E. coli cells. Recently, improvements in acetate tolerance have been achieved through the use of genetic strategies and medium supplementation with certain amino acids and pyrimidines. The aim of our study was to evaluate an alternative to improve the acetate tolerance of E. coli BL21 (DE3), a popular strain used to express recombinant proteins. In this work we reported the cultivation of BL21 (DE3) in complex media containing acetate at high concentrations. In the presence of 300 mM acetate, compared with pH 6.5, pH 7.5 improved cell growth by approximately 71%, reduced intracellular acetate by approximately 50%, and restored the expression of glutathione S-transferase (GST), green fluorescent protein (GFP) and cytochrome P450 monooxygenase (CYP). Further experiments showed that alkaline pHs up to 8.5 had little inhibition in the expression of GST, GFP and CYP. In addition, the detrimental effect of acetate on the reduction of 3--2,5-diphenyl tetrazolium bromide (MTT) by the cell membrane, an index of cellular metabolic capacity, was substantially alleviated by a shift to alkaline pH values of 7.5–8.0. Thus, we suggest an approach of cultivating E. coli BL21 (DE3) at pH 8.0±0.5 to minimize the effects caused by acetate stress. The proposed strategy of an alkaline pH shift is a simple approach to solving similar bioprocessing problems in the production of biofuels and biochemicals from sugars.Excess acetate has long been an issue for the production of recombinant proteins in  At 25°C the dissociation constant of acetic acid (pKa) is 4.75. Based on the Henderson-Hasselbalch equation, at pH 7.0 HAc is estimated to be 0.56 mM per 100 mM total acetate (Ac– + HAc). As is well known, the HAc molecule is able to diffuse freely across the E. coli cell membrane and dissociates into Ac– and H+ intracellularly E. coli, ActP, with an affinity Km for acetate of 5.4 µM, suggesting that it is an acetate scavenger that can enhance cellular survival under conditions of carbon source starvation The use of glucose or glycerol as the carbon source in a complex medium may result in acetate accumulation at high levels, often up to 50–100 mM, particularly in high-density fermentations E. coli cells produce acetate when they have surpassed a threshold value of the specific rate of glucose consumption and that only under glucose limitation is the specific growth rate directly related to acetate production ptsHI operon in wild-type E. coli strain GJT001 and found that in 2×LB broth with 2% glucose the mutant TC110 was able to grow quickly and produced much less acetate (9.1±6.6 mM in TC110 vs. 90.4±1.6 mM in GJT001) E. coli strain VH32 lacking PTS with a modified glucose transport system and found that strain VH32 cultured at glucose concentrations of up to 100 g/L produced a maximum concentration of only 2 g/L (33 mM) acetate, while its parental strain W3110 accumulated a maximum of 13.6 g/L (227 mM) acetate E. coli  E. coli was dependent on the specific growth rate and decreased by 12-fold around the overflow switch growth rate of 0.27±0.02 h−1acs-yjcH-actP operon) repression and acetyl-CoA synthetase (Acs) down-regulation, which results in decreased assimilation of acetate produced by phosphotransacetylase (Pta), and disruption of the Pta-Acs node. The above studies shed light on a future when we can use a PTS-deficient and Acs-overexpressing E. coli strain in a simple batch cultivation with no need to restrict the glucose concentration to avoid acetate accumulation.For decades many researchers have focused on strategies that reduce acetate accumulation. Briefly, the approaches developed involve process optimization and host organism modification E. coli tolerance towards acetate because of its promising applications in biofuel and chemical production from lignocellulose feedstocks E. coli K-12 caused by acetate at up to 8 g/L E. coli K-12 caused by acetate in a defined medium based upon citrate-phosphate buffer at pH 6.0 and concluded that acetate caused the depletion of the intracellular methionine pool and the concomitant accumulation of the intermediate homocysteine, which was found to inhibit the growth of E. coliE. coli strains improved the cell growth in M9 glucose medium (pH 6.0) supplemented with 20 mM sodium acetate E. coli DH5α, which was constructed by knocking out its original crp gene, and isolated a mutant A2 with the greatest tolerance to acetate vs. 0.016 h−1) when exposed to the stress of 15 g/L (183 mM) sodium acetate (NaAc) in M9 minimal medium On the other hand, a few studies have turned to improving E. coli BL21 (DE3) is a popular strain used for recombinant protein expression and its growth in complex media containing glucose or glycerol tends to result in acetate accumulation at different levels, depending on the specific strategy for process control. It is well documented that recombinant protein expression in E. coli cells is decreased by acetate accumulation E. coli BL21 (DE3) was further confirmed, and specific conditions for the cultivation of and protein expression in this strain were investigated in complex media at high acetate concentrations (up to 300 mM). A comparison of cell growth at pH 6.5 and pH 7.5 and intracellular acetate accumulation and recombinant protein expression of GST, CYP and GFP under acetate stress suggested that an alkaline pH shift would be an alternate approach for improving the tolerance to acetate. Further experiments showed that alkaline pH up to 8.5 supported bacterial growth well and inhibited the expressions of GST, CYP and GFP very little. In addition, we also studied the effects of up to 300 mM NaCl and NaAc on the MTT-reducing activity of the E. coli cell membrane to show the differences in inhibition of cell growth and protein production caused by salt and acetate stress. The alleviation of the detrimental effects of acetate on the performance of this strain by a shift to alkaline pH (7.5–8.5) suggests a simple approach to solving such bioprocessing problems.In this study, the acetate tolerance of E. coli BL21 (DE3) (F–Bdcm ompT hsdS (rB– mB–) gal) was used in all experiments. The strains E. coli BL21 (DE3), Origami (DE3) and DH5α and the plasmids pET-28a (+), pET-32a (+) and pRSET-B were purchased from Novagen, USA. Gene gst was from pET-4T-1  and expressed glutathione S-transferase  using pET-32a(+). An enhanced green fluorescent protein  and a cytochrome P450 monooxygenase  were expressed using pET-28a(+). TAT-survivin (T34A) (12 kDa), a mutant of tumor suppressor protein, was expressed using pRSET-B 2HPO4•12H2O and 1 g/L KH2PO4, and its pH was set at 7.0±0.2 before sterilization. Media 2×BYT and 4×BYT contained two- and four-fold BYT ingredients. Other stock solutions, including sterile water, 250 g/L NaOH, 25% phosphoric acid , 600 mM NaAc, 600 mM NaCl, 500 g/L glycerol and 500 g/L glucose, were autoclaved seperately. BYT-glycerol medium contained 10 g/L glycerol, and BYT-glucose medium contained 10 g/L glucose. YTA medium contained 10 g/L yeast extract , 10 g/L tryptone , 33 g/L (NH4)2SO4, 4.5 g/L Na2HPO4•12H2O and 0.5 g/L KH2PO4. All media and stock solutions were autoclaved at 121°C for 15 min.Buffered YT medium (BYT) contained 5 g/L yeast extract , 10 g/L tryptone , 9 g/L NaE. coli cells harboring various plasmids were grown for 10 h before inoculation. To grow E. coli strains harboring plasmids, 100 mg/L ampicillin was used in the case of pET-32a(+) and pRSET-B, and 50 mg/L kanamycin in the case of pET-28a(+). To express recombinant proteins, 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was used. Solutions of 250 g/L NaOH and 25% (v/v) phosphoric acid were used to adjust pH in situ during cultivation. To reduce the effect of sampling on cell growth, all shake flask experiments were conducted on a 30 mm orbital shaker at 200 rpm  in an incubation room at 37°C.Other media in our study were prepared using the above stock solutions and incubated at 37°C for at least 1 h before inoculation. BYT-glycerol medium (pH 7.0) was used for preculture preparation, unless otherwise specified. All precultures of E. coli BL21 (DE3) harboring pRSET-B-TAT-survivin (T34A) was inoculated into 50 mL medium in a 250-mL Erlenmeyer flask. Baterial cells were cultivated for 4 h and induced by 1 mM IPTG for 4 h. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Laemmli using a separating gel of 12.5% acrylamide and approximately 15 µg of cellular proteins per lane In the case of shake flask cultivation, TAT-survivin (T34A) expression was carried out in YTA-glucose medium containing 0, 50, 100, 200 and 300 mM NaAc at pH 7.5. Glucose was used to lower auto-induction In the case of fermentor cultivation, the inoculum volume was 20 mL of preculture per liter of medium. The fed-batch cultivations were carried out in a 4-L BIOSTAT B fermentor  and a 30-L fermentor  with initial volumes of 3.7 L and 25 L, respectively. Medium glucose was monitored offline using a GOD-POD kit  according to the manufacturer's instructions and was maintained at 10±5 g/L by using a glucose stock solution of 500 g/L. The temperature was maintained at 37°C, and the pH was controlled at 7.5±0.2 using 25% NaOH solution. The dissolved oxygen (DO) was monitored using a polarographic oxygen electrode and the airflow rate was kept at 2.0 L/min per liter broth.E. coli BL21 (DE3) preculture grown for 10 h was inoculated into 20 mL of BYT-glycerol medium in a 250-mL Erlenmeyer flask at 37°C. Samples were taken in situ every 30 min to monitor cell growth by measurement of the optical density (OD) at 600 nm (OD600). To avoid oxygen limitation on cell growth, the initial OD600 was set at 0.15∼0.2. The specific growth rate (h−1) was calculated according to the formula X2 = X1·eμΔt using OD600 values from the first 3 h of cultivation. Experiments were conducted in duplicate for each NaAc and NaCl gradient at each pH.The experiments were conducted in the BYT-glycerol medium containing 0, 50, 100, 200 and 300 mM NaAc or NaCl at pH 6.5 and pH 7.5 To start the cultivation, g for 5 min, washed with deionized water and dried at 115°C for about 24 h to constant weight. OD600 of 1.0 corresponded to approximately 0.385 g of DCW per liter culture of E. coli.To determine the dry cell weight (DCW), 10 mL of culture were collected by centrifugation at 4000 E. coli BL21 (DE3) cells were first grown for 10 h, inoculated into 100 mL of BYT-glycerol medium (pH 7.0) in a 500-mL Erlenmeyer flask, cultivated to an OD600 of approximately 2.5 and collected by centrifugation at 8000 g for 5 min at room temperature. The cells were then resuspended thoroughly in 50-mL centrifuge tubes by vigorous vortexing in the BYT-glycerol medium (pH 6.5 and 7.5). To prepare the acetate-stressed medium, 4×BYT solution was adjusted to pH 6.5 and pH 7.5 and mixed with 600 mM NaAc solution of the same pH, 500 g/L glycerol solution, and the resuspended cells in a 250-mL Erlenmeyer flask. Sterile water was then added to adjust the final volume to 20 mL. All stock solutions and flasks were incubated at 37°C for at least 1 h in advance. In order to obtain a sufficient amount of cells for the determination of intracellular acetate concentration, the E. coli cells were adjusted to an OD600 of 5.0 and 1.0-mL samples were taken at 2, 10 and 40 min.The experiments were performed in the BYT- glycerol medium containing 50, 100, 200 and 300 mM NaAc at pH 6.5 and pH 7.5. E. coli cells, 1.0 mL of sample was centrifuged in a 1.5-mL tube at 10,000 g for 25 sec, the supernatant medium was pipetted away, the cell pellet together with the tube bottom was separated by cutting the tube, and the residual extracellular liquid medium in the pellet was then removed by absorbance using tissue paper. Using the above sampling protocol, the extracellular medium remaining was estimated to be 2.5±0.5 µL determined by trypan blue dye exclusion To collect g for 10 min and the supernatants were further removed by 0.22-µm filtration. Potassium hydroxide (KOH) was used as the eluent  at a flowrate of 1.0 mL/min and the column temperature was set at 30°C.Acetate determination was performed on an ion chromatography system (Dionex ICS 1500) equipped with a guard column IonPac AG11-HC (4×50 mm) and an analytical column IonPac AS11-HC (4×250 mm). To dissolve the above cell extracts, 1000 µL of ultrapure water (18.2 MΩ•cm at 25°C) were added to the 5-mL centrifuge tubes, mixed by vortexing and allowed to stand for 10 min. Then, the samples were centrifuged at 5000 E. coli BL21 (DE3) cells were grown in 20 mL of BYT-glycerol medium at pH 6.5, 7.0, 7.5, 8.0 and 8.5 in 250-mL Erlenmeyer flasks at 37°C. Experiments were performed in duplicate. To avoid side-effects of sterilization at alkaline pH, BYT medium was autoclaved at pH 6.5 and then adjusted to set-up pH values. For recombinant protein expression, GST, CYP and GFP were expressed by E. coli BL21 (DE3) in the BYT-glycerol medium at pH 6.5, 7.5 and 8.5 under the same conditions. At inoculation, 100 mg/L of ampicillin was used in the case of pET-32a(+)-GST and 50 mg/L kanamycin was used in the case of pET-28a(+)-GFP and pET-28a(+)-CYP. Baterial cells were cultivated for 2 h and induced by 1 mM IPTG for 4 h. The medium pH decreased during the 6-h cultivation and was adjusted in situ using 20–100 µL of 25% NaOH solution.For bacterial growth, E. coli BL21 (DE3) in BYT-glycerol medium containing 300 mM NaAc at pH 6.5 and 7.5; 300 mM NaCl was used as a control. Other conditions were the same as in the experiments of recombinant protein expression at different pHs.Recombinant proteins GST, CYP and GFP were expressed by E. coli strains, BL21 (DE3), Origami (DE3) and DH5α were used in the experiment. BYT-glycerol medium (pH 7.0) was used for the preculture preparation of E. coli BL21 (DE3) and DH5α, and BYT-glycerol medium (pH 7.0) supplemented with 50 mg/L kanamycin and 50 mg/L tetracycline was used for the preculture preparation of E. coli Origami (DE3). Specific growth rates were determined at 20, 25, 30, 35 and 40°C in the BYT-glycerol medium containing 300 mM NaAc at pH 6.5 and pH 8.0, using 300 mM NaCl as a control. To start the cultivation, 500 µL of E. coli preculture grown for 10 h was inoculated into 20 mL of BYT-glycerol medium containing NaAc or NaCl in a 250-mL Erlenmeyer flask and pre-incubated for 30 min to allow temperature recovery. Samples were then taken in situ every 1 h to monitor cell growth. To avoid oxygen limitation on cell growth, OD600 values from the first 3 h of cultivation were used. Experiments were performed in duplicate for each temperature.Three E. coli cellular viability was determined according to the MTT method as previously described E. coli BL21 (DE3) cells were first grown for 10 h, then inoculated into 20 mL of BYT-glycerol medium (pH 7.0) in a 250-mL Erlenmeyer flask and cultivated for 4–5 h to the mid-exponential growth phase (to an OD600 of approximately 2.0). The cells were diluted to an OD600 of 0.100 using 2×BYT-glycerol solution  before MTT assay. Then, 100 µL of cell dilution were mixed with 100 µL of NaAc or NaCl solution of the same pH in 1.5-mL centrifuge tubes. To start MTT reduction, 20 µL of 5 g/L MTT solution were added to the above mixtures and the tubes were incubated at 37°C for 20 min. The complex of formazan crystals and cells was collected by centrifugation and dissolved in alkaline dimethyl sulfoxide (DMSO) 600 per mL culture.E. coli BL21 (DE3) cells could produce acetate of up to 250 mM even on a medium containing glycerol as the sole carbon source and that the accumulation of medium acetate was not responsible for poor rtPA expression in the scale-up from shake flasks to fermentors E. coli BL21 (DE3) was further examined on acetate of up to 300 mM, a concentration not reported in previous studies. 600 during cultivation, compared with the control without exogenous NaAc (−1 at 6–10 h to 0.01∼0.03 h−1 at 16–18 h (E. coli BL21 (DE3).Our previous study demonstrated that ous NaAc . In the ous NaAc , althougous NaAc . Bacteri 16–18 h . These rWe also observed that the cellular respiration activity declined during the induction . From 10E. coli cells on NaAc was determined at pH 6.5 and 7.5  on 50, 100, 200 and 300 mM NaAc, respectively. In the case of pH 7.5, it was 30, 50, 147 and 171 µM/(OD600 • mL). The intracellular acetate concentrations at pH 6.5 were approximately 2∼3-fold those at pH 7.5 over the 40-min incubation  cells were grown in BYT-glycerol medium at various pH values from 6.5 to 8.5  grown on 300 mM NaAc in BYT-glycerol medium  and DH5α. Under the stress of 300 mM NaAc, the specific growth rates of the three strains tested at pH 8.0 were remarkably higher than those at pH 6.5, especially at 30–40°C (E. coli Origami (DE3) at 30°C was approximately 0.52 h−1 at pH 8.0 but 0.17 h−1 at pH 6.5  and Origami (DE3) were estimated at 0.25∼0.31 h−1 (E. coli strains BL21 (DE3) and Origami (DE3) showed quite similar growth profiles under the conditions in our experiments  when the medium pH decreased from 7.5 to 6.5 (600 • mL) at pH 6.5. In contrast, for the medium contained 50–300 mM NaAc, the shift from pH 7.5 to 6.5 caused a sharp drop in electron reduction, by approximately 55–75%, depending on acetate concentrations (600 • mL) at pH 7.5 and 220±23 MRU/(OD600 • mL) at pH 6.5, a drop of 69%. Our results show that in the presence of acetate, a one-unit drop in pH from 7.5 to 6.5 causes a drop in bacterial MTT activity, although pH 7.5 and 6.5 are both suitable for cell growth in the BYT-glycerol medium without any acetate  cells, a popular strain currently used in recombinant protein expression.Accumulation of acetate has long been an issue for recombinant protein production by – always accompanies the molecule HAc and their relative proportions may change with adjustment of the medium pH. HAc can diffuse freely across cell membranes, resulting in the collapse of the transmembrane pH gradient, and dissociates intracellularly into Ac–, causing an Ac– accumulation if the intracellular pH is higher than the medium pH (E. coli BL21 (DE3) cells, an alkaline medium pH (7.5 to 8.5) reduces the concentration of toxic extracellular HAc molecules and thus reduces the intracellular Ac– accumulation, which may help to alleviate growth inhibition, increase cell viability and preserve the expression of recombinant proteins  and found that, in HKSII medium (pH 7.2) containing 5 g/L glucose, acetate addition resulted in inhibition of cell growth at concentrations from 6 g/L (100 mM), but a significant decrease in the specific production rate of MAE-hGH at concentrations from 2.4 g/L (40 mM) E. coli F-122 to express HIV582-β-galactosidase fusion protein in a modified M9 minimum medium (pH 7.0) supplemented with L-tryptophan, L-leucine, L-proline and vitamin B1 Although the adverse effects of acetate have been widely reported, the results of available studies on acetate tolerance in E. coli cell cultivation and recombinant protein expression is around 7.0, ranging from 6.5 to 7.5. Although pH 6.5 and pH 7.5 are both suitable for cell growth and protein expression in a medium without acetate, there may be a great difference in the presence of acetate. Our results showed that pH 7.5 significantly reduced the intracellular acetate concentration and improved the specific growth rate  and found that 100 mM acetate had little effect on the growth rate or proton motive force across the cell membrane –It is a long-held view that the undissociated form of the weak acid HAc is an uncoupler and is able to cause a collapse of the transmembrane ΔpH (the proton-motive force across the cytoplasmic membrane) E. coli cell growth, recombinant protein expression or cellular MTT-reducing activity  shows that an appropriate increase in extracellular pH drove the intracellular acetate out of cells, changed the intracellular toxicity of acetate into the extracellular osmotic pressure of Ac–, and thus, improved the resistance of cells to acetic acid (E. coli Origami (DE3) and DH5α  at pH not less than 7.5 to combat acetate stress. However, it also should be noted that sterilization of the complex medium at alkaline pH tends to cause cell growth inhibition (data not shown), and thus it is necessary to adjust the medium pH using NaOH solution on inoculation. In brief, the proposed strategy of an alkaline pH shift introduces an efficient paradigm for cultivating E. coli cells, which was shown to improve the expression of recombinant proteins and may help in the production of biofuels and biochemicals from lignocellulosic hydrolysates containing high concentrations of acetate.In conclusion, in this study we addressed the effects of the crucial factor of medium pH on the performance of


Taken together, all these aspects highlight S100 proteins as potential therapeutic targets and as a promising panel of cancer biomarkers. In this work, we have developed a mass spectrometry (MS)-based method for the multiplexed and specific analysis of the entire S100 protein family in tumour tissues and have applied it to investigate the expression of S100 isoforms in the context of thyroid cancer, the main endocrine malignancy.Mounting evidence demonstrates a causal role for S100 proteins in tumourigenesis and several S100 isoforms have shown utility as biomarkers of several types of cancer. The S100 family is comprised of 21 small isoforms, many of them implicated in important cellular functions such as proliferation, motility and survival. Furthermore, Selected Reaction Monitoring (SRM)-MS and stable isotope labelling/label-free analysis were employed to investigate the expression of the 21 S100 protein isoforms in thyroid tissue samples. Specimens included 9 normal thyroid tissues and 27 tumour tissues consisting of 9 follicular adenomas (FA), 8 follicular carcinomas (FTC) and 10 papillary carcinomas (PTC).The multiplexed and targeted mass spectrometry method led to the detection of eleven S100 protein isoforms across all tissues. Label- and label-free analyses showed the same significant differences and results were confirmed by western blot. S100A6, S100A11 and its putative interaction partner annexin A1 showed the highest overexpression in PTC compared to normal thyroid. S100A13 was also elevated in PTC. Reduced S100A4 expression was observed in FA compared to all other tissues. FA and FTC showed reduction of S100A10 and annexin A2 expression.Targeted mass spectrometry allows the multiplexed and specific analysis of S100 protein isoforms in tumour tissue specimens. It revealed S100A13 as a novel candidate PTC biomarker. Results show that S100A6, S100A11 and Annexin A1 could help discriminate follicular and papillary tumours. The diagnostic and functional significance of S100A4 and S100A10 reduction in follicular tumours requires further investigation.The online version of this article (doi:10.1186/s12885-015-1217-x) contains supplementary material, which is available to authorized users. This prgression .in vivo [Dysregulation of S100 isoform expression has been observed in numerous cancers ,5  were obtained from the neuroendocrine tumour bank at the Kolling Institute of Medical Research, Sydney. Normal samples were obtained from dissection of non-neoplastic tissue from the contralateral lobe of patients who had undergone thyroidectomy or from patients with benign disease where thyroidectomy was indicated. Clinicopathological features are shown in Table 13C615N4) and 40 mg/mL of L‐lysine‐2HCl (13C615N2) (Cambridge Isotope Labs), 10% dialysed FBS (Life Technologies) and 1% Penicillin-Streptomycin (Gibco). The medium was sterile filtered through a 0.22 micron filter. TPC1 thyroid cancer cells were cultured for at least eight doublings at 37°C in a humidified atmosphere with 5% CO2. Upon reaching 70-90% confluence, cells were washed with phosphate buffered saline (PBS), harvested and stored at -80°C until use. Heavy amino acid incorporation was verified by MS.SILAC RPMI 1640 media (Thermo Scientific Pierce) was supplemented with 240 mg/mL of L-arginine‐HCl , 0.1% SDS, 150 mM NaCl, 1 mM EDTA, pH 8) with complete, EDTA-free protease inhibitor cocktail (Roche), phosphatase inhibitor tablet (Thermo Scientific Pierce) and lysed on ice with sonication (three cycles of 20 s pulses). Lysates were centrifuged  and the supernatants retained. Protein quantitation was performed with a BCA protein assay kit (Thermo Scientific).. Labelled TPC1 cells allowed the quantitative analysis of candidate reference proteins and S100 proteins S100A2, S100A6, S100A10, S100A11 and S100A13. It is not unexpected that a particular cell line does not express detectable levels of all S100 isoforms, therefore additional stable isotope-labelled S100 synthetic peptides were spiked into the samples before tryptic digestion (see below for details). While it is conceivable to perform the entire analysis using stable isotope-labelled peptides from reference proteins and S100 proteins, using the SILAC spike-in represents a less expensive approach that does not rely on prior knowledge of the most appropriate peptide sequence to represent the target analyte.40 ug of protein from tissue samples and 20 ug of protein from metabollically labelled TPC1 cells were mixed in triplicate before SDS-PAGE. A ratio 2:1 of sample protein to labelled protein was employed given that previous tests measuring the abundance of candidate reference proteins showed varying levels of actual tumour protein amount in different samples due to the presence of blood proteinsIn addition, label-free MS analysis to quantitate all S100 proteins was also carried out as we previously described . To thisCorresponding aliquots of specimens and reference samples were mixed with NuPAGE LDS sample buffer (Invitrogen) and 50 mM dithiothreitol (DTT), incubated at 70°C for 10 min and loaded onto 4-12% Bis-Tris NuPAGE gels (Invitrogen). Proteins were separated using MES running buffer , fixed into the gel with 50% ethanol/10% acetic acid for 1 h and then stained for 1 h with colloidal Coomassie G-250 (Sigma). Gel bands from three mass regions were excised , cut into approximately 1 × 1 mm cubes and destained with 50% ACN in 50 mM ammonium bicarbonate . Gel pieces were dehydrated with 100% ACN, speed-vac dried and incubated in 10 mM DTT/100 mM Ambic at 56°C for 1 h followed by treatment with 55 mM iodoacetamide/100 mM AmBic at RT in the dark for 45 min. Gel pieces were dehydrated again, speed-vac dried, rehydrated with 120 uL of 13 ng/uL trypsin solution (sequencing grade modified trypsin (Promega) in 50 mM AmBic and 0.1% SDOC) and kept on ice for 1 h.13C, 15N stable-isotope labelled peptide standards ELPSFLGK (S100A4), ALNSIIDVYHK (S100A8), NIETIINTFHQYSVK (S100A9) and 1 pmol of SFWELIGEAAK (S100A14) and AVIVLVENFYK (S100A16) . This step allowed the inclusion of additional labelled S100 peptides not detected in TPC1 cells according to preliminary tests. All samples were then incubated overnight at 37°C. Peptides were extracted thrice with 0.1% SDOC in ultrasonic waterbath for 10 min and then acidified with 1 uL of formic acid. The precipitate was removed by centrifugation  and the samples were dried in a speed-vac, ready for LC-SRM-MS.Before tryptic digestion, gel pieces from the region 7-14 kDa were spiked with 2 pmol of The selection of S100 peptides, transitions and optima MS parameters was based on our previous work . The seqSamples were analysed using a Waters nanoACQUITY UPLC system coupled to the QTRAP 5500 mass spectrometer (AB Sciex). Weak (A) and strong (B) elution solvents were 0.1% FA (v/v) in water and 0.1% FA (v/v) in ACN, respectively. Samples were loaded onto a Waters Symmetry C18 trapping column  and peptides were separated on a Waters BEH C18 nano UPLC column  at 35°C under constant flow rate of 0.4 uL/min. Different elution gradients were used depending on the regions from the gel: a) 7-14 kDa: 3% solvent B for 1 min, 3-40% B in 30 min and 40-85% B in 2 min, b) 14-32 kDa: 3% solvent B for 1 min, 3-40% B in 60 min and 40-85% B in 2 min, c) 32-100 kDa: 3% solvent B for 1 min, 3-40% B in 110 min and 40-85% B in 2 min.13C15N stable isotope-labelled peptide ESDTSYVSLK from human C-reactive protein , used as LC loading control for label-free experiments. Blank injections using a 30 min gradient were performed between each sample. The lists of peptide sequences in Table m/z + 8.857 and CE(3+) = 0.0544*m/z-2.4099, taken from Skyline. Peptide identities were corroborated by SRM-triggered IDA MS/MS and searched in the UniProt human database using Mascot v. 2.3.0.Peptide samples were reconstituted with a solution of 2% ACN/0.1% TFA (v/v) and 17.8 fmol/uL of The potential for peptide interferences was monitored with AuDIT software  and signIn the case of label-free analyses, co-elution of fragment ions and consistency of retention times were used as a guide for peak identity assignment. Signals with CV less than 20% in transition ratios across samples and replicates were considered to represent interference-free conditions. Summed raw peak areas were first divided by the total peak area of the spiked CRP peptide and then against the peak areas of the same peptides present in a reference pool sample that was prepared in the same batch along with the samples of interest. This was done to compensate for differences in sample preparation and system response as detailed in . Again, et al. [Identification of the most stably expressed endogenous reference proteins across all the thyroid tissue samples  was performed using the geNorm algorithm, developed by Vandesompele et al. . The geoTaking into account the normalisation factors derived from the reference proteins, 10 ug of protein from tissue lysates of normal, FA, FTC and PTC samples were separated on 4-12% Bis-Tris NuPAGE gels and electroblotted onto nitrocellulose (Bio-Rad). The membrane was blocked with 5% skim milk in TBS for 1 h at RT. For S100A4 analysis, the membrane was cut in three sections from around 5-17 kDa, 17-32 kDa and >32 kDa. The sections were incubated overnight at 4°C with a) 5-17 kDa - rabbit polyclonal anti-S100A4 , b) 17-32 kDa - rabbit monoclonal anti-Rab7a  and c) rabbit monoclonal anti-β-Tubulin . For S100A13 analysis, the membrane was cut in two sections (5-17 kDa and >32 kDa) and incubated overnight with either rabbit monoclonal anti-S100A13  or rabbit monoclonal anti-Rab7a . The secondary antibody was fluorescence-labelled IRDye 800CW goat anti-rabbit IgG (H + L) , incubated at RT for 1 h and visualized with the Odyssey infrared imaging system.Because tumour specimens contain naturally variable amounts of blood proteins, and in the case of thyroid specimens, thyroglobulin, a simple protein quantitation assay would be insufficient for normalising loading quantities which is a requirement for accurate quantitation. Therefore, our first task was to identify endogenous reference proteins within thyroid tumours that could be used for loading normalisation. Traditionally, ‘housekeeping genes/proteins’ like GAPDH and β-actin have been used as normalisation loading controls; however, increasing evidence shows that these may not be the best reference proteins as their expression stability is compromised when compared across different groups of samples or disease states ,50. We uet al. [In the geNorm method by Vandesompele et al. , pairwiset al. -55. In oFrom the selected subset, Rab7a and cyclophilin A were the most stable proteins identified Figure a and paiK, S100A2; ELPSFLGK, S100A4; LQDAEIAR, S100A6; DPGVLDR, S100A11; LIGELAK, S100A13 and AVIVLVENFYK, S100A16) demonstrated linear response with up to 3 pmol on-column [Our targeted MS assay for all members of the S100 protein family revealed that the following eleven S100 protein isoforms were detected across all normal, FA, FTC and PTC tissues: S100A1, S100A2, S100A4, S100A6, S100A8, S100A9, S100A10, S100A11, S100A13, S100A14 and S100A16  compared to normal thyroid. Additionally, increased expression of S100A13 was generally observed in FA and FTC specimens, although this varied considerably amongst these samples. Analysis of four publicly available gene expression datasets, three of them comparing matched normal-PTC tumour samples, confirmed the overexpression of S100A13 mRNA transcripts in PTC   and well correlated with S100A11 expression , as previously reported ,58. ANXA2fc = - 2.47) and FTC (log2fc = -1.84) when compared against normal thyroid samples. A similar result was obtained with reference to PTC (Figure 2: 0.93, Figure Significantly decreased abundance of S100A10 and its known binding partner ANXA2  was showC Figure f. The exNo significant expression differences were observed for S100A1, S100A2, S100A8, S100A9, S100A14 and S100A16 in FA, FTC and PTC specimens with respect to normal thyroid tissue, except for S100A2 and S100A8 in FA. S100A8 and S100A9 are known to form heterodimers and their coordinated expression was observed in all tissues examined (Additional file The analysis of S100 proteins by label-free SRM provided essentially the same results as those found by stable-isotope labelling (see Figure The S100 protein family represents a promising panel of biomarkers for cancer and other diseases. However, methods are lacking for the quantitative screening of multiple S100 protein isoforms in clinical specimens. Here, we have showed that targeted MS using SRM enables the specific and multiplexed quantitation of S100 proteins in a large-scale study with 37 tissues of thyroid origin. As our method provided relative quantitation between samples it was necessary to investigate which proteins in thyroid tissues could act as stable endogenous reference proteins, which in this case were Rab7a, cyclophilin A and PSMB2. To the best of our knowledge, this is the first study to identify these loading control proteins for thyroid cancer specimens. Of note, others have already detected dysregulation of β-actin and tubulin isoforms in FA, FTC and PTC , hence sThe MS method harnesses the high accuracy and precision of SILAC-based MS quantitation and expands the number of targeted S100 protein isoforms through the use of label-free SRM. Although the quantitation with the SILAC spike-in achieved greater precision than the label-free experiments, both approaches identified the same significant changes in the tissue samples and were well correlated. Isotope-labelled peptide standards offer an advantage in that LC retention time and non-specific peptide interferences can be readily determined. Moreover, the SRM technique can readily be configured to measure protein concentration through the inclusion of quantified stable isotope peptide standards . While wOur experiments revealed the novel observation of increased S100A13 in PTC compared with FA, FTC and normal thyroid tissue, and this was supported by Western blot and previous studies of mRNA transcript levels. S100A13 gene silencing in lung cancer cells has been observed to decrease the invasive potential in vitro . MoreoveOverexpression of S100A6 in PTC has been observed previously , but morFA and FTC cannot be readily distinguished by cytologic evaluation of FNA biopsies, which poses an important diagnostic issue. Our findings using the MS assay show the loss of S100A4 in follicular adenoma tissue compared with follicular thyroid carcinoma, so this difference in expression has diagnostic potential. A previous study did not report the decrease of S100A4 in FA . Our resThe reduced expression of S100A10 in FA and FTC was well correlated with the expression of its interaction partner ANXA2. The S100A10-ANXA2 complex anchors to the plasma membrane via the ANXA2 subunits and has an active role in the trafficking of ion channels . A previWe have demonstrated the use of targeted mass spectrometry for the multiplexed and specific analysis of S100 protein isoforms in thyroid tumour specimens. This approach provides unambiguous structural evidence of S100 protein isoform expression thereby overcoming potential problems of antigen cross-reactivity that can interfere with affinity detection methods. This study led to the global characterisation of S100 protein expression in thyroid tumours and highlighted several S100 protein isoforms for potential diagnostic and therapeutic evaluation. S100A13 emerged as a novel candidate PTC biomarker. Our results indicate that S100A6, S100A11 and ANXA1 have utility to discriminate follicular and papillary thyroid tumours. The diagnostic and functional significance of S100A4 and S100A10 reduction in follicular tumours warrants further investigations.


O-galactosyltransferase (Hyp-O-GALT) enzymes that remain to be fully characterized.Arabinogalactan-proteins (AGPs) are ubiquitous components of cell walls throughout the plant kingdom and are extensively post translationally modified by conversion of proline to hydroxyproline (Hyp) and by addition of arabinogalactan polysaccharides (AG) to Hyp residues. AGPs are implicated to function in various aspects of plant growth and development, but the functional contributions of AGP glycans remain to be elucidated. Hyp glycosylation is initiated by the action of a set of Hyp-GALT3-At3g06440, GALT4-At1g27120, and GALT6-At5g62620) were identified as Hyp-O-GALT genes by heterologous expression in tobacco leaf epidermal cells and examined along with two previously characterized Hyp-O-GALT genes, GALT2 and GALT5. Transcript profiling by real-time PCR of these five Hyp-O-GALTs revealed overlapping but distinct expression patterns. Transiently expressed GALT3, GALT4 and GALT6 fluorescent protein fusions were localized within Golgi vesicles. Biochemical analysis of knock-out mutants for the five Hyp-O-GALT genes revealed significant reductions in both AGP-specific Hyp-O-GALT activity and β-Gal-Yariv precipitable AGPs. Further phenotypic analysis of these mutants demonstrated reduced root hair growth, reduced seed coat mucilage, reduced seed set, and accelerated leaf senescence. The mutants also displayed several conditional phenotypes, including impaired root growth, and defective anisotropic growth of root tips under salt stress, as well as less sensitivity to the growth inhibitory effects of β-Gal-Yariv reagent in roots and pollen tubes.Three members of the GT31 family  contains supplementary material, which is available to authorized users. O-linked AG polysaccharides. AGPs are found in cell walls, plasma membranes, and extracellular secretions of virtually all plant cells, tissues and organ types . Previously characterized GALT2 and GALT5 were used as positive controls for this assay, while GALT1 effectively served as a negative control, given its involvement with N-glycan biosynthesis 14, and d[AO]51, consisting of non-contiguous peptidyl Hyp residues, were used to examine the effect of these model AGP peptide sequences of various lengths on GALT activity. [AP]7, consisting of alternating Ala and Pro residues, was tested for the requirement of peptidyl Hyp for galactosylation. ExtP, a chemically synthesized extensin peptide consisting of contiguous peptidyl Hyp residues, tested whether contiguous peptidyl Hyp residues act as potential acceptors. Two commercially available pectic polysaccharides, Rhamnogalactan I from potato and Rhamnogalactan (a mixture of RGI and RGII) from soybean, were also tested as potential substrates acceptors. All the non–AGP substrate acceptors, including [AP]7, failed to incorporate [14C]Gal, indicating the GALT activity was specific for AGP sequences containing non-contiguous peptidyl Hyp  indicated that all five Hyp-O-GALT transcript levels displayed unique expression patterns in the seed coat during embryogenesis , GALT4 , and GALT6  compared to WT , GALT5 , and a galt2galt5 double mutant and were used here as positive controls [O-GALT activity  demonstrated no such reductions and were indistinguishable from WT  caused a significant reduction in AGP content, with most significant effects being exhibited by galt5 in stems, galt4 in siliques, and galt6 in senescent leaves  compared with WT  compared to WT.Prior evidence for the involvement of AGPs (SOS5) and GALT2/GALT5 in seed coat mucilage prompted an examination of the potential functions of mucilage , 37, 38.s pectin . The galgalt mutant seeds were analyzed. Sequential extraction of seeds with ammonium oxalate, 0.2 N NaOH, and 2 N NaOH was performed to assess changes in the soluble and adherent mucilage  compared to wild type seeds . Less significant differences were observed in galt2, galt4, and galt5 single mutants, whereas more significant differences were observed in galt2galt5 mutants. All the galt mutants except for galt1 displayed a decrease in total sugars in the 2 N NaOH extracts, which represent the majority of the adherent mucilage and contain strongly linked pectins and cross-linking glycans/hemicelluloses [In order to confirm and quantify the changes in non-adherent (soluble) and adherent mucilage, WT and ge Table . Both galluloses , 41.TablGALT6 mutants  displayed early onset of senescence compared to WT and the other galt mutants. This was visualized by premature yellowing of leaves and was correlated with a slightly greater reduction in chlorophyll content and protein content in GALT6 mutants compared to WT  and GALT6  mutants, and to a lesser extent the GALT4  mutants, exhibited significant reductions in root elongation compared to WT when grown in the presence of 100 and 150 mM NaCl , GALT5 , and the galt2galt5 double mutant [GALT1 mutants did not show salt hypersensitive growth and were indistinguishable from WT in this assay. The galt single and double mutants were not sensitive to osmotic stress as illustrated by mannitol  in the presence of 100 mM NaCl, which was not observed in WT and GALT1 mutants  and GALT6  mutants, and to a lesser extent the GALT4  mutants were slow to reorient their root growth compared to WT when grown in the presence of 100 mM NaCl. Such delayed reorientation was previously reported for knock-out mutants of GALT2 , GALT5 , and the galt2galt5 double mutant; these mutants were used here as positive controls [GALT1 mutants  reoriented quickly and were indistinguishable from WT in this assay.A root bending assay was used as another means to evaluate salt hypersensitivity of the controls . As expeO-GALTs for AGPs [N–linked glycans [Bioinformatic approaches were previously used to identify a small, six-membered gene family within the GT 31 family of the CAZY database as potential candidates for encoding Hyp-for AGPs , 18, 42. glycans , while G glycans , 16. In GALT1-GALT6 in tobacco epidermal cells demonstrated a significant increase in Hyp-GALT activity compared with various tobacco control plants, with the notable exception of GALT1  present in Arabidopsis microsomal membranes and to GALT2 and GALT5 expressed in Pichia, specifically all require UDP-Gal as the sugar donor [2+ followed by Mg2+ for their optimal activity, in contrast to Mg2+ followed by Mn2+ in Pichia microsomes.Heterologous transiently expressed ar donor , 45, 46.galt knock-out mutants for all these genes exhibit reduced  Hyp-GALT activity and contain considerably less  glycosylated  AGPs. In addition, AGP profiling of the galt3, galt4, and galt6 mutants extends these findings and indicates that their activity is not limited to a particular AGP or a small subset of AGPs, but instead broadly acts on coexpressed AGPs, similar to that previously reported for galt2 and galt5 mutants  biosynthesis , 45–49,  located .GALT3, GALT4, and GALT6 with that of GALT2 and GALT5  and were analyzed as described by Breton et al. [www.genemania.org) using GALT2 and GALT5 as query genes.Protein sequences from GALT1, GALT3, GALT4 and GALT6 were run through several prediction programs  to obtain information on their putative subcellular localization and topology , 26, 74.n et al. . The coeAt1g26180-, At3g06440 , At1g127120 , and At5g62620 galt6-1, Sail_59_D08 and galt6-2, Sail_70_B02) were obtained from the Arabidopsis Biological Resource Center . All plants used in this study were germinated after 4 days of stratification in the dark at 4 °C and were grown under long-day conditions  in growth chambers or growth rooms. Sequencing of the amplified PCR products from the mutant plants led to the accurate determination of the T-DNA insertion site.Arabidopsis thaliana accession Columbia-0 (Col-0) and two T-DNA insertion lines for http://www.dna.ohio.edu/). To analyze transcript levels of GALT1, GALT3, GALT4, and GALT6, total RNA was isolated from 2 week old seedlings of WT and mutant plants. For tissue specific expression profiling, tissue was harvested at different growth stages as defined by Boyes et al. [Genomic DNA was isolated from leaves of the mutants and WT plants and was extracted using the 2× CTAB method described by Murray and Thompson . Subsequs et al. . In bothUBQ10, At4g05320) as a reference. Primer sequences are listed in Additional file For quantitative real-time PCR (qRT-PCR), cDNAs were amplified using Brilliant II SYBR Green qRT-PCR Master Mix with ROX  in an MX3000P real-time PCR instrument (Agilent Technologies). PCR was optimized and reactions were performed in triplicate. The transcript level was standardized based on cDNA amplification of ubiquitin 10 . N-terminal 6x-His tag fusion gene constructs were amplified using Q5 high fidelity DNA Taq polymerase , initially cloned into the pENTR/D-TOPO vector , and eventually cloned into the destination vector pMDC32 gateway vector using LR clonase enzyme mix . Primers used for amplification are listed in Additional file Agrobacterium strain GV3101 by the freeze thaw method, and the transformants were grown overnight in Luria-Bertani (LB) medium. Bacterial cells were harvested by centrifugation and suspended in a buffer containing 10 mM MES, 10 mM MgCl2, and 50 μM acetosyringone (OD600 = 0.2). Leaves from 6-week-old WT N. tabacum cv. Petit Havana were used for Agrobacterium-mediated transient expression. Four days after infiltration, leaves were harvested, and microsomes were prepared according to the method described by Liang et al. [Coding regions of g et al.  with minGALT3, GALT4, and GALT6 devoid of stop codons was cloned into the pENTR/D-TOPO vector  and sequenced. The resulting plasmids were cloned in the destination vector pEarlyGate 101 by a gateway cloning strategy, using LR clonase enzyme mix  to generate the YFP N-terminal fusion constructs. These gene constructions were transformed into Agrobacterium strain GV3101 and infiltrated into tobacco leaves as described in the above section except that the bacterial concentration was lower (OD600 = 0.05). The GALT3-YFP, GALT4-YFP, and GALT6-YFP constructions were co-expressed with either the ER marker HDEL-GFP or the Golgi marker sialic acid transferase (ST)-GFP to ascribe subcellular localization. Transformed plants were incubated under normal growth conditions and imaged 2 days post-infiltration using an upright Zeiss LSM 510 META laser scanning microscope , with a 40 × oil immersion lens and an argon laser. For imaging the expression of YFP constructs, the excitation line was 514 nm, and emission data were collected at 535–590 nm; whereas for GFP constructs, the excitation line was 458 nm, and the emission data were collected at 505–530 nm.Full length GALT1, GALT3, GALT4, and GALT6. Three permeabilized microsomal membranes were included as controls, one from the WT tobacco leaves, one from WT tobacco leaves infiltrated with Agrobacterium GV3101 transformed with the empty expression vector (pMDC32), and one with tobacco leaves infiltrated with ST-GFP constructs as negative controls.The standard GALT reaction was performed as described in Basu et al.  using deThe GALT reaction products were purified by RP-HPLC as described by Liang et al. .GALT2-GALT6 constructs were used for determination of substrate specificity as described by [Microsomal fractions from tobacco leaves expressing ribed by , 16.galt1-1, galt1-2, galt3-1, galt3-2, galt4-1, galt4-2, galt6-1 and galt6-2 according to Liang et al. [Plant microsomal membranes were extracted from WT, g et al.  with mingalt1-1, galt1-2, galt3-1, galt3-2, galt4-1, galt4-2, galt6-1, and galt6-2 mutant plants as described by Schultz et al. [7 was used as a control to monitor the retention time of a pure AGP peptide. Samples were eluted from the column following a linear gradient with solvent B (0.1 % trifluoroacetic acid in 80 % acetonitrile): 0–30 % solvent B in 30 min, then 30–100 % in 30 min at a flow rate of 0.5 ml/min. Chromatography was monitored by absorption at 215 and 280 nm.AGPs were extracted from the WT, z et al.  precipitz et al.  and Yariz et al. . AGP proz et al.  with mod2 (2 % w/v) at a volume of 2 ml for each gram of tissue, and stirred for 3 h at room temperature. Samples were centrifuged for 30 min at 10,000 g at room temperature. The supernatant was freeze-dried overnight and resuspended in 1 ml of 2 % CaCl2 and transferred to 2 ml microcentrifuge tubes. AGPs were precipitated overnight at 4 °C with an equal volume of the β-Gal - Yariv reagent (2 mg/m in 2 % w/v CaCl2). The insoluble β-Gal-Yariv–AGP complex was collected by centrifugation at 10,000 g in a microcentrifuge for 1 h. The β-Gal-Yariv was removed by washing the pellet three times in 2 % (w/v) CaCl2 and then twice in methanol. The pellet was dried, dissolved in 100 μl of water mixed with 25 mg of solid sodium dithionite and incubated for 30 min at 50 °C until the mixture decolorized. The resulting solution was then desalted on a PD-10 column (Pharmacia) that had been equilibrated with water, and the eluate was freeze-dried.AGPs from siliques, flowers, inflorescence stem, and senescent leaves were extracted as described by Lamport , with migalt1, galt3, galt4, and galt6 plants were collected, and silique length and weight were measured. For seed number, siliques were decolorized by incubation in 100 % ethanol at 37 °C overnight before dissection of the siliques. For reciprocal cross-pollinations, 10 flowers from WT, galt4-1, galt4-2, galt6-1, and galt6-2 were selected at stage 12. These flowers were emasculated before pollinating them with fresh pollen obtained from flowers at stage 13. After 10 days, siliques were collected from these flowers to examine seed set.Mature siliques from 6-week old WT, galt1-1, galt1-2, galt3-1, galt3-2, galt4-1, galt4-2, galt6-1 and galt6-2 seedlings were grown on MS plates for 7 days before they were transferred to MS plates supplemented with 50 μM α-Gal-Yariv reagent or 50 μM β-Gal-Yariv reagent. For seedling growth in salt, 7-day-old seedlings of WT, galt1-1, galt1-2, galt3-1, galt3-2, galt4-1, galt4-2, galt6-1, and galt6-2 were transferred to MS medium containing 1 % agar and 100 mM or 150 mM NaCl. Root length was determined on low-magnification (×10) digital images captured using a CCD camera and image analysis freeware . For analysis of salt hypersensitivity of the mutant plants, root growth was monitored using a root bending assay [For monitoring root growth in response to β-Gal-Yariv reagent, WT, ng assay , and imagalt1-1, galt1-2, galt3-1, galt3-2, galt4-1, galt4-2, galt6-1, and galt6-2 plants 1 to 2 weeks after bolting were used for the examination of pollen tube phenotypes. Individual open flowers were germinated in vitro as described by Boavida and McCormick [3BO3, 1 mM MgSO4, 5 mM KCl, 5 mM CaCl2, 10 % sucrose, and 1.5 % low-melting agarose, pH 7.5 and 30 μM β-Gal-Yariv reagent or 30 μM α-Gal-Yariv reagent) at 22 °C and 100 % humidity in the dark. Pollen tube germination rates were calculated by dividing the total number of germinated tubes by the number of grains. Images and measurements of pollen tubes were done at 20× magnification in a Nikon Phot-lab2 microscope coupled with a SPOT RT color CCD camera and SPOT 4.2 analysis software.Flowers collected from WT, cCormick , on solihttp://rsb.info.nih.gov/ij/). To ensure comparable results, the area 3–5 mm behind the root tip was analyzed. Plants grown on agar plates were carefully removed in 100 μl of half-strength MS medium on microscope slides for analysis. Quantification of root hairs length and density was performed using 10 seedlings for each genotype, and 25 root hairs from each root were measured.Root hair length from 8-day-old plants grown on agar plates was determined on low-magnification (×10) digital images captured using a CCD camera and image analysis freeware  and sampling started at 19 DAG and continued every other day until full senescence was reached (39 DAG) as described by Breeze et al. . Becausegalt6-1, and galt6-2 leaves by immersion in 1 ml of N, N-dimethylformamide for 48 h in the dark at 4 °C. Absorbance was recorded at 664 and 647 nm, and total chlorophyll concentration was calculated as described by Xiao et al. [Chlorophyll was extracted from WT, o et al. . The totgalt mutant seeds were extracted sequentially with 0.2 % ammonium oxalate, 0.2 and 2 N sodium hydroxide for 1 h each with vigorous shaking at 37 °C. Both sodium hydroxide extractions were neutralized with acetic acid. Total sugar (μg/mg seed) was determined with a phenol-sulfuric assay based on Dubois et al. [One-hundred milligram of WT and s et al. . In shorFor each analysis, both enzyme activity and phenotypic differences in mutants were compared with the WT using one-way ANOVA. The P values were derived from post hoc tests using Dunnett’s adjustment for multiple comparisons. Statistical analyses were performed with Prism 6 software .GALT1: At1g26810; GALT2: At4g21060; GALT3: At3g06440; GALT4: At1g27120; GALT5: At1g74800; GALT6: At5g62620.The data sets supporting the results of this article are included within the article and its additional files. The accession numbers of the genes analyzed in this study are as follows:


All of them exhibited much higher photocatalytic activity than the pure AgBr in photodegradation of methyl orange (MO) under visible light irradiation. When the loading amount of Fe3O4 was 0.5 %, the hybrid materials displayed the highest photocatalytic activity, and the degradation yield of MO reached 85 % within 12 min. Silver halide often suffers serious photo-corrosion, while the stability of the Fe3O4/AgBr hybrid materials improved apparently than the pure AgBr. Furthermore, depositing Fe3O4 onto the surface of AgBr could facilitate the electron transfer and thereby leading to the elevated photocatalytic activity. The morphology, phase structure, and optical properties of the composites were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), UV–visible diffuse reflectance spectra (UV–vis DRS), and photoluminescence (PL) techniques.Magnetically separable Fe Parti2 , Ag–AgBr2 [2/RGO , AgBr(I)2 [2/RGO , Fe(III)2 [2/RGO , and Ag/2 [2/RGO  have bee3O4 has excellent conductivity, so it could act as an electron transfer channel and acceptor, which could suppress the photo-generated carrier recombination. For instance, Ye et al. reported that the hierarchical core–shell-structured Fe3O4/WO3 has a more effective photoconversion capability than pure WO3 or Fe3O4 [2@Fe3O4 magnetic supports, they exhibited faster degradation rates for 4-chlorophenol than N-TiO2 [For the nanosized or microsized photocatalysts, effective separation from the mixed system and recycle using are important problems to restrain their real applications , 17. Immor Fe3O4 . The Ag n N-TiO2 . However3O4 has high conductivity and its CB level (1 V vs. NHE) makes it become a good candidate for coupling with AgBr. Based on the above reason, we prospect their combination could improve the photocatalytic performance by enhancing charge transport. Herein, conductive Fe3O4 particles and visible light active AgBr were coupled together to prepare the magnetically recyclable Fe3O4/AgBr composites with visible light activity. Studies of their photocatalytic performance in the decomposition of methyl orange (MO) indicated that Fe3O4/AgBr photocatalysts exhibited excellent catalytic activity under visible light illumination. Meanwhile, the stability of AgBr was improved when it was coupled with Fe3O4.The motivation of the present research originated from the idea that Fe3O4 nanospheres were prepared according to the literature reported previously [3 · 3H2O, 3.0 g NaAc, and 10 mL oleic acid were added to 30 mL ethylene glycol into a three-necked flask, and then a red solution was formed. The mixture was stirred vigorously at 50 °C for 20 min until all reagents were dissolved completely. Then, the mixture was transferred into a Teflon-lined autoclave and heated at 200 °C for 20 h. The products were cooled down to room temperature, washed with ethanol for several times, and dried under vacuum to give a black solid.The Feeviously . In a ty3O4 nanospheres (0.01 g) were dispersed in 20 mL deionized water and then ultrasonically dispersed evenly. AgNO3 (1.18 g) was added into the solution, and then NaBr (0.1 mol/L) was added dropwise slowly. The resulting suspensions were filtered, washed several times with distilled water, and finally dried in vacuum. Different Fe3O4/AgBr samples were obtained by adjusting the mass ratio of Fe3O4 and AgBr, and the sample was denoted as Fe3O4/AgBr-x (x means the percentage of Fe3O4).Feθ range 10°–90°, step size 0.08°, accelerating voltage 40 kV, applied current 40 mA). The morphology of the samples was taken on a Hitachi S-4800 scanning electron microscope (SEM). UV–visible diffuse reflectance spectra (UV–vis DRS) were obtained on a Shimadzu U-3010 spectrometer, using BaSO4 as a reference. The photoluminescence (PL) spectra were recorded on a F-7000 FL spectrophotometer.X-ray diffraction (XRD) patterns were measured on an X’Pert Philips diffractometer . Prior to irradiation, the suspension was magnetically stirred in the dark for 30 min to establish an adsorption–desorption equilibrium. A 300-W Xe arc lamp with a 420 cutoff filter was used as the light source . At 2-min intervals, 5 mL of the suspension was collected and centrifuged for 3 min to remove the catalyst particulates for analysis. The residual MO concentration was detected at 464 nm using a UV–vis spectrophotometer .MO was selected as the model pollutant to evaluate the photocatalytic activity of the Fe3O4 nanospheres was about 100 ~ 200 nm. The surface of Fe3O4 particles was rough, and each magnetic microsphere was constructed with many small magnetic grains. From Fig. 3O4 was coupled with AgBr, the particle size of the composite increased apparently than the pure AgBr particles. The magnetic property of the surface Fe3O4 would result in the agglomeration of the particles. The EDS spectrum of Fe3O4/AgBr-0.5 hybrid materials indicates that the atomic ratio of Fe and Ag is approximately 1:134, which is a little larger than the designed value.Figure 3O4/AgBr hybrid materials with different Fe3O4 contents, which matched well with those of Fe3O4  [3O4 content, no characteristic peaks were ascribed to Fe3O4 emerging with AgBr phase, which should be due to the lower content of Fe3O4.Figure 85-1436) . The dif3O4/AgBr hybrid materials are illustrated in Fig. 3O4 particles show strong absorption both in ultraviolet and visible light regions, which may be attributed to its small band gap. The absorption band edge of AgBr was about 470 nm, so the calculated band gap was 2.64 eV. AgBr was often used as a good visible light sensitizer because it exhibited a strong absorption in the visible light. After loading Fe3O4 on AgBr particles, the visible light absorption increased significantly. And as the increase with the loading content of Fe3O4, the visible absorption of the composites enhanced gradually, indicating that the existence of Fe3O4 could promote visible light absorption effectively.The UV–vis spectra of Fe3O4 nanospheres were loaded on AgBr particles, the photocatalytic activity increased apparently than the pure AgBr. The photocatalytic mechanism of Fe3O4/AgBr composites for MO degradation under visible light is illustrated in Fig. 3O4 (1 V vs. NHE) is much lower than that of AgBr (−1.1 V vs. NHE) [3O4. And the conductivity of Fe3O4 is as high as 1.9 × 106 S m−1; the electrons on Fe3O4 particles would transfer out quickly and react with the surface pollutants. Meanwhile, Ag nanoparticles on the surface of AgBr can act as electron capture traps to improve the separation efficiency of the charge carriers and thereby improving the photocatalytic efficiency. These should be the main reason for the enhancement of the photocatalytic activity for Fe3O4/AgBr composites. In addition, the loading amount of Fe3O4 particles has an effect on the activity of the composites. The sample Fe3O4/AgBr-0.5 has the best photocatalytic activity; the degradation yield of MO reached nearly 85 % within 12 min. In order to clarify the reasons for this result, the active species in photodegradation process of MO were detected. Methanol, silver nitrate, and terephthalic acid solution were added into MO dye solution to capture electrons, holes, and · OH, respectively. As can be seen from Fig. The photocatalytic performances of the photocatalysts were evaluated by photoinduced decolorization of MO aqueous solution, as shown in Fig. vs. NHE) –25, so t3O4/AgBr-0.5 was evaluated. As shown in Fig. + to the metallic Ag, and the small Ag nanoparticles would cover on the surface of AgBr. And the surface Ag nanoparticles would prohibit the photo-absorption of the inner AgBr. When the amount of Ag was enough, the photo-excitation of the inner AgBr would be hold back, and as a result, the photocatalytic activity decreased remarkably as the reaction proceeding. However, for the Fe3O4/AgBr hybrid materials, the stability was much better. The degradation yield of MO on Fe3O4/AgBr composites was 0.83, 0.78, and 0.71 in the consecutive cycles, respectively. The Fe3O4 particles on the surface could transfer the photo-excited electrons out quickly, which inhibit the self-reduction of AgBr. So, the long-term stability of Fe3O4/AgBr composites was obtained than the pure AgBr. In the photocatalytic application, effective recycling of catalyst is very important. Thanks to the existence of Fe3O4, the catalyst has magnetism, which is favorable for recycling. As shown in the inset of Fig. 3O4/AgBr-0.5 composites could be easily separated from the suspension by an external magnetic field. As expected, the as-prepared Fe3O4/AgBr composites exhibited a certain magnetic response.As well known, AgBr is not stable, and it often suffers photo-corrosion. So, the stability of AgBr and Fe3O4. No characteristic peaks were ascribed to Fe3O4 emerging with Fe3O4/AgBr composites, which should be due to the lower content of Fe3O4. Moreover, Fig. 3O4/AgBr samples, indicating that an appropriate amount of Fe3O4 could significantly reduce the recombination rate of photo-generated electrons and holes of AgBr. The PL intensity of the Fe3O4/AgBr-0.5 sample was the lowest, which indicated that the separation efficiency of charge carriers was the highest. That was in accord with the photocatalytic activity result very well.The fluorescence spectrum can provide much more information about carrier capture, migration, conversion, separation, etc., so it has been used for measuring the separation of the photo-generated electron–hole pairs . The emi3O4/AgBr hybrid materials with high photocatalytic efficiency under visible light were prepared through the precipitation method. The Fe3O4/AgBr samples showed much higher photocatalytic activity than the pure AgBr, which was due to the matched band structure of two components and the higher conductivity of Fe3O4. When the loading amount of Fe3O4 was 0.5 %, the highest photoactivity was obtained, and the degradation yield of MO reached 85 % within 12 min. The PL spectra indicated that Fe3O4/AgBr hybrid materials had the higher separation efficiency of the photo-excited charge carriers, and that was in accordance with the photocatalytic activity very well. In addition, the stability of Fe3O4/AgBr composites was improved comparing with the pure AgBr. The photo-excited electrons would transfer out quickly from the surface Fe3O4, so the self-reduction of AgBr to metallic Ag was prohibited, and as a result, the long-term stability of Fe3O4/AgBr was obtained.Fe


Peripheral blood samples (50 ml) were obtained from three healthy volunteers and peripheral blood mononuclear cells (PBMCs) were obtained from each via Ficoll-Conray density gradient centrifugation. Each suspension of PBMCs (1×106/ml) was divided into two parts; CAPRI cells were obtained from one part through a series of induction, amplification and cytokine cultures, while CIK cells were obtained from the other part through induction with different cytokines. During the culture process, the proliferation and morphological changes were observed for the two cell types using Trypan blue staining. At day 14, the cytotoxic activity of the two cell types was examined through determining lactate dehydrogenase release in the presence of K562 leukemia cells and MCF-7 breast cancer cells. In addition, secretory levels of interferon (IFN)-γ and interleukin (IL)-2 were detected using enzyme-linked immunospot (ELISPOT) technology. The results revealed that at day 5 and 14 of culture, there were significantly fewer CAPRI cells compared with CIK cells (P<0.001), although the survival rate of each cell type was >95%. The cytotoxic activity of CAPRI cells towards the K562 cell line was effector-target ratio-dependent (40:1 and 20:1) with values of 55.1±3.25 and 35.0±2.65%, respectively, which were significantly reduced compared with the corresponding data in CIK cells, 60.0±3.03 and 39.7±3.42% . Furthermore, the cytotoxic activity of CAPRI cells towards MCF-7 cells were 71.5±3.06, 56.0±3.76 and 40.2±2.90% at effector-target ratios 40:1, 20:1 and 10:1, respectively. These data were significantly higher than the corresponding values in CIK cells, 65.4±3.86, 49.5±3.91 and 36.1±3.73% . As determined using ELISPOT technology at different cell concentrations (1×106/ml and 5×105/ml), IFN-γ secretion levels, determined by the number of spot-forming cells, of CAPRI cells were 126.2±10.31 and 48.8±10.99, respectively, which were significantly reduced compared with those of CIK cells, 409.3±7.76 and 159.3±15.45, respectively (P<0.001). IL-2 secretion levels in CAPRI cells were 325.1±16.24 and 113.8±11.29 at 1×106/ml and 5×105/ml, respectively, which were significantly increased compared with CIK cells, 212.0±16.58 and 70.7±10.57, respectively (P<0.001). In conclusion, the present study demonstrated that CAPRI cells had a reduced proliferation rate compared with CIK cells as well as a less potent cytotoxic effect on K562 cells; however, the two cell types had potent cytotoxic activity towards solid tumor MCF-7 cells. In addition, CAPRI cells secreted lower levels of IFN-γ and increased levels of IL-2 compared with CIK cells. These results indicated that antitumor activities of CAPRI and CIK cells proceeded via different mechanisms.The present study aimed to compare the antitumor effects of cascade primed immune (CAPRI) cells and cytokine-induced killer (CIK) cells  Cancer is a prominent public health problem worldwide, which has increasing incidence and mortality rates . Progresin vitro, through investigating cell morphology, proliferation, cytotoxic activity to tumor cells and the ability of these cells to secrete cytokines. These methods of comparison may be extended for the future detection of a variety of cell lines and cytokines in order to better guide clinical treatment.Cascade primed immune (CAPRI) cells and cytokine-induced killer (CIK) cells have been used as novel adoptive immunotherapy cells and are known to have different strengths and biological characteristics . These c2. Low molecular weight heparin injections were purchased from Qilu Pharmaceutical Co., Ltd . The substrate mix (BCIP/NBT Color Development Substrate) was purchased from Promega Corp. . 96-well plates and cell culture flasks were obtained from Beijing Aibo Biological Engineering Co., Ltd. Recombinant human (rHu) interferon (INF)-γ and rHu interleukin (IL)-2 were purchased from Peprotech Co. . Mouse monoclonal anti-human CD3 antibodies were provided by Beijing Tonglihaiyuan Biotechnology Co., Ltd. . CAPRI cell culture patented reagents were obtained from the Institute of Immunology, University of Munich . Lymphocyte stratified fluid was purchased from Tianjin Haoyang Biological Products Co., Ltd . IFN-γ and IL-2 ELISPOT kits were purchased from Shenzhen Dakewei Biotechnology Co., Ltd. .K562 human leukemia cells and MCF-7 human breast cancer cells were purchased from the cell library of Cancer Institute of Chinese Medical Sciences Academy . K562 and MCF-7 cells were cultured in RPMI 1640 medium  with 10% fetal bovine serum  at 37°C in 5% CO2 incubator was obtained from Sanyo Electric International Trading Co., Ltd.  and the micropipettes were purchased from Thermo Fisher Scientific Co., Ltd. . The low-speed centrifuge (B40) was obtained from Hebei Anxin Baiyang Centrifugal Machinery Factory  and enzyme-linked immunospot  analyzers were provided by Cellular Technology, Ltd . The inverted microscope (BDS200) was obtained from Chongqing Auto Optical Instruments Co., Ltd.  and the ELISA analyzer (RT2100c) was obtained from Rayto Life and Analytical Science Co. .Clean benches, portable autoclaves and the constant temperature water bath were purchased from Shanghai Boxun Industry and Commerce Co., Ltd. . The CO6cells/ml. Samples were labeled  and the PBMC suspensions from each source were divided into two parts as follows: a1, b1 and c1 for CAPRI cell induction; and a2, b2 and c2 for CIK cell induction.The current study was approved by the Ethics Committee of the Affiliated Hospital of Weifang Medical University  and written informed consent was obtained from the patient/the patient’s families. A total of 50 ml blood was extracted from three healthy volunteers and heparin  was added to prevent coagulation. The three specimens were then stratified with lymphocyte fluid. Ficoll-Conray density gradient centrifugation  was performed to obtain three separate suspensions of peripheral blood mononuclear cells (PBMCs). Following washing with 0.9% NaCl, RPMI 1640 medium was used to adjust the PBMC suspension to 1×106 cells/ml) of a1, b1 and c1 were added to the flasks and incubated at 37°C in a 5% CO2 incubator for 3 h. CAPRI patent reagent A (0.4 ml) was added to each culture flask for 3 h at 25°C and the stimulated culture substances were subsequently obtained.A total of 12 ml coating solution  was added to three flasks, which were then incubated at 4°C for 24 h. The coating solution was removed and 20 ml saline  was added to each flask, which were then mixed uniformly and left to stand for 2 min prior to saline removal. A total of 18 ml complete medium (RPMI 1640 and 10% FBS) was added to each flask. PBMC suspensions .Lymphocyte stratified fluid (12 ml) was added to each stimulated culture medium and then cultured at 37°C in a 5% CO2 incubator for 72 h. CAPRI cells were then collected and used for subsequent experiments. One part were further cultured  to study the cells proliferative capacity, the other groups (3×106 cells/ml) were frozen (−80°C) till 14 days and the cytotoxic activity and cytokine secretion levels of them were used to perform a comparative study with CIK cells.Complete medium was used to resuspend the cells and CAPRI patent reagent B (0.4 ml) was added to each cell suspension, which were then dispensed into flasks and cultured at 37°C in a 5% CO2 incubator for 2 h of static culture. IFN-γ  was added and continuously cultured for 24 h, 37°C, 5% CO2. IL-2 cytokines (300 U/ml) were then added with 50 ng/ml CD3 monoclonal antibody. Cells passaged every 3 days and a further 300 U/ml IL-2 was added with each passage. Samples were cultured at 37°C in a 5% CO2 incubator for 14 days to collect CIK cells. These cells were subsequently used for determining proliferation, cytotoxic activity and cytokine secretion.The three PBMC suspensions  were transferred into sterile culture flasks and 18 ml complete medium containing 10% FBS was then added. Samples were then incubated at 37°C in a 5% COAn inverted microscope was used to dynamically observe the proliferation of three PBMC suspensions , which were induced using the methods described above. On days 1, 3, 5 and 14 of culture, cells were stained using Trypan blue  and counted in order to determine proliferation and morphology. Experiments were performed in triplicate.CIK cells cultured for 14 days  and the thawed CAPRI cells  were the effector cells. K562 leukemia cells and MCF-7 breast cancer cells were used as the target cells. All experiments were performed in triplicate and plating of the treatment groups is shown in µl) concentrations of 4×106, 2×106 and 1×106/ml were added to each well with 100 µl target cells at a concentration of 1×105/ml. CIK cells, B1-B9; and CAPRI cells, C7-C12 and D1-D3.Three different effector-target ratios of 40:1, 20:1, 10:1 were used; effector cells (100 µl/well) were plated at concentrations of 4×106, 2×106 and 1×106/ml for each group  with 100 µl culture medium in order to detect spontaneous cytotoxic activity. CIK cells, B10–B12 and C1–C6; and CAPRI cells, D3–D12.Effector cells (100 µl/well) were plated at a concentration of 1×105/ml into three wells (A1–A3) with 100 µl culture medium. These cells were incubated at 37°C in a 5% CO2 culture incubator, at 45 min prior to the end of incubation, 20 µl/well cell lysate was added. In addition, to determine the spontaneous cytotoxicity of target cells, 1×105/ml target cells (100 µl/well) were plated into three wells (A4–A6) with 100 µl culture medium.In order to determine maximum cytotoxic activity, target cells , which were then incubated 20 µl/well cell lysate, which was added at 45 min prior to the end of culture at 37°C in a 5% CO2 incubator.Blank control wells (A7–A9) consisted of 200 2 incubator for 4–6 h. At 45 min prior to the end of culture, the culture plates were removed and 20 µl cell lysate was added to wells in the target cells maximum cytotoxicity group and the corrected volume control group. Cells in these two groups were then centrifuged at 250 × g for 4 min and further cultured for 45 min.Following centrifugation at 250 × g for 4 min, cells were cultured at 37°C in a 5% COµl supernatant per well was transferred into a separate 96-well plate with 50 µl/well substrate mix and incubated for 30 min at room temperature in the dark. Stop solution  was then added and pigmented particles were broken up using an oscillator . Absorbance was measured at 490 nm using an ELISA analyzer.Each group was centrifuged at 250 × g for 4 min and 50 Mean absorbance values were calculated for each group. In order to obtain the corrected values for A (test), T (target cells spontaneous release) and E (effector cells spontaneous release), the mean absorbance value for the blank control group was subtracted from the mean absorbance values for the test, target cells spontaneous release and effector cells spontaneous release groups. The mean absorbance values in target cell maximum release group minus the mean absorbance values in corrected volume group, and the corrected Tmax was obtained. Cytotoxic activity (%)=(A−E−T)/(Tmax−T)×100%.CAPRI cells  and CIK cells  were transferred and the stimulating substance was added, under aseptic conditions.µl/well RPMI 1640 medium and incubated at room temperature for 10 min. CAPRI or CIK cells at concentrations of 1×106/ml and 5×105/ml were then plated (100 µl/well). The positive and negative control groups for each cell type were plated as follows: Negative control, 1×106 and 5×105 cells/ml with 10 µl/well RPMI 1640 medium with 10% FBS; positive control, 1×106 and 5×105 cells/ml with phytohemagglutinin cell stimulator working fluid. The well plate was then covered and incubated at 37°C in a 5% CO2 incubator for 20 h. Following incubation, the cells and medium were removed and added to ice-cold deionized water (200 µl/well). Hypotonic cell lysis was performed with Hypotonic Lysis Buffer  at 4°C for 10 min. Cells were then washed five times for 60 sec each with 1X washing buffer  and then dried on absorbent paper.ELISPOT plates were precoated with 200 µl; in ELISPOT kit) was added to each experimental well and incubated at 37°C for 1 h. Cells were then washed, as above, and dried on absorbent paper. Horseradish peroxidase-avidin diluted antibody working solution (100 µl) was added to each experimental well and incubated at 37°C for 1 h. Cells were then washed, as above, and dried on absorbent paper.A biotin-labeled diluted antibody solution  and incubated at room temperature in the dark for 25 min. In order to terminate staining, liquid was poured out in each well, the base plate was opened and deionized water  was used to wash the wells five times. The plates were then placed at room temperature in the dark, with the base closed, until they dried.Each experimental well was stained with 3-amino-9-ethylcarbazole working solution  was used for data analysis. Values are expressed as the mean ± standard deviation. Independent samples t-tests were used for comparisons between two groups. P<0.05 was considered to indicate a statistically significant difference between values.7. Cultured CIK cells began to proliferate within 3 days and entered the proliferation stage within 5 days. Following 14 days, cultured cells reached a density of (60.21±6.08)×107. Following 3 days of culture, the proliferation rate of CIK cells was significantly faster compared with CAPRI cells; of note, at day 5, the proliferation rate of CIK cells was ~3 times that of CAPRI cells (P<0.001); at day 14, CIK cells had proliferated ~60 times compared with day 1, which was ~10 times that of CAPRI cells at day 14 (P<0.001) ×10P<0.001) .Cells were harvested and examined under an inverted microscope, which revealed that cells grew as colonies in suspension  and 3. CThe results of the cytotoxic activity of CAPRI and CIK cells against K562 cells are shown in The results of the cytotoxic activity of CAPRI and CIK cells against MCF-7 cells are shown in 6 and 5×105 cells/ml). In CAPRI cells, the number of IFN-γ SFCs detected were 126.2±10.31 and 48.8±10.99, respectively, which were significantly lower compared with the corresponding values in CIK cells, 409.3±7.76 and 159.3±15.45 (P<0.001)  (As shown in P<0.001) . The numP<0.001) .Developments in the fields of molecular tumor biology, cell biology and immunology of cancer have resulted in a more comprehensive and in-depth understanding of tumors . Adoptiv+ T helper (Th) cells and CD8+ cytotoxic T lymphocytes (CTLs) -restricted antitumor activity of NK cells  cells, NK-like T cell lymphocytes (NKT cells), dendritic cells, CD4s (CTLs) . Th cells (CTLs) . CIK celNK cells . The potNK cells ; inducedNK cells ; inducedNK cells ; and proNK cells .The antitumor mechanisms of CIK cells have been established; however, CAPRI cells are a novel method of treatment, for which the antitumor mechanisms are relatively undetermined . It was CAPRI cells were reported to require synergy of human leukocyte antigen (HLA)-I and HLA-II expression for successful tumor cell lyses . This de+ and CD8+ T cells, which make up 80% of the CAPRI effector cells, while IFN-γ is predominantly produced by CD8+ T cells and NK cells, of which NK cells are the major effector cells of CIK cells ×107, respectively, following treatment for 14 days. The cell proliferative activity of CAPRI cells was significantly lower than that of CIK cells (P<0.001). Compared with the 1st day, the proliferative rate of CAPRI was 3 times higher at the 5th and 6 times higher at the 14th days, while CIK was 10 times higher at the 5th and 60 times higher at the 14th days.The results of the current study demonstrated that the cell density of CAPRI and CIK cells was (6.32±1.23)×10In conclusion, the experimental results of the present study demonstrated that CAPRI cells have a weaker cytotoxic effect on NK target cells of K562 leukemia cells compared with their potent cytotoxic effect on the NK-insensitive solid tumors MCF-7 cells. These experimental methods may be used as a model to further explore the cytotoxic activity of tumor cells in other entities and to provide guidance for future clinical studies and treatment options. The cytotoxic activity of CIK cells is not MHC restricted, however CAPRI cells filter MHC of cells, which remedies the deficiency of CIK cells treatment. The ELISPOT technique may be used to detect the secretion of cytokines for these two cell types prior to and following treatment for clinical efficacy assessment. Since the culture time and antitumor mechanisms between CAPRI and CIK cells differed, this may offer the possibility for combination of the two cell types for antitumor therapy; however, further studies and clinical trials are required in order to confirm the effectiveness of this combined therapy.


Escherichia coli expressing three clones that were derived from the enrichment culture that used crystalline cellulose showed activity against crystalline cellulose. In addition, these three enzyme solutions generated a reducing sugar from cedar wood powder. From these results, the construction of a metagenomic library from cultures that were repetition enriched using crystalline cellulose demonstrated that this technique is a powerful tool for obtaining cellulases that have activity toward crystalline cellulose.To obtain cellulases that are capable of degrading crystalline cellulose and cedar wood, metagenomic libraries were constructed from raw soil sample which was covered to pile of cedar wood sawdust or from its enrichment cultures. The efficiency of screening of metagenomic library was improved more than 3 times by repeating enrichment cultivation using crystalline cellulose as a carbon source, compared with the library constructed from raw soil. Four cellulase genes were obtained from the metagenomic libraries that were constructed from the total genome extracted from an enrichment culture that used crystalline cellulose as a carbon source. A cellulase gene and a xylanase gene were obtained from the enrichment culture that used unbleached kraft pulp as a carbon source. The culture supernatants of The online version of this article (doi:10.1186/2193-1801-3-365) contains supplementary material, which is available to authorized users. Lignocellulosic pools are a renewable source of feedstock for biofuel production. Cellulose, hemicellulose, and lignin are closely associated, and covalent cross-linkages have been suggested to occur between lignin and polysaccharides. Cellulose is the most abundant biomass in nature. Cellulose has great potential for a number of applications, including biofuel production. The cost of ethanol production from lignocellulosic materials is high, and the main challenge is high cost of the hydrolysis process. Numerous studies have been performed to improve the hydrolysis of lignocelluloses by pretreatment  are necessary for cost reduction of biorefinery process. Metagenomics is the study of genetic material recovered directly from environmental samples and it is a powerful tool to identify novel biocatalysts, natural products, and new molecular structures  and its enrichment cultures (1st and 2nd enrichment culture with Avicel or unbleached kraft pulp as the carbon source) was extracted, and the 16S rRNA genes were amplified. The purified rRNA gene fragments were cloned into the pGEM-T easy vector. Recombinants were randomly selected, and clonal plasmids were extracted. The sequences of a few bacterial 16S rRNA gene clones (500–600 bp) were determined. A total of 16 clones : AB921992 ~ AB921995], 8 clones from 1st enrichment culture with Avicel [AN: AB21996 ~ AB22003], and 4 clones from 2nd enrichment culture with Avicel [AN: AB22004 ~ AB22007]) were analyzed to estimate the bacterial diversity in the original soil and Avicel enrichment cultures. The genes from the original soil bacteria were from several bacterial groups. However, 5 of 8 sequences from the 1st Avicel enrichment culture and all 4 clones from the 2nd Avicel enrichment culture were within the gamma-proteobacteria group. It was expected that the diversity of bacteria has lowered considerably by 2nd enrichment cultivation. Thus, Enrichment cultivation was stopped at 2nd time enrichment.C1A2) and encoded part of an identical genomic sequence (1A2). Two cellulase genes C1A1 and C1A2 contained in respective sequence 1A1 (AN: AB92208) and 1A2 (AN: AB922009) were identified from 1st Avicel enrichment culture. Four active cellulase clones, p2a1–p2a4 were isolated from the 2nd Avicel enrichment culture library . The sequences of p2a2 and p2a4 were the 1A2 gene. Clone p2a1, and p2a3 have independent cellulase  within geomic sequence 2A1(AN: AB9220010) and 2A3 (AN: AB9220011), respectively. Although no active clone was obtained from the 1st pulp enrichment culture, an active cellulase  and a xylanase  clone were identified in the 2nd pulp enrichment culture library .Five metagenomic expression libraries were constructed. Each library clone contained a 2–7 kb insert (average 4.3 kb), and 23,000–40,000 clones were screened Table , corresp1A1 cellulase gene were cellulase B (622 amino acids) of Cellvibrio mixtus  and endoglucanase (619 amino acids) of Hahella chejuensis KCTC 2396 . These homologs have a [glycosyl hydrolase family 5 (GH5)]-[carbohydrate-binding module 6 (CBM6)]-[CBM6] domain structure; therefore, C1A1 is predicted to have a [GH5]-[CBM6]-[CBM6] domain structure . One ORF (C1A2) had a conserved putative cellulase (endoglucanase)-like sequence with [CBM] and]-[merpin/A5-protein/PTPmu (MAM)] domain  which only have a catalytic domain. Sequence 2A3 was an analog of C1A1 with 98.4% similarity at the amino acid level.Sequence analyses of the cloned cellulase and xylanase genes revealed no significant nucleotide homology to known cellulase genes in the databases Table . The bes2P3 contained a putative glycoside hydrolase gene (C2P3) and 3 other ORFs, and sequence 2P1 consisted of a putative endo-1,4-β-xylanase (X2P1) and 2 other ORFs. Cellulase C2P3 (414 amino acids) consisted only of a glycosyl hydrolase domain. Phylogenetic analyses of the isolated cellulases revealed that the isolated glycosyl hydrolases were grouped in four independent branches where they clustered with typical GH family members, while xylanases were branched from the cellulase cluster  had one or two CBMs, and their cellulase activity was measured using crystalline cellulose (Avicel) and wood powder (extract-free cedar powder). The three crude cellulases generated reducing sugar from crystalline cellulose (Avicel). In addition, they generated the equivalent level of reducing sugar from extract-free cedar wood powder (Table Six glycosyl hydrolase genes (5 cellulases and a xylanase) were obtained from enrichment cultures. Three genes (C1A2) has been obtained from metagenomic libraries of 1st and 2nd enrichment culture. When unbleached pulp was used as a carbon source for enrichment, glycoside hydrolase genes (cellulase and xylanase) were provided with hit rate of 0/100 Mb (1st enrichment) and 2/172 Mb (2nd enrichment). Although the screening efficiency improved by repeating enrichment, the efficiency was greatly different between enrichment cultures using Avicel and unbleached pulp. It was presumed that non-cellulolytic bacteria have grown vigorously in enrichment culture with unbleached pulp than enrichment culture with Avicel, because unbleached pulp has more various components that are easy to utilize such as amorphous cellulose and hemicellulose than avicel as crystaline cellulose. The efficiency of screening was improved by repeating enrichment and was also possible to improve more than three times by enrichment cultivation with strong selective pressure. Because the 16S rRNA gene diversity suggested that bacterial diversity decreased by the enrichment and the positive cellulase clones contained the same cellulase gene were identified from 1st and 2nd enrichment culture with Avicel, repeating the enrichment cultivation reduced the cellulase gene diversity  has been investigated  was the vector for cloning experiments.C. japonica sawdust sediment was collected from a backyard, in Kyushu University, Fukuoka, Japan. Enrichment was performed in modified M9 minimal medium with microcrystalline cellulose (Avicel®) or unbleached hardwood kraft pulp as a carbon source. Enrichment cultures from the soil sample were prepared by adding 1.0 g soil to 40 mL sterile minimal medium containing 20 g/L carbon source, 6.0 g/L Na2HPO4, 3.0 g/L KH2PO4, 0.5 g/L NaCl, 1.0 g/L Yeast Extract (Difco), 1 mM MgSO4, 0.1 mM CaCl2, and 0.04 mM FeSO4. Cultures were grown at 37°C for 7 days at 120 rpm (1st enrichment culture). Then, 0.5 mL culture medium was transferred to new minimal medium and incubated for an additional 7 days (2nd enrichment culture). Bacteria were recovered from each enrichment culture by centrifugation , and the obtained pellets were washed with 50 mM Tris-HCl (pH 8.0). The resulting precipitates were used for DNA extraction.To construct metagenomic libraries, a soil sample under g for 10 min. The pellet was re-extracted, and the combined supernatants were extracted with an equal volume of phenol-chloroform . The aqueous phase was recovered by centrifugation, and the DNA was precipitated with 0.6 volume of isopropanol at room temperature for 20 min. A pellet of crude nucleic acids was obtained by centrifugation at 12,000 × g for 10 min. After washing with 70% (v/v) ethanol, the DNA was re-suspended in 1 mL 10 mM Tris-HCl (pH 8.5) and purified by electrophoresis in a 1.0% low melting point agarose gel . Then, DNA fragments over 22 kb were collected and extracted. Recovered DNA was precipitated with an equal volume of isopropanol, washed with 70% ethanol, and air-dried. Dried DNA was dissolved with 50 μL TE buffer .DNA was extracted using a protocol based on the direct lysis method of Zhou et al.  with min2 and 7.5% polyethylene glycol 6000. The DNA fragments were ligated into the pUC19 vector, which had been digested with BamHI and phosphatase treated with the TaKaRa Ligation Kit v2.1. E. coli DH5α was transformed with ligated pUC19, and transformants were selected on Luria-Bertani medium (LB) agar plates containing ampicillin (100 μg/mL) and 0.2% (w/v) rimazol brilliant blue dyed CMC or xylan . All sequences (500–600 nucleotides) were compared with those in the GenBank database (http://www.ncbi.nlm.nih.gov/BLAST) using BLAST. Multiple sequence alignment was carried out using ClustalX software, and a phylogenic tree was generated by the neighbor-joining method . The supernatants were concentrated 7.5 times by ultrafiltration. To measure the cellulase activity, crude concentrates were incubated with 1.0% (w/v) Avicel or cedar wood powder (extract free) in 100 mM Tris-HCl (pH 6.8) for 2 h at 50°C. The released reducing sugars were measured as D-glucose equivalents