 3 μM gabazine and by 70 ± 13%  in the presence of 1 μM strychnine  a significant (p < 0.001) increase in AP frequency from 0.1 ± 0.02 to 1.1 ± 0.27 Hz  to 0.19 ± 0.02 Hz. A comparable effect was observed in slices without SP, in which the frequency of PSCs increased by 697 ± 170% (n = 6) to 0.26 ± 0.05 Hz  these taurine-induced GABAergic PSCs mediated an excitatory effect; (iv) taurine, acting mainly on glycine receptors, induced an inward current and repetitive APs in GABAergic interneurons; and (v) GABAergic SP neurons do not substantially contribute to the taurine-induced activity. We conclude from these results that taurine can enhance the activity of excitatory GABAergic networks in the immature neocortex.The main findings of this study can be summarized as follows: (i) bath application of 300 μM taurine increased the frequency of PSCs in a considerable fraction of pyramidal neurons; (ii) taurine, but also glycine and isoguvacine specifically increased the frequency of GABAPassive and active membrane properties of the pyramidal neurons from P2–P4 mice are in accordance with previous results (Kriegstein et al., A receptor antagonist gabazine. This finding may be explained by the hypothesis that GABAA receptor activation on presynaptic cells is required for the activation of the PSCs, but the observation that bath-application of glycine induced a similar gabazine-sensitive increase in PSC frequency suggests that a taurine-mediated glycine receptor activation is sufficient to induce these PSCs. Second, the taurine-induced PSCs reversed around ECl, which would, however, also match to a glycinergic neurotransmission. But the observation that the glycine-induced increase in PSC frequency was completely blocked by gabazine indicates that under this condition only GABAergic PSCs were induced and suggests that this is also the case for the taurine-induced PSCs. These suggestions are supported by the observation that an adequate glycinergic stimulation of presynaptic cells is required to provoke taurine-induced PSCs. And finally, the observation that a specific inhibition of synpatically GABAA receptors by low gabazine concentrations completely abolished the taurine-induced PSCs also indicates that these PSCs were GABAergic. In summary, these results strongly suggest that bath-application of taurine, but also of glycine or isoguvacine, selectively activated GABAergic PSCs.One major conclusion of our study is that taurine specifically increased the frequency of GABAergic PSCs. Several lines of evidence support this hypothesis. First, the taurine-mediated increase in PSC frequency was unaffected by glutamatergic antagonists but completely suppressed by the GABAA receptors mediate depolarizing membrane responses in immature neurons, due to the high [Cl-]i in these cells (Owens et al., A variety of previous studies demonstrated that activation of GABAA and glycine receptors can potentially contribute to the taurine effect. The puzzling observation that in interneurons strychnine abolished responses to focal taurine application, while the tonic current mediated by bath applied taurine was only reduced by 70%, may be caused by distinct activation kinetics of glycine and GABA receptors upon fast application and by a differential desensitization of both receptors in the continuous presence of taurine. While GABAA receptors had already been described in immature interneurons (Gozlan and Ben-Ari, A receptors was insufficient to activate the presynaptic GABAergic networks.Theoretically, the GABAergic PSCs can rely on an increased spontaneous release probability of GABA vesicles or from an excitation of presynaptic GABAergic interneurons (Hessler et al., The subtype of the GABAergic interneurons that mediate these excitatory synaptic inputs could not be defined in our study. We can exclude that a GABAergic population of SP neurons (Hanganu et al., A receptors (Garaschuk et al., A receptors to spontaneous activity has been reported in the first postnatal week (Conhaim et al., In the immature hippocampus early generated pioneer GABAergic interneurons synchronize network activity transients (Bonifazi et al., Finally, it has to be considered that the taurine concentration of 300 μM used in the present study most likely exceeds the interstitial taurine levels that can be expected in the immature neocortex. However, in rat and human fetal brains taurine is the most abundant neurotransmitter (Das and Ray, Werner Kilb designed the experiments, Bogdan A. Sava, Rongqing Chen and Haiyan Sun performed the experiments, Bogdan A. Sava and Werner Kilb analyzed the data, Heiko J. Luhmann and Werner Kilb wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Calochortus (Liliaceae) displays high species richness, restriction of many individual taxa to narrow ranges, geographic coherence of individual clades, and parallel adaptive radiations in different regions. Here we test the first part of a hypothesis that all of these patterns may reflect gene flow at small geographic scales. We use amplified fragment length polymorphism variation to quantify the geographic scales of spatial genetic structure and apparent gene flow in Calochortus albus, a widespread member of the genus, at Henry Coe State Park in the Coast Ranges south of San Francisco Bay. Analyses of 254 mapped individuals spaced 0.001–14.4 km apart show a highly significant decline in genetic identity with ln distance, implying a root-mean-square distance of gene flow σ of 5–43 m. STRUCTURE analysis implies the existence of 2–4 clusters over the study area, with frequent reversals among clusters over short distances (<200 m) and a relatively high frequency of admixture within individuals at most sampling sites. While the intensity of spatial genetic structure in C. albus is weak, as measured by the Sp statistic, that appears to reflect low genetic identity of adjacent plants, which might reflect repeated colonizations at small spatial scales or density-dependent mortality of individual genotypes by natural enemies. Small spatial scales of gene flow and spatial genetic structure should permit, under a variety of conditions, genetic differentiation within species at such scales, setting the stage ultimately for speciation and adaptive radiation as such scales as well. Calochortus  within species at small spatial scales, and in some cases ultimately resulting in speciation and endemism over limited scales as well  on an experimental slope after a heavy storm moved <2 m. In natural populations of Calochortus westonii, most seeds spill from the fruit within 15 cm of the parent plant, producing a tight clump of 20–30 seedlings the following spring , and the size of its genetic neighborhoods (Nb). This article is the first step of an investigation into genetic differentiation, introgressive gene flow, and phylogenetic relationships in the Bay Area clade of Calochortus.Here we use amplified fragment length polymorphism (AFLP) markers test the hypothesis that total gene flow in us albus  over scaCalochortus albus is herbaceous, with one ribbonlike basal leaf developing early each spring from a perennial bulb. Later in the spring, a single flowering stalk is produced, from which hang nodding, globe-shaped flowers  and, although they are self-compatible, protandry, and pollinator observations by Jokerst , a 350-km2 California state park located southeast of San Jose in the interior South Coast Ranges . Coordinates of all other individuals were determined based on their bearing and distance from the focal plant using a compass and a sonic range-finder . In each study population, and at 10 sites randomly located between populations, reproductive and nonreproductive individuals were counted along three 1 m × 10 m transects, to produce estimates of the density of reproductives and nonreproductives.During spring 2006, 20 populations of t Ranges . Sixteent Ranges , oak-wooEcoRI and MseI in a 10 μL reaction containing 83 ng DNA, 0.05 μL 100 ng/100 μL BSA, 5 U EcoRI, and 5 U MseI and incubated at 37°C for 2 h. Double-stranded adapters were then ligated to the resulting digestion fragments. Double-stranded adapters were produced by mixing equal volumes of equimolar solutions of two oligos for both the M (5′ GAC GAT GAG TCC TGA G 3′ and 5′ TAC TCA GGA CTC AT 3′) and the E (5′ CTC GTA GAC TGC GTA CC 3′ and 5′ ATT TGG TAC GCA GTC TAC 3′) adapters. These solutions were then incubated at 95°C for 5 min and allowed to cool 1°C per minute at room temperature. A volume of 0.19 μL of both the M and the E adapters, along with 3.52 μL ddH2O, 1 μL 10 × ligase buffer, and 0.4 U T4 DNA ligase were added to the 5 μL digestion reaction and incubated at 16°C overnight. Ligation products were then diluted at a ratio of 17 μL ligation product to 70 μL ddH20.High-quality genomic DNA was extracted using a DNeasy® 96-well plant extraction kit . AFLP data were generated following the protocols of Myburg et al. , with mi2, 2 μL 2.5 mmol/L (each) dNTPs, 0.38 μL M+C primer, 0.38 μL E+A primer, 1.5 U Taq, 5 μL dilute ligation product, and 13 μL ddH20. Reactions were then cycled under the following conditions: 72°C for 60 sec; 20 cycles of 94°C for 50 sec, 56°C for 60 sec, 72°C for 120 sec; 72°C for 120 sec. Pre-selective amplification products were then diluted with ddH2O at a ratio of 40 μL: 720 μL.Pre-selective amplification reactions were carried out using the dilute ligation products and primers complementary to the adapters but extending one additional, specified base in the 3′ direction. These 25 μL reactions contained 2.5 μL 10 × buffer, 1.5 μL 25 mmol/L MgCl2, 3 μL 2.5 mmol/L (each) dNTPs, 0.5 μL deionized formamide, 1.25 μL M+C— primer, and 0.25 μL labeled E+A– primer, 1.25 U Taq, and 10.75 μL ddH2O. The reactions were then exposed to the following cycling conditions: 10 cycles of 94°C for 50 sec, 65°C for 60 sec (decreasing by 1°C each cycle), 72°C for 120 sec; then 20 cycles of 94°C for 50 sec, 56°C for 60 sec, 72°C for 120 sec; then 72°C for 10 min.Three rounds of selective amplifications were performed using dilute pre-selective amplification products and three different primer pair combinations. All of the primers used in this final selective amplification were complementary to those used in the pre-selective amplification but extended an additional two or three bases in the 3′ direction. The primer pair combinations used were as follows: M+CCAG and E+ATT, M+CTT and E+ACT, and M+CCCG and E+AGC. In each primer pair the E+A– primer was fluorescently labeled. These 25 μL reactions contained 2.5 μL 10 × buffer, 1.5 μL 25 mmol/L MgClSelective amplification products were cleaned using magnetic beads  and run on an ABI 3100 capillary sequencer using a fluorescent internal standard in each lane . Chromatograms were analyzed using GeneMarker  to generate 0/1 matrices of fragments 100–300 bp in length for the M+CCAG/E+ATT and M+CCCG/E+AGC primer pair combinations and 100–400 bp in length for the M+CTT/E+ACT primer pair combination.SPAGeDi 1.3 , we calculated the Sp statistic  = the average kinship between adjacent plants. The average kinship between plants falling into the first distance category (0–3 m) was used to estimate F(1). The Sp statistic was then used to estimate the root-mean-square distance of gene dispersal (σ) aswhere bDe is effective population density and σ is the root-mean-square distance of gene dispersal (Vekemans and Hardy Nb)  Wright , 1969 waquations  hold besquations . Therefoequation . A restrDe as the density D of reproductive individuals times 0.5, 0.3, and 0.1, given that effective densities of natural plant populations often fall within this range  animal pollination/gravity dispersal; (2) animal pollination/animal dispersal; (3) wind pollination/gravity dispersal; and (d) animal pollination/mixed animal and gravity dispersal. The latter permitted us to assess whether C. albus had exceptionally short dispersal distances given its ecology of pollen and seed dispersal.We compared the values of the nd Hardy , to deteK, and then compare the estimated log probability of data under each K, ln Pr(X|K). We conducted 20 replicate runs for all proposed values of K between 1 and 10, assuming dominant AFLP markers, admixture among clusters and individuals (α = 1), default allele frequency distribution (λ = 1), and correlated allele frequencies. Each run used 104 iterations following a burnin period of 5 × 104 iterations. We estimated the number of clusters as the value of K with the greatest Pr(X|K), and then tested that using the ΔK procedure of Evanno et al.  loci scored.The number of pairs of individuals at various distances ranged from 96 in the smallest distance class (<3 m) to 12,160 in the largest distance class (>6561 m) . The perr2 = 0.883, P < 0.002 for two-tailed t-test with 7 df) or for just the 16 northern sites  (r2 = 0.044) or for just the 16 northern sites ; both patterns were significant under Mantel tests.Kinship values, averaged over each distance class, declined in a highly significant fashion with the logarithm of distance between individuals, for all sites  A and B. D of reproductive C. albus was 0.50 ± 0.33 individuals m−2; that of nonreproductives, 1.57 ± 1.47 individuals m−2. Assuming that D = 0.16, 0.50, and 0.83 individuals m−2, and that De = 0.5D, 0.3D, and 0.1D, we estimated that the root-mean-square distance of gene dispersal σ was 16.7 ± 10.6 m, averaged across the study region, with a range of 5.2–42.9 m . For all sample sites, we estimated that neighborhood size Nb was 140 ± 47 individuals, with a range of 69–193; the Sp statistic was 0.00404 ± 0.00157, with a range from 0.00259 to 0.00722 (Nb = 125 ± 53 (range: 52–194) and Sp = 0.00575± 0.00277 (range: 0.00275–0.00967)  .Sp = 0.00404 for C. albus is the lowest for any herbaceous species tabulated by Vekemans and Hardy  dispersed herbs. The latter category includes 17 of the 24 herbs tabulated by Vekemans and Hardy  is strikingly short in absolute terms, providing prima facie evidence of gene flow restricted to small spatial scales.The average estimate of nd Hardy , althougnd Hardy , and by K) curves between K = 2 and K = 5 (K = 2 in both cases (K) after the burnin period for all but the largest number of implied clusters. Plots of cluster membership against the spatial order of the sample sites for K = 2 to K = 5 indicated a repeated set of rather steep transitions from populations dominated by one cluster to the other, on the scale of tens of meters, and a substantial fraction of admixed individuals at several sample sites , the hiCalochortus. Our aggregate estimate of gene flow in Calochortus based on AFLP variation almost surely overestimates the spatial scale of seed movement, which Patterson and Givnish  coupled with a two-generation analysis of adults and maternal and biparental tissue in dispersed seedlings produce the most powerful estimates of both pollen and seed dispersal , which may cause it to appear to have less intense genetic structure than other taxa  increas


Adrenal insufficiency is a serious complication of AIDS. Usually the integrity of HPA  axis is assessed by measuring cortisol response to 250µg of ACTH. Recent studies have shown that lower ACTH dose increases the sensitivity of the procedure. In the present study we have tried to estimate prevelance of adrenal insufficiency using low dose ACTH test (1µg).To estimate the prevelance of adrenal insufficiency in AIDS patients using low dose ACTH test.50 patients with confirmed diagnosis of HIV were included in the study. History and physical examination were recorded. Lab investigations included Cd4 count, serum cortisol and low dose acth stimulation test. Data of patients with adrenal insufficiency (GROUP 1) was compared with those without adrenal dysfunction (GROUP 2). Stastical analysis was done using appropriate tests.37/50 (74%) of study subjects had adrenal insufficiency. Basal cortisol in (GROUP 1) and (GROUP 2) was 10.09µg/dl and 21.95µg/dl (P < .05). Cortisol post Acth stimulation test in (GROUP 1) and (GROUP 2) was 9.49 µg/dl and 19.93µg/dl (P < .05). Mean Cd4 count in (GROUP 1) and (GROUP 2) was 138.7±56.17 cells/µl and 171.8.7±25.41cells/µl (P < .05). Blood glucose, serum sodium was low and serum potassium, eosinophil counts were high in (GROUP 1) when compared to (GROUP2).Adrenal insufficiency in patients with acquired immunodeficiency syndrome is a common problem in clinical practice.


Escherichia coli, but disulfide-rich proteins are here often misfolded, degraded, or found in inclusion bodies.Although disulfide bond formation in proteins is one of the most common types of post-translational modifications, the production of recombinant disulfide-rich proteins remains a challenge. The most popular host for recombinant protein production is in vitro wheat germ translation system for the expression of an immunological important eukaryotic protein that has to form five disulfide bonds, resistin-like alpha (mFIZZ1). Expression in combination with human quiescin sulfhydryl oxidase (hQSOX1b), the disulfide bond–forming enzyme of the endoplasmic reticulum, results in soluble, intramolecular disulfide bonded, monomeric, and biological active protein. The mFIZZ1 protein clearly suppresses the production of the cytokines IL-5 and IL-13 in mouse splenocytes cultured under Th2 permissive conditions.We optimize an The quiescin sulfhydryl oxidase hQSOX1b seems to function as a chaperone and oxidase during the oxidative folding. This example for mFIZZ1 should encourage the design of an appropriate thiol/disulfide oxidoreductase-tuned cell free expression system for other challenging disulfide rich proteins. E. coli, the Dsb (disulfide bond)-family  of proteins are involved de novoResistin is part of the FIZZ (found in inflammatory zones) family of genes, and was first characterized in murine models where it has been extensively studied as a potential link between type II diabetes and obesity We have chosen for a fast and easy to tune expression system, the eukaryotic cell-free translation system based on the wheat germ embryo (WGE) E. coli SHuffle™ T7 Express, Origami™ DE3 and BL21 DE3. Soluble and insoluble fractions were evaluated on non-reducing 15% SDS-PAGE . The Dsb disulfide bond formation machinery of E. coli seems to be unable to cope with this multiple cysteine containing polypeptide chain. Also the expression of recombinant resistin (mFIZZ3) using a pQE-31 vector resulted in the expression in inclusion bodies in E. coli JM109 We first decided to target the expression of mFIZZ1 with an N-terminal His-tag to the cytoplasm of SDS-PAGE  and on iSDS-PAGE . On a noWe decided to use a fast and easy to tune expression system, the eukaryotic cell-free translation system based on the wheat germ embryo In our attempt to increase the amount of soluble mFIZZ1, we evaluated the co-expression of mFIZZ1 and mFIZZ1′ with the helper proteins hQSOX1b and hPDI, both eukaryotic thiol/disulfide oxidoreductases. We cloned hQSOX1b and hPDI into a pEU-GST vector and evaluated the co-expression of hQSOX1b, hPDI and both thiol/disulfide oxidoreductases together with mFIZZ1 or mFIZZ1′ under the same experimental cell free expression conditions. The plasmid DNA of the four constructs (2 µg) was transcribed for 6 h at 37°C using SP6 RNA polymerase, the mRNA of the plasmids were cooled down and checked on agarose gel . In all Interestingly, when we compared the expression of mFIZZ1 and mFIZZ1′ by immunoblot , we obse2+ Immobilized Metal Affinity Chromatography (IMAC). We obtained a final yield of ∼300 µg mFIZZ1′ in a 6 ml wheat germ reaction. Co-expression with hQSOX1b resulted in a final yield of ∼120 µg for a 6 ml reaction mixture, which might be due to the translation from two plasmids with the same and limited amount of compounds. For mFIZZ1 the yield was ∼340 µg, while in the presence of hQSOX1b it was ∼160 µg.In the next step, we purified mFIZZ1 and mFIZZ1′ in the presence and absence of hQSOX1b using NiPurified proteins were evaluated on 15% SDS-PAGE under reducing and non-reducing conditions followed by immunoblot using an anti-His antibody . The samEscherichia coli. Our results strongly indicate that mFIZZ1 and mFIZZ1′ are monomeric in solution. For the experiment in the absence of hQSOX1b similar results were obtained. In mFIZZ2 and mFIZZ3, an extra N-terminal cysteine is present  We checked secondary structure of recombinant purified mFIZZ1′ in the presence and absence of hQSOX1b with far-UV circular dichroism (CD). The CD-spectra of mFIZZ1′ produced in the presence and absence of hQSOX1b showed a double minimum at 208 and 222 nm characteristic for α-helical proteins, indicating that the protein produced contains a significant amount of secondary structure . We usedWe next evaluated the ability of purified mFIZZ1′ and mFIZZ1 to suppress Th2 cytokine expression by splenocytes in vivoIn contrast, recombinant mFIZZ1 and mFIZZ1′ co-expressed with hQSOX1b significantly decreased IL-5 and IL-13 secretion, and the same values for the bacterially derived protein (rRA) were obtained . The conE. coli RNase I activity assay in vitro folding of RNase I with DsbA and DsbC under these condition From the previous results, it is not clear whether hQSOX1b works as an oxidase, an isomerase, or a chaperone. We used an E. coli.  (We next tested the isomerase activity of hQSOX1b on scrambled RNase I of RNase I) . hQSOX1bin vitro oxidase activity of DsbC 2 to form H2O2, and hQSOX1b does not need to be recycled by a downstream pathway like for the Dsb-proteins. As 50 nM of hQSOX1b has no chaperone effect on unfolded RNase I  incubated for 3 min at 25°C. The combination of the folding catalysts hQSOX1b + hPDI and DsbA + DsbC results in the highest oxidative folding efficiency, with the largest contribution for the isomerases hPDI and DsbC . The in  RNase I , hQSOX1b RNase I . These rIn vivo, the signal peptide of mFIZZ1 is removed by signal peptidase enzymes All together, well-folded, soluble full-length monomeric mFIZZ1 is produced in a wheat germ cell-free expression system. The protein mFIZZ1 forms five disulfides and the protein mFIZZ1′ with its signal peptide forms six disulfides. Both mFIZZ1 proteins are only biologically active in the presence of hQSOX1b. Indeed, in the absence of hQSOX1b, all disulfide bonds are formed, but no bioactivity was detected. We might conclude that these non-active forms are disulfide-scrambled with a higher helical content (CD-spectrum) and a different structural fold. The fact that mFIZZ1′ with its predicted signal peptide gave a similar result came as a surprise. The quiescin sulfhydryl oxidase hQSOX1b seems to have chaperone and oxidase activity.2O2 from the formation of disulfide bonds by hQSOX1b may have increased the chaperone activity of hPDI. In this case hPDI might correct disulfide bonds as an isomerase and its chaperone activity will increase the moment more disulfide bonds are introduced by the oxidase hQSOX1b, which would result in a clear working together of those two thiol/disulfide oxidoreductase. At this stage, however, it is not clear which of the properties of hQSOX1b and hPDI are needed to correctly fold mFIZZ1 and mFIZZ1′ into a biological active protein. In vivo, the functional role of hQSOX1b in the folding of mFIZZ1 protein is highly unlikely due to different cellular locations.The chaperone activity of hQSOX1b might prevent incorrect disulfide formation or might even prevent post-translational over-oxidation. As an oxidase, hQSOX1b needs to work in concert with the isomerase hPDI. To our knowledge, this is the first time that chaperone activity has been described for hQSOX1b. Perhaps not a complete surprise as this quiescin sulfhydryl oxidase consists N-terminal Trx-domains like in PDI. For PDI, molecular chaperone activity has been reported for the folding of procollagen E. coli might also be explored. This method is certainly cheaper and higher yields might be obtained, but here, the endotoxin levels are a drawback. When we tested the endotoxin level on purified expressed mFIZZ1′ in the limulus amebocyte lysate (LAL) assay, the same values as for PBS (negative control) were obtained (data not shown). So, the use of a bacteria-free expression system also overcomes the potential effects of LPS and other endotoxin contaminants, which is certainly an advantage for follow-up animal and/or cell culture testing.The final total protein yield of purified mFIZZ1 varies from 200 to 300 µg, which is relatively low due to the short translational life span of the wheat germ extract compared to cell-based expression 2O2, and as a consequence oxidative stress, quiescin sulfhydryl oxidases are the most competent catalysts known for the de novo insertion of protein disulfide bonds in the endoplasmic reticulum et al.E. coli with the help of sulfhydryl oxidase/disulfide isomerase Erv1p. They showed how Erv1p is capable of introducing multiple disulfide bonds in a fragment of tissue plasminogen activator (vtPA) and the homodimeric resistin (mFIZZ3). We use the open cell free expression system. Our wheat germ cell free expression method is simple and effective for the in vitro production of soluble and active recombinant eukaryotic proteins that have to form multiple disulfides. Moreover, an open system makes it easier to screen for the correct folding partners as the addition of multiple oxidases and/or isomerases becomes in vision.We would like to emphasize that with the addition of quiescin sulfhydryl oxidase hQSOX1b we have introduced a new method for the folding of disulfide rich eukaryotic proteins. The sulfhydryl oxidase hQSOX1b does not require any additional partners to introduce disulfides into proteins. Although, the reaction with substrate proteins results in the formation of HMGSSHHHHHHSSGLVPRGSHM-mFIZZ1. The coding sequence of mFIZZ1 was amplified by PCR and introduced in pET14b restricted with NdeI and BamHI. The construct was sequenced at the VIB Genetic Service Facility (GSF). For periplasmic expression mFIZZ1 was fused to the PeIB signal sequence. Plasmid DNA of mFIZZ1 was transformed into E. coli SHuffle™ T7 Express (BioLabs), Origami™ DE3 and BL21 DE3 (Novagen), grown in LB medium supplemented with 25 µg/ml ampicillin, induced at a cell density (OD600 nm) of 0.7 with 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG), and cultured for 6 h at 37°C except for SHuffle™ T7 cells, the expression done at 30°C. Harvested cells were resuspended in 50 mM potassium phosphate, pH 7.5, 300 mM NaCl, 0.1 mg/ml lysozyme, 0.1 mg/ml AEBSF, 0.1 µg/ml leupeptine. Cells were broken by sonication at 4°C and centrifuged for 30 min at 15,000 rpm. For identification, protein fractions of total (5 µl), supernatant (7.5 µl) and pellet (7.5 µl) were analyzed on non-reducing 15% SDS-PAGE and immunoblot using anti-His monoclonal antibody .The mFIZZ1 gene without signal peptide  was cloned into the pET-14b vector (Novagen) with an N-terminal His-tag MGHHHHHHLE-mFIZZ1. This plasmid vector is specially designed for the wheat-germ cell-free expression system ). The hQSOX1b  without signal peptide and hPDI  without signal peptide genes were cloned with a GST-tag at the N-terminal position into the pEU-GST-MCS vector. The coding sequence of hQSOX1b and hPDI were amplified by PCR and introduced into the pEU-GST-MCS vector digested with BamHI and SmaI, or the XhoI and SmaI, respectively.mFIZZ1 (D24–S111) and mFIZZ1′ were cloned into the pEU-vector  with an N-terminal His-tag All constructs were sequenced at the VIB Genetic Service Facility (GSF).Plasmid DNA of mFIZZ1, mFIZZ1′, hPDI and hQSOX1b (2 µg) was transcribed using SP6 RNA polymerase, 25 mM NTP mix, RNase inhibitor and 5× transcription buffer  for 6 h at 37°C. The mRNA was cooled down to avoid degradation, and checked on 1% agarose gel. For translation, 10 µl of mRNA was mixed with the same amount of the wheat germ extract WEPRO 7240  and 0.1 mg of creatine kinase to make the bottom layer, and incubated with 206 µl of 1× SUB-A MIX SGC (upper layer) at 15°C for 20 h without shaking in a 6-well plate  in a Thermomixer . The reaction mixture was centrifuged  for 30 min at 4°C. For identification, protein fractions, total (5 µl), soluble (7.5 µl) and pellet (7.5 µl) of the expressed proteins were visualized on immunoblot using as primary antibody anti-His or anti-GST antibody  and as secondary anti mouse polyclonal antiserum . The same samples were ran on a non-reducing 15% SDS-PAGE followed by Coomassie Brilliant Blue staining.http://www.labimage.com). The experiments were repeated three times for reproducibility.pEU GST-tag hQSOX1b, GST-tag hPDI and His-tag mFIZZ1 or mFIZZ1′ (2 µg each) were separately transcribed using SP6 RNA polymerase, 25 mM NTP mix, RNase inhibitor and 5× transcription buffer. The mRNA of respectively mFIZZ1, mFIZZ1′, hQSOX1b and hPDI were translated for 10 min using the wheat germ extract WEPRO 7240 at 26°C. mRNA of mFIZZ1 or mFIZZ1′ (10 µl each) was then mixed with the same amount of mRNA from hQSOX1b, hPDI, and hQSOX1b + hPDI and incubated with 206 µl of the SUB-A mixture for each reaction at 15°C for 20 h without shaking in a 96-well plate. After the incubation, the reaction mixture was centrifuged at 15,000 rpm for 30 min at 4°C. The protein concentration of the soluble and pellet fractions was determined using a Bradford assay For up-scaling, plasmid DNA of His-tag mFIZZ1 and mFIZZ1′ (25 µg) and (25 µg) GST-tag hQSOX1b were transcribed separately using SP6 RNA polymerase, 25 mM NTP mix, RNase inhibitor and 5× transcription buffer for 6 h at 37°C. For translation, 250 µl of mRNA was mixed with the same amount of wheat germ extract (WEPRO 7240), and with 1 mg of creatine kinase to make the bottom layer. 5.5 ml of 1× SUB-A MIX included a mixture of amino acids were used to make the upper layer. Translation was performed at 15°C for 20 h without shaking in a 6-well plate using a Thermomixer. The expressed proteins were detected by immunoblotting using anti-His antibody  and on CBB-stained 15% SDS-PAGE prior to purification.For co-expression, mRNA from mFIZZ1 or mFIZZ1′ and hQSOX1b were translated for 10 min at 26°C using wheat germ extract WEPRO 7240H. After 10 min incubation, mRNA of mFIZZ1 or mFIZZ1′ were mixed with the same amount of mRNA from hQSOX1b and translated at 15°C for 20 h without shaking.−1 cm−1 at 280 nm. Protein aliquots were stored at −20°C.For purification, 2 batches (mFIZZ1 or mFIZZ1′) of 6 ml reaction (with and without hQSOX1b) were centrifuged at 15,000 rpm for 15 min at 4°C. The supernatant was separately loaded on 1 ml His-trap Ni-NTA resin equilibrated with 50 mM potassium phosphate buffer solution pH 7.5, 150 mM NaCl containing 10 mM imidazole. The column was washed with 5 column volumes and, the protein was eluted with a linear imidazole gradient from 50 to 500 mM in the same buffer solution. The purity of the elution peak fractions was evaluated on 15% SDS-PAGE under reducing and non- reducing conditions. Pure fractions were collected and dialyzed against PBS for 4 h at 4°C with two buffer changes. Protein concentrations were spectrophotometrically determined with a molar extinction of 18,740 Mhttp://wolfson.huji.ac.il/purification/Protocols/PAGE_Basic.html). Briefly, the samples of mFIZZ1 (pI 4.81) and mFIZZ1′ (pI 5.18) expressed with and without hQSOX1b were mixed on ice with sample buffer solution containing 100 mM Tris/HCl, pH 6.8, bromophenol blue, and glycerol. For the reduced conditions, the samples were first incubated for 30 min with 20 mM DTT. Samples were loaded on a polyacrylamide native gel (5% stacking gel (pH 6.8) and 15% resolving gel (pH 8.9)). The running buffer solution contained 50 mM Tris/HCl, pH 8.9, and 380 mM glycine. As marker the PageRuler™ pre-stained Protein Ladder (Fermentas) is used, which contains SDS. After a 5 h run at 4°C, gels were CBB stained.For the basic-native gel conditions, a method for acidic and neutral proteins was used (Corynebacterium glutamicum mycoredoxin-1 (Cg_Mrx1) Escherichia coli DsbG (Ec_DsbG) Samples of mFIZZ1 (10 µM), mFIZZ1′ (5.3 µM), mFIZZ1 + hQSOX1b (20 µM), and mFIZZ1′ + hQSOX1b (9.2 µM) were were incubated with the cross-linker mixture of 1 mM EDC and 1 mM NHS  for 3 h at room temperature in PBS, pH 7.4 buffer solution. The reaction was stopped by addition of SDS-PAGE sample buffer. After boiling for 10 minutes, the mixture was evaluated on 15% SDS-PAGE under non-reducing conditions. As negative control, monomeric http://dichroweb.cryst.bbk.ac.uk) CD-spectra were recorded at 25°C using a spectropolarimeter . Protein was dissolved in water at 0.2 mg/ml. CD-spectra were recorded at a scan speed of 50 nm/min, 1 nm bandwidth and with a response time of 1 s. Spectra were recorded between in 0.01 cm path length quartz cuvette from 190 to 260 nm. The mFIZZ1 protein band was excised from the gel and the tryptic peptides were analyzed by tandem mass spectrometry (MS/MS) as described tm, Luminos) The number of free thiols in samples was determined using a Thiostar assay . Protein purity was confirmed by SDS-PAGE and HPLC analyses. Cells were cultured at the concentration of 2×105 cells/well in 96-well round-bottom plates. Th2-permissive conditions were: αCD3/αCD28 , rIL-4 , anti-IL-12  and anti-IFNγ . Four days later, supernatants were recovered for quantification of IL-5 and IL-13 by standard sandwich ELISA protocols (antibodies from eBioscience). Single cell suspensions from C57BL/6 mouse spleens were cultured under Th2 permissive conditions with the addition of PBS (control), bacterial recombinant mFIZZ1/RELMα (Peprotech), or mFIZZ1′ expressed with or without hQSOX1b at concentrations indicated. Peprotech RELMα was generated in E. coli RNase I was added. The absorbance change was followed in function of time at 659 nm, the wavelength with a maximum spectral difference between methylene blue intercalated RNA with and without RNase I.The RNA hydrolysis activity was performed as described For the chaperone activity, unfolded RNase I was prepared by mixing guanidine hydrochloride (GuHCl), 50 mM Tris/HCl pH 8.0 and RNase I wild type to obtain the final concentration of 1.2 µM RNase I, 6.7 M GuHCl, 56 mM Tris/HCl pH 8.0. After 2 h incubation at 25°C, the mixture was dialyzed for 2 h against 0.6 M GuHCl, 50 mM Tris pH 8.7, 50 mM NaCl. Unfolded RNase I (500 nM) was pre-incubated with varying concentrations of (5 µM–50 nM) hQSOX1b, 5 µM DsbA or 5 µM DsbC in 50 mM Hepes/NaOH, pH 7.4, 150 mM NaCl for 3 min at 15°C before RNase activity was measured.E. coli scrambled RNase I (scRNase I) For the isomerase activity, For the oxidase assay, reduced and unfolded RNase I (ruRNase I)


Trichoderma reesei is the preferred organism for producing industrial cellulases. However, a more efficient heterologous expression system for enzymes from different organism is needed to further improve its cellulase mixture. The strong cbh1 promoter of T. reesei is frequently used in heterologous expression, however, the carbon catabolite repressor CREI may reduce its strength by binding to the cbh1 promoter at several binding sites. Another crucial point to enhance the production of heterologous enzymes is the stability of recombinant mRNA and the prevention of protein degradation within the endoplasmic reticulum, especially for the bacteria originated enzymes.cbh1 promoter were replaced with the binding sites of transcription activator ACEII and the HAP2/3/5 complex to improve the promoter efficiency. To further improve heterologous expression efficiency of bacterial genes within T. reesei, a flexible polyglycine linker and a rigid α-helix linker were tested in the construction of fusion genes between cbh1 from T. reesei and e1, encoding an endoglucanase from Acidothermus cellulolyticus.In this study, the CREI binding sites within the cbh1 and e1 were successfully expressed in T. reesei under the control of promoter pcbh1m2. The higher enzyme activities and thermostability of the fusion protein with rigid linker indicated that the rigid linker might be more suitable for the heterologous expression system in T. reesei. Compared to the parent strain RC30-8, the FPase and CMCase activities of the secreted enzyme mixture from the corresponding transformant R1 with the rigid linker increased by 39% and 30% at 60°C, respectively, and the reduced sugar concentration in the hydrolysate of pretreated corn stover (PCS) was dramatically increased by 40% at 55°C and 169% at 60°C when its enzyme mixture was used in the hydrolysis.The modified promoter resulted in an increased expression level of the green fluorescent protein reporter by 5.5-fold in inducing culture medium and 7.4-fold in repressing culture medium. The fusion genes of T. reesei.This study shows that optimizations of the promoter and linker for hybrid genes can dramatically improve the efficiency of heterologous expression of cellulase genes in  Trichoderma reesei (teleomorph Hypocrea jecorina)  and incubated at 28°C and 200 rpm. The minimal medium contained 0.4% KH2PO4, 0.28% (NH4)2SO4, 0.06% MgSO4·7H2O, 0.05% CaCl2, 0.06% urea, 0.3% tryptone, 0.1% Tween 80, 0.5% CaCO3, 0.001% FeSO4·7H2O, 0.00032% MnSO4·H2O, 0.00028% ZnSO4·7H2O, 0.0004% CoCl2 [Transformants were selected using hygromycin B (10 μg/ml) and cefotaxime (300 μM) on potato dextrose agar (PDA). Each positive transformant was used to create monoconidial cultures for genetic stability and confirmed single-copy integration of 4% CoCl2  and was ®-24  instrument in combination with TRIzol® Reagent  was successfully used for total RNA extraction. Reverse transcription was carried out using the PrimeScript® RT reagent Kit . Relative expression levels of egfp were calculated in comparison with the expression of act encoding actin by RT-PCR with primers GFPrtF, GFPrtR, actF and actR.After being cultured for 2 days, mycelia of transformants were collected for fluorescence observation or for total RNA extraction, after rinsing with sterilized water three times. The FastPrepg for 10 min. The fusion proteins TCE1-FLE or TCE1-RIG in the collected culture filtrate were purified using Novagen Ni-NTA His•Bind® Resin  with step gradient elution. The 40 mM imidazole washouts were collected, and the buffer was exchanged with 20 mM pH 7.4 NaH2PO4 using a Vivaspin™ ultrafilter  to remove the imidazole. Fusion protein production was examined by SDS-PAGE and Western blotting using an anti-His antibody . Proteins were quantified using the DC protein assay kit , according to the manufacturer's instructions.After being cultured for 7 days, the culture filtrate of transformants was collected by a centrifugation at 4°C and 8,000 × T. reesei RC30-8 or its transformants was collected by a centrifugation at 4°C and 8,000 × g for 10 min after being cultured for 7 days) or the purified TCE1-FLE and TCE1-RIG were used to examine substrate specificity and to characterize its properties. CMCase activity was assayed by measuring the amount of reducing sugar released from CMC  using the DNS method [540 using a Varioskan Flash microplate reader .The crude secreted enzymes of fungal strains  containing 1 mg/ml D-glucono-1,5-σ-lactone was incubated with 20 μl of diluted culture supernatant at 50°C for 30 min. Then, 100 μl of each sample was mixed with 100 μl 2% sodium carbonate, and photometric assays were carried out at OD400.Cellobiohydrolase activity was measured as reported by Deshpande  with sliT. reesei RC30-8 or its transformants or the purified fusion proteins on FPase activity were measured using a modified IUPAC method [pNP released per minute per milligram protein or per milliliter fermented culture. Student's t-test was performed with Excel 2007 , employing a two-tailed test.The specific activities of the crude enzymes secreted by C method . The assThe thermostability of the purified proteins or the secreted crude enzymes was assayed by the similar enzyme activity assay methods mentioned above. Samples were exposed to thermal stress in water baths at 60°C, 70°C and 85°C for up to 24 h. Three aliquots were pipetted out for enzyme activity assays at intervals of 15 min-12 h during the exposure.T. reesei RC30-8 or its transformants as crude enzyme preparations, 3% PCS (30 mg) and 30 μl different enzyme preparations were incubated in 50 mM saline sodium citrate (pH 5.0) at 50°C, 55°C, 60°C, 70°C and 75°C for 24 h, and the total volume of the saccharification mixture was 1 ml. The hydrolysates were then centrifuged at 4°C, 12000 × g for 20 min and the concentrations of glucose and cellooligosaccharides in the supernatant were measured by high-performance liquid chromatography , using a refractive index detector . The hydrolysates were filtered using a 0.22 μm pore size membrane  and then separated on a Shodex Asahipak NH2P-50 4E column  with isocratic elution  at 1 ml/min.In the hydrolysis of dilute sulfuric acid pretreated corn stover , using tThe authors declare that they have no competing interests.T. reesei. JB and RV commented on and polished the manuscript. JZ carried out enzyme assays. LC and LM participated in transformation. ZZ and CW supervised the study and revised the manuscript. All authors read and approved the manuscript.GZ performed main experiments and data analysis, and drafted the manuscript. SS performed the modification of promoters. YJ participated in the expression of e1 directly in


Human papillomavirus (HPV) type 16 is associated with oropharyngeal carcinomas (OPC). Antibodies (Abs) to HPV16 E6 and E7 oncoproteins have been detected in patient sera; however, Abs to other early HPV-derived proteins have not been well explored.N=40), partners of patients with HPV16+ OPC (N=11), and healthy controls (N=50).Antibodies to the HPV16 proteome were quantified using a novel multiplexed bead assay, using C-terminal GST-fusion proteins captured onto Luminex beads. Sera were obtained from untreated patients with OPC . In a validation cohort, HPV16 E1, E2, and E7 antibody levels were significantly elevated compared with healthy control samples (P⩽0.02) and partners of OPC patients (P⩽0.01).Oropharyngeal carcinomas patients with known virus-like capsid particle+ Abs had elevated serum Abs to HPV16 E1, E2, E4, E6, and E7, and L1 antibody levels, but not E5. The ratios of specific median fluorescence intensity to p21-GST compared with controls were E1: 50.7 Patients with HPV16+ OPC have detectable Abs to E1, E2, and E7 proteins, which are potential biomarkers for HPV-associated OPC. Approximately 20 million Americans are currently infected with human papillomavirus (HPV), and another 6.2 million people become newly infected each year  arrays  or HPV18 (N=10) Abs defined by the competitive Luminex immunoassay  developed by Merck  (N=20) were obtained from Brown University and were age (±5 years), residence, and gender matched to cases and processed simultaneously. Serum samples were collected using a standardised sample collection protocol and stored at −80 °C until use.Human papillomavirus serology was performed using retrospectively identified sera from 20 head and neck cancer patients (‘training set’) collected at Brown University that were known to be positive for HPV16 (N=17) and Johns Hopkins University  as well as healthy anonymous blood donors (N=30) and partners of OPC patients (N=11). Of these samples, 55 were sera and 16 were plasma (6 of these were obtained at the time of leukopheresis).To confirm the findings from the training set, serum from 30 pre-therapy OPC cases and 30 controls, collected as part of a larger research study, were evaluated. This included blood from OPC patients at Dana-Farber Cancer Institute  products were each captured onto 2000 microspheres at 40 microspheres per μl in PBS-1% BSA. Protein-bound microspheres were pooled together, then re-aliquoted to a 96-well filter plate . Microspheres were blocked with 10% each of normal sera from mouse, rabbit, goat, and rat; 0.5% polyvinyl alcohol ; 0.8% polyvinylpyrrolidone (Sigma-Aldrich); and 2.5% Chemicon (Millipore) in PBS-1% BSA. Test sera were diluted 1 : 80 in blocking buffer and incubated with the washed beads overnight at 4 °C while shaking. Biotin-conjugated goat anti-human IgG antibody  and streptavidin-R-PE  were added. To control for non-specific and GST-specific autoantibody background, the ratio of median fluorescence intensity (MFI) for individual HPV-specific Abs to the MFI for the control p21-GST antigen was measured. To determine protein expression, GST-protein tags were detected with anti-GST monoclonal Ab  and PE-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories).SeroMAP carboxylated microspheres  from regions #35–44 were coupled at a ratio of 5 Taq polymerase , 10 mmol l–1 Tris–HCL, pH 9.0, 50 mmol l–1 KCl,4.6 mmol l–1 MgCl2, 0.02 mmol l–1 of each deoxyribonucleotide triphosphate, 11 pmol of each primer, and 100–500 ng of DNA. The reactions were cycled at 95 °C for 2 min, followed by 20 s each of 95 °C then 55 °C and 30 s at 72 °C. The final cycle was 72 °C for 5 min and held at 15 °C until analysed. As a control for DNA quality, the DNA was separately amplified with primers to the β-globin gene . The DNA samples that were positive for HPV were genotyped by restriction endonuclease fragment analysis. The HPV PCR products were digested in three separate reactions using PstI, Rsa I, and Hae III. All PCR products were resolved on 1 mm, 5% acrylamide gels, stained with ethidium bromide, and visualised by transillumination. The pattern of restriction fragment-length polymorphism of each sample was compared with the Access Genetics database to determine the HPV genotype . Polymerase chain reaction was performed using a procedure developed by Access Genetics , which amplifies the HPV L1 domain with degenerate forward and reverse primers (N=20) and the validation control sera (N=30) were used. An MFI ratio > (the average + 3 s.d.) of all control samples was designated positive. These levels were E1: 5.4, NE2: 8.5, CE2: 6.8, E4: 2.3, E5: 4.2, E6: 9.0, E7: 6.6, L1: 9.5, and L2: 8.0. To determine the variability of serum detection for each HPV antigen, test serum from a single OPC patient (tumour HPV16+ confirmed by PCR) was assayed in duplicate on four separate days for each of the HPV16 antigens. Intra-assay and inter-assay variability was determined by calculating the coefficient of variation  × 100). Heat maps and cluster analysis were done using MultiExperiment Viewer software . For all subsequent studies, the MFI ratio of specific HPV Ab to p21-GST was used to control for non-specific protein binding.IgG Abs specific for HPV-derived proteins were detected in two representative serially diluted OPC patient sera . For botWe initially selected a retrospective training set of sera from patients known to have Abs to HPV16 VLP or HPV18 VLP by the highly sensitive Merck cLIA assay, which were predicted to have higher levels of Abs to HPV-derived early genes. The characteristics of patients with HPV-associated OPC and age- and gender-matched controls (training set) are shown in P⩽0.05)  ; Table 2P=0.914). No correlation between HPV16L1 bead array signal intensities and VLP titre were observed (R=0.638), likely related to structural differences in epitope display, which has been observed for L1-specific epitopes   ; Table 4vs 1 out of 30 controls), NE2 (25 out of 30 (83%) of cases vs 1 out of 30 controls), CE2 (24 out of 30 (80%) of cases vs 1 out of 30 controls), and HPV16E7 (19 out of 30 (63%) of cases vs 0 out of 30 controls), P<0.001. Compared with E1, E2, and E7 Abs, fewer patients had E4 Abs (13 out of 30 (43%) vs 0 out of 30 controls) or E6 Abs (15 out of 30 (50%) vs 1 out of 30 controls). All 16 patients with known HPV16+ tumours by PCR had detectable E1, E2, or E7 Abs. Four of the OPC patients had HPV16-negative tumours by PCR; of these, two had detectable Abs to both E1 and E7. This may reflect cross-reactivity with other HPV types, limitations of the HPV PCR assay, or represent HPV16 infection at other sites. As this cohort included a mix of sera and plasma, a direct comparison of the subset of serum cases and controls was performed, with the specificity of detection of HPV16 E1, E2, E6, and E7 Abs maintained (P<0.002).The HPV Abs most strongly associated with OPC were HPV16E1 (22 out of 30 (73%) of cases Given the high proportion of adults (>70%) having ever been infected with high-risk (HR) HPV in most populations , the HPV16 antibody responses in the validation set detected by bead array ELISA were grouped by unsupervised hierarchical clustering analysis . DistincIt is estimated that 75–80% of sexually active persons in the United States will be infected with HR HPV in their lifetime , 16E6 and 16E7-specific IgG were detected in 7% of cases with a relative risk (odds ratio) of 2.7, found 0.5–5 years before diagnosis (In cervical cancer, Abs to 16E6 and 16E7 develop late in the course of disease and have been shown to correlate with disease response (The previously unreported E1- and E2-specific Abs in our cohort is striking because of both the high titre and high frequency in OPC patient sera, which improves the sensitivity of detection from 70% to 100% of HPV16+ OPC when combined with E6 and/or E7 Abs. Early studies demonstrated low immunoreactivity to an E1-derived peptide in cervical cancer sera . As a reHuman papillomavirus antibody signatures may provide a rapid, convenient and inexpensive screening method for assessing the HPV status of suspected or known malignancy. Human papillomavirus+ OPC is a strong, independent factor for improved prognosis (