The isoprenoid family of compounds is estimated to contain ∼65,000 unique structures including medicines, fragrances, and biofuels. Due to their structural complexity, many isoprenoids can only be obtained by extraction from natural sources, an inherently risky and costly process. Consequently, the biotechnology industry is attempting to genetically engineer microorganisms that can produce isoprenoid-based drugs and fuels on a commercial scale. Isoprenoid backbones are constructed from two, five-carbon building blocks, isopentenyl 5-pyrophosphate and dimethylallyl 5-pyrophosphate, which are end-products of either the mevalonate or non-mevalonate pathways. By linking the HMG-CoA reductase pathway  to the mevalonate pathway, these building block can be synthesized enzymatically from acetate, ATP, NAD(P)H and CoA. Here, the enzymes in these pathways are used to produce pathway intermediates and end-products in single-pot reactions and in remarkably high yield, ∼85%. A strategy for the regio-specific incorporation of isotopes into isoprenoid backbones is developed and used to synthesize a series of isotopomers of diphosphomevalonate, the immediate end-product of the mevalonate pathway. The enzymatic system is shown to be robust and capable of producing quantities of product in aqueous solutions that meet or exceed the highest levels achieved using genetically engineered organisms in high-density fermentation. Enzymes have been honed over evolutionary time to accomplish specific catalytic tasks Improved access to large numbers of pure proteins, and a rapidly increasing repertoire of well characterized enzymes, isoenzymes and mutants have substantially increased the potential to utilize The medicinal values of isoprenoids have been documented as early as 168 BC The carbon backbones of isoprenoids are assembled from two fundamental building blocks, isopentenyl 5-pyrophosphate and dimethylallyl 5-pyrophosphate R, S)-[2H3]methyl-mevalonolactone, -mevalonolactone, acetyl-CoA, glutamate dehydrogenase (bovine liver), acetyl-CoA synthetase (Baker's yeast), myokinase (rabbit muscle) and lysozyme (bovine) were purchased from Sigma. Sodium acetate , sodium acetate  and D2O (99%) were purchased from Cambridge Isotope Laboratories, Inc. All other chemical reagents were of the highest grades available. Plasmids pET28efTR , pET28efS2 A100G , and pET28-efR  were generous provided by Prof. V. W. Rodwell Staphylococcus aureus), phosphomevalonate kinase (Streptococcus. pneumoniae), and diphosphomevalonate decarboxylase (Streptococcus. pneumoniae) were expressed and purified as described previously Lactate dehydrogenase (rabbit muscle), pyruvate kinase (rabbit muscle), and inorganic pyrophosphatase (Baker's yeast) were purchased from Roche Applied Science. (E. coli BL21(DE3) freshly transformed with the expression plasmid of interest. The cells were cultured to an OD595 of 0.8, protein expression was induced by the addition of isopropyl-1-thio-β-D-galactopyranoside , and the incubation was continued for 4 h at 37°C. The culture temperature was then shifted to 18°C and incubation was continued for 16 h. The cells were then harvested by centrifugation . The MVK 9-GST-tag to the N-terminus of the enzyme; whereas, the acetoacetyl-CoA thiolase 6-tag to the N-terminus. Dual-tag proteins were purified using a GST resin followed by a His resin. All buffers and columns were equilibrated at 4°C prior to use. Purification began by suspending cell pellets (5.0 ml/g cell paste) in Buffer A  supplemented with lysozyme (0.10 mg/ml), PMSF (290 µM), and pepstatin A (1.5 µM). EDTA (1.0 mM) was added to Buffer A when purifying dual-tag systems. After suspension for 1 hr at 4°C, cells were disrupted by sonication and debris was removed by centrifugation . Supernatants containing dual-tag proteins were loaded onto a Glutathione Sepharose 4 FF column equilibrated with Buffer A + EDTA, the column was then washed with three column volumes of the same buffer, and protein was eluted using Tris/Cl , KCl (500 mM) and reduced glutathione (10 mM). Supernatants containing singly tagged proteins, or GST-resin eluants containing dual-tag proteins, were loaded onto a Ni-NTA column equilibrated with Buffer B . The column was washed with Buffer C , and fusion protein was eluded with Buffer D . Glycerol was then added to the singly-tagged eluants (5% v/v) and stored (see below). Tags were removed from the dual-tag proteins by incubation with PreScission protease +  containing DTT  and KCl (100 mM). Following proteolysis, the dialysate was passed over a GSTrap column to remove the GST-tagged protease. The purity of the single- and double-tags proteins was estimated, using SDS-PAGE, at >85 and >95%, respectively. Eluants containing purified proteins were frozen rapidly and stored at −80°C.37°C LB/ampicillin media was inoculated with Acetyl-CoA synthetase activity was monitored by coupling the production of AMP to the oxidation of NADH 398 = 0.136 mM−1 cm−1), acetate (2.0 mM), CoA (2.0 mM), ATP (4.0 mM), PEP (6.0 mM), MgCl2 (1.0 mM + [nucleotide]), KCl (50 mM), β-ME (10 mM). Acac-Coa thiolase activity was monitored by following the appearance of acac-CoA (acetoacetyl-CoA) at 302 nm 2 (2.0 mM). HMG-CoA synthase activity was monitored at 386 nm (−1 cm−1) by coupling the production of 3-hydroxy-3-methyl glutaryl-CoA to the oxidation of NADPH using HMG-CoA reductase. The conditions were: HMG-CoA reductase (1.0 µM), acac-CoA (1.0 mM), Ac-CoA (1.0 mM), NADPH (1.5 mM), KCl (50 mM), β-ME (10 mM). HMG-CoA reductase activity was monitored by following oxidation of NADPH. The conditions were: 3-hydroxy-3-methyl glutaryl-CoA (0.50 mM), NADPH (0.20 mM), KCl (50 mM), β-ME (10 mM). Mevalonate kinase activity was monitored by coupling the production of ADP to the oxidation of NADH 339 = 6.22 mM−1 cm−1), PEP (7.0 mM), mevalonate (135 µM), ATP (5.0 mM), MgCl2 (1.0 mM + [nucleotide]), KCl (50 mM), β-ME (10 mM). Phosphomevalonate kinase activity was monitored by coupling the production of ADP to the oxidation of NADH DPM Decarboxylase activity was monitored by coupling the production of ADP to the oxidation of NADH + (50 mM), pH 8.0, and T = 25±2°C.To establish conditions for the synthesis of DPM, the activity of each enzyme was assessed under the synthesis conditions. Apparent kinetic constants were extracted from reaction progress curves 2 (1.0 mM + [ATP]), β-ME (10 mM), Hepes/K+ (50 mM), pH 8.0, T = 25±2°C. Reactions progress was monitored by following the oxidation of NADPH associated with the HMG-CoA reductase reaction. DPM formation was assayed by adding an aliquot the DPM-synthesis reaction into a DPM decarboxylase assay mixture (DPM-DC (0.10 µM), PK (4.0 U/ml), LDH (8.0 U/ml), NADH (200 µM), PEP (4.0 mM) ATP (2.0 mM), MgCl2 (1.0 mM + [nucleotide]), KCl (50 mM), β-ME (10 mM), Hepes/K+ (50 mM), pH 8.0, T = 25±2°C) and monitoring NADH oxidation at 340 nm. The assay-reaction dilution was sufficient (330-fold dilution) to prevent the HMG-CoA reductase reaction from contributing significantly to the measurement. The reactions yielded essentially quantitative conversion of acetate to the endproduct, DPM.DPM was synthesized in a one-pot reaction using the following conditions: Ac-CoA synthetase (2.0 µM), acac-CoA thiolase (2.0 µM), HMG-CoA synthase (4.0 µM), HMG-CoA reductase (2.0 µM), mevalonate kinase (2.0 µM), phosphomevalonate kinase (3.0 µM), pyruvate kinase (5.0 U/mL), myokinase (2.0 U/ml), inorganic pyrophosphatase (2.0 U/ml), ATP (5.0 mM), PEP (10 mM), acetate (12 mM), CoA (5.0 mM), NADPH (10 mM), KCl (50 mM), MgCl2 (5.0 mM), Hepes/K+ (50 mM), pH 8.0. The reactants were mixed gently for 10 hr at T = 25±2°C. Reaction progress was monitored by assaying aliquots of the reaction for AMP synthesis using the Ac-CoA synthetase assay described above. The conversion of CoA to labeled Ac-CoA was >95%.The synthesis of regiospecifically labeled DPM requires appropriately labeled Ac-CoA. Labeled Ac-CoA precursors were synthesized using the following conditions: acetyl-CoA synthetase (2.0 µM), pyrophosphatase (2.0 U/ml), labeled acetate (4.0 mM), CoA (4.0 mM), ATP (4.0 mM), MgClsee, The synthesis of acac-CoA was achieved using the conditions identical to those described for the synthesis of Ac-CoA with the exception that acac-CoA thiolyase (2.0 µM) and DTNB  were present. DNTB reacts with CoA and was used to draw the acac-thiolase reaction forward. The DTNB reaction was monitored at 412 nM 13C or 2H) was prepared from CoA and labeled acetate as described above , HMG-CoA synthase (4.0 µM), HMG-CoA reductase (2.0 µM), MVK (2.0 µM), PMK (1.0 µM), PEP (10 mM), NADPH (5.0 mM), unlabelled acac-CoA (2.0 mM), ATP (5.0 mM), KCl (50 mM), and β-ME (10 mM). The unlabeled acac-CoA was prepared as describe above , β-ME (10 mM), Hepes/K+ (50 mM), pH 8.0, T = 25±2°C. Reaction progress was monitored as described above , acac-CoA thiolase (7.0 µM), HMG-CoA synthase (10 µM), HMG-CoA reductase (7.0 µM), mevalonate kinase (5.0 µM), phosphomevalonate kinase (3.0 µM), pyruvate kinase (10 U/mL), myokinase (5.0 U/ml), inorganic pyrophosphatase (5.0 U/ml), ATP (100 mM), PEP (800 mM), acetate (340 mM), CoA (5.0 mM), NADPH (300 mM), MgClove see, . Under t2 (220 mM), β-ME (10 mM), Hepes/K+ (50 mM), pH 8.0, T = 25±2°C. The conversion of acetate to IPP yields nine IPP-equivalents of pyruvate , two for conversion of Mev to DPM and one for the decarboxylation of DPM to IPP). Pyruvate was quantitated by adding an aliquot of the IPP-synthesis reaction to a lactate dehydrogenase assay mixture: (LDH (8.0 U/ml), NADH (200 µM), KCl (50 mM), β-ME (10 mM), Hepes/K+ (50 mM), pH 8.0, T = 25±2°C). Dilution of the synthesis reaction was sufficient (>500-fold) to prevent enzymes from the reaction from contributing significantly to the pyruvate measurements. Sixty-nine percent of the acetate was converted to DPM.IPP synthesis was accomplished in a one-pot reaction using the following conditions: Ac-CoA synthetase (7.0 µM), acac-CoA thiolase (10 µM), HMG-CoA synthase (12 µM), HMG-CoA reductase (10 µM), mevalonate kinase (7.0 µM), phosphomevalonate kinase (5.0 µM), diphosphomevalonate decarboxylase (3.5 µM), pyruvate kinase (20 U/mL), myokinase (7.0 /ml), inorganic pyrophosphatase (7.0 U/ml), ATP (200 mM), PEP (800 mM), acetate (340 mM), CoA (5.0 mM), NADPH (300 mM), MgClR/S)-mevalonate (370 mM), ATP (50 mM), PEP (350 mM), MgCl2 (60 mM), β-ME (7.0 mM), Hepes/K+ (50 mM), pH 8.0, T = 25±2°C. Reaction progress was monitored as described above -mevalonate is 1∶1 R)-mevalonate in the (R/S)-mixture was converted to DPM.DPM synthesis was accomplished in a reaction using the following conditions: mevalonate kinase (5.0 µM), phosphomevalonate kinase (3.0 µM), pyruvate kinase (10 U/mL), -mevalonate (375 mM), ATP (50 mM), PEP (450 mM), MgCl2 (60 mM), β-ME (7.0 mM), Hepes/K+ (50 mM), pH 8.0, T = 25±2°C. The conversion of (R/S)-mevalonate to IPP was monitored by following the formation of pyruvate using lactate dehydrogenase -mevalonate in the (R/S)-mixture was converted to IPP.IPP synthesis was accomplished in a reaction using the following conditions: mevalonate kinase (5.0 µM), phosphomevalonate kinase (3.0 µM), diphosphomevalonate decarboxylase (1.6 µM), pyruvate kinase (10 U/mL), , and the column was “washed” with five volumes of equilibration buffer. The compounds were eluted using a 750 ml, linear salt gradient (0–1.0 M KCl) at 2.0 mL/min. DPM eluted at 0.32 mM KCl and contained <1% nucleotide. To remove excess KCl and concentrate the DPM, the purified compound was loaded onto a 5.0 ml bed of AG MP-1 equilibrated with NH4HCO3 . The column was then “washed” with five volumes of NH4HCO3  before eluting the DPM with 1.8 volumes of NH4HCO3 . Excess NH4HCO3 was removed by rotary evaporation at 45°C. The desalted compounds were dissolved in ultra pure water (2.0 mL) and the solution was adjusted to pH 7.5 with KOH. NH4HCO3 in the desalted, purified DPM was measure using an enzymatic assay that couples the reduction of NADP+ to the synthesis of glutamate from + (0.20 mM), glutamate dehydrogenase (14 U/mL), Hepes/K+ (45 mM) pH 8.0 at T = 25±2°C. The +  at −80°C.To maximize the purity and recovery of DPM, PEP (which chromatographs near DPM) was converted to pyruvate by adding one PEP-equivalent of ADP to the synthesis reaction mixture. Small and large molecules were separated by ultrafilitration (10-kDa cutoff). The small-molecule filtrate was passed through a 35 mL bed of anion exchange resin (AG MP-1) equilibrated with Hepes/KOne dimensional NMR was used to confirm the structure and isotopic labeling of the DPM isotopomers. A Bruker DRX 300 MHz spectrometer equipped with a 5 mm broadband probe was used to acquire data. Sample temperature was 25±2°C. Proton spectra were the average of 32 scans (64K points each) acquired over 20 ppm using a 1.0 s recycle delay. The residual water signal was suppressed by presaturation of the HOD resonance. Spectra were processed with 1.0 Hz line broadening, and proton chemical shifts were referenced to 3-(trimethylsilyl) propionate i - vi, i, and regenerated in reactions ii, iii and iv  at a fixed concentration and titrating each preceding enzyme successively until the DPM-synthesis rate was 80–90% of the maximum rate achievable at each step. For example, MVK (v) was titrated at a fixed concentration of PMK until the rate of DPM synthesis became independent of MVK concentration – the maximum rate. The MVK concentration was then adjusted to allow 80–90% of the maximum rate, and an analogous procedure was performed with HMG-CoA reductase (iv). The procedure was performed in succession for each enzyme in the pathway to determine the relative enzyme concentrations to be used in the synthesis. Once relative concentrations were established, the absolute concentrations were set to achieve the desired reaction times, which ranged from 8–72 hr. Mevalonate kinase from S. aureus was selected because, unlike the S. pneumoniae enzyme, it is not allosterically inhibited by DPM The relative enzyme concentrations used in the DPM syntheses were determined empirically by adjusting concentrations such that flux through the pathway was not rate-limited by any single step. This was accomplished by setting PMK , was set at 5.0 mM, which ranges from 5.8–68×Km. Typical substrate concentrations of the other reactants were as follows: acetate ; CoA ; NADPH ; and PEP . Under these conditions, and using the enzyme concentrations detailed in Synthesis of (R)-diphosphomevalonate . Each step adds a single acetate to the CoA thioester R-group. The pattern of acetate incorporation into the R-group, and ultimately DPM, is shown in via solvent exchange with exchange-sensitive intermediates. Achieving certain labeling patterns required removal of enzymes by ultrafiltration at intermediate stages of the synthesis, and/or that reactions were run in D2O (see below).The regiospecific incorporation of isotopes has proven extremely valuable in the elucidation of metabolism 2H3]acetate or -[6-2H3]mevalonolactone as starting material . Reactions were complete after 22 hrs, and virtually quantitative conversion of starting material to DPM was achieved in all cases. Approximately ∼86% of the maximum theoretical maximum yield of DPM was obtained after purification  and inorganic pyrophosphatase (ix)  and DTNB (in excess over Ac-CoA) were then added to form acac-CoA. DTNB reacts quantitatively with CoA 2O. 1H NMR confirmed that exchange was complete and occurred exclusively at the C4-position of DPM . The reactions were essentially quantitative and the production of DPM was ∼96% of the theoretical maximum.The synthesis of [4-n of DPM . It is n2H2]DPM. Unlabeled acac-CoA was synthesized, the enzymes used in the synthesis were “strained” from the reaction by ultrafiltration, labeled Ac-CoA was then added along with the enzymes, and reagents needed to complete the synthesis of DPM  is split to into an ABX pattern by the incorporation of 13C  was observed at 0.50 M acetate. PEP and NADPH could be increased to near saturation  without significant decrease in velocity, and ATP could be added to 0.15 M without inhibition or noticeable precipitation. The concentration-optimized system contained acetate, ATP, PEP and NADPH at 0.35, 0.10, 0.40, 0.30 M, respectively, and yielded DPM and IPP at 22 and 18 g/liter, respectively −63% and 69% conversions of acetate to product (R/S) mevalonate, ATP, and PEP at 0.370, 0.05, 0.35 M, respectively, and yielded DPM and IPP at 42 and 35 g/liter, respectively −73% and 76% product yields (Reactions that yield highly concentrated product).Given the considerable societal value of isoprenoids, the difficulties obtaining them, and the current efforts to bio-synthesize these compounds at commercial scale, it was of interest to assess the potential of the  product . Productt yields . The reaThe syntheses outlined in the preceding paragraph are highly scalable. Reaction yields were independent of volume from 0.10 ml–1 liter and are expected to be similar at larger volumes. Under the high ionic strength conditions of these assays, the enzymes proved to be quite stable. The majority lost ≤20% of their activity over 2 days at room temperature. The exceptions were acetoacetyl-CoA thiolyase and inorganic pyrophosphatase, which lost 63% and 72% of their activity, respectively, over this time period.quinghao plant (Artemisia annua) at ∼3 mg/g dry weight. In contrast, genetically engineered tobacco produces artemisinin at ∼0.8 mg/g dry weight E. coli as the host, pathway optimization has yielded ∼0.3 g/liter of artemisinic acid in shaking flasks E. coli has produced yields as high as 23g/liter E. coli cultures E. coli have produce taxadiene  at ∼1g/liter in shaking flasks E. coli grown in shaker flasks Attempts to genetically engineer plants and bacteria to produce commercial quantities of isoprenoids have met with variable success. Artemisinin, a potent antimalarial, is currently isolated from the While these efforts have helped define the complexities associated with expressing and controlling the isoprenoid biosynthetic pathway in living organisms, only fermentation in conjunction with genetic engineering is yielding product quantities required for successful commercial application. The cell-free approach described here yields product quantities that are comparable to, or exceed those achieved in high-density fermentation and have the advantage that product is formed in a simple aqueous system from which it can be recovered readily.The enzymes that comprise the HMG CoA reductase and mevalonate pathways have been used along with enzymatic substrate-recycling and product-removal systems to efficiently synthesize intermediates and end products of these pathways in high yield. Strategies for using these enzymes to regio-specifically position isotopes in the isoprenoid backbone are described and used to synthesize and purify isotopomers of DPM, the immediate end product of the mevalonate pathway. The enzymatic platform is robust and produces isoprenoid precursors, DPM and IPP, in quantites ranging from 20–40 g/liter. These values meet or exceed all published values for isoprenoid production using genetically engineered organisms. The platform produces product in simple aqueous solutions, which, in most cases, will make isoprenoid isolation far simpler than extraction from high-density fermentums. These favorable attributes recommend the enzymatic platform as a valuable alternative to cell-culturing methods as a clean, sustainable, non-biocompetitive method for the production of isoprenoids.Figure S11H NMR spectra of DPM isotopomers. The specificity and efficiency of labeling of the isotopomers were estimated based on the integration of the 1H signals. The results were as follows: [4-2H2]DPM , [6-2H3]DPM  and DPM .(TIF)Click here for additional data file.Figure S231P NMR spectrum of DPM. The resonance positions, splitting pattern and nearly identical integrated intensities of the <- and -resonances indicate an intact pyrophosphoryl-moiety. The asterisk identifies the resonance of phosphocreatine, which was added as an internal standard. The absence of a peak at ∼5 ppm indicates that phophomevalonate is undetectable.(TIF)Click here for additional data file.Text S1(DOCX)Click here for additional data file.


The results showed good correlation (R2 > 0.99) between time-resolved fluorescent immunochromatographic strip test and high performance liquid chromatography (HPLC). Soybean sauce samples analyzed using time-resolved fluorescent immunochromatographic strip test revealed that 64.2% of samples contained aflatoxin B1 at levels ranging from 0.31 to 12.5 µg·kg−1. The strip test is a rapid, sensitive, quantitative, and cost-effective on-site screening technique in food safety analysis.Rapid and quantitative sensing of aflatoxin B1 with high sensitivity and specificity has drawn increased attention of studies investigating soybean sauce. A sensitive and rapid quantitative immunochromatographic sensing method was developed for the detection of aflatoxin B1 based on time-resolved fluorescence. It combines the advantages of time-resolved fluorescent sensing and immunochromatography. The dynamic range of a competitive and portable immunoassay was 0.3–10.0 µg·kg Aflatoxins are a group of extremely toxic metabolites produced by Aspergillus flavus and A. parasiticus, which grow on foods . Althoug−1 in the European Union and 5.0 µg·kg−1 in China [Soybean sauce is a widely consumed condiment worldwide and used extensively as a seasoning due to its unique taste and aroma ,7. It isin China ,9.Rapid, quantitative, low-cost, and reliable tests for AFB1 detection in soybean sauce are urgently needed. Generally, AFB1 is detected via high performance liquid chromatography (HPLC), ultrahigh performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS), enzyme-linked immunosorbent assay (ELISA) and membrane matrix immunoassays ,11,12. IQuantitative fluorescent immunosensing methods have been developed recently for the analysis of aflatoxins. They include fluorescence polarization immunoassay and time-resolvedfluorescence immunoassay (TRFIA). Liu et al. reported an extract-free immunochromatographic assay based on fluorescent microsphere probes for qualitative AFB1 detection in soybean sauce, resulting in a visible LOD of 2.5 mg/L . HoweverWe have developed a competitive and quantitative immunoassay in which Eu-nanospheres were conjugated with antibodies against AFB1, serving as the immunoreagent. The portable assay was used to record the time-resolved fluorescence. The limit of detection (LOD), dynamic range, and recovery were recorded. This proposed method facilitates rapid, sensitive and extensive quantification of AFB1 and other toxins in soybean sauce.2a-BSA and rabbit anti-mouse IgG were purchased from Sigma-Aldrich . Mannitol, sucrose, bovine serum albumin (BSA) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and polyvinyl pyrrolidone (PVP) were obtained from Roche Applied Science . The nanospheres enclosing fluorescent europium (Eu-nanospheres) measuring 190 nm in diameter and 1.02% solid content were supplied by Shanghai Uni Bio-tech Company Limited . The 95 NC membranes were purchased from Millipore Corporation . All aqueous solutions were prepared in ultra-pure water of 18.2 MΩ purified using a Milli-Q quality water system . All chemicals were of analytical grade unless otherwise stated.AFB1 standard solutions, AFB3G1 showed high specificity to aflatoxin B1, with the following cross-reactivities to aflatoxin B1, B2, G1 and G2: 100%, 6.4%, <0.02% and <0.02%, respectively. The sensitivity (expressed by IC50) of MAb 3G1 to aflatoxin B1 was 1.6 ng/mL. Soybean sauce samples were purchased from the local market in Wuhan, China.The antibody (Anti-AFB1 mAb3G1) was produced in our laboratory and further purified with a protein G immunoaffinity column ,23. The The portable time resolved fluorescent immunochromatographic (TRFIA) apparatus was homemade. The signal was detected under the exciting light at a wavelength of 365 ± 10 nm, and signal was acquired at 613 ± 10 nm using the photomultiplier tube (PMT) . A typic−1, pH 7.2). Next, 200 μL of Eu-nanospheres was added to 800 μL of boric acid buffer and ultrasonicated  at 20 °C for 2 min. Then, 40 μL of EDC (15 mg/mL) was added and stirred for 15 min, followed by centrifugation at 17,000 g for 10 min. The precipitate was re-suspended in 1.0 mL of boric acid buffer by ultrasonication. A gradient of antibody concentrations was added, and the solution was vortexed for 12 h before centrifugation at 17,000 g for 10 min. Subsequently, the supernatant was obtained for protein quantification. The residue was re-suspended in 1.0 mL of boric acid buffer (containing 0.5% BSA), and vortexed for 2 h. The solutions were diluted and lyophilized in the penicillin bottle, and stored at 4 °C before use. The pH of the buffer solutions and the antibody dosage were optimized before the next step.EDC was used to develop active nanospheres with carboxyl groups in aqueous media. EDC is the most effective reagent used to link amino and carboxyl groups. The probe was synthesized according to the published method with slight modifications ,26,27. F2a-BSA and rabbit anti-mouse IgG were coated on NC membrane as T line and C line, respectively, using Bio Dot XYZ Platform at an appropriate jet rate. The distance between the T line and the C line was 10 mm. The NC membrane, sample pad and absorbent pad were laminated and pasted onto a plastic backing plate. The whole assembly of scale board was cut longitudinally and divided into strips using a guillotine cutter (CM 4000) measuring 4.0 mm × 70 mm and stored at 4 °C.The immunochromatographic (IC) strip contained an NC membrane, plastic backing plate, sample pad, and absorbent pad. The IC was prepared as previously described . The samAn indirect competitive immunoassay was performed using the IC strip. The Eu- probe was diluted 50, 100, 200, 300 and 500 times with protective reagents . The optimal Eu-probe concentration was similarly screened as a checkerboard titration in an ELISA using the IC strip. AFB1-free samples were analyzed during the development of the technique as well as in the validation study to verify the stand curve, the LOD, limit of quantitation (LOQ), recovery, and repeatability.−1, were analyzed with the IC strip test to estimate the sensitivity and linear range of the method, respectively. The fluorescent intensities from the T and C lines were recorded as (T) and (C), respectively, using portable TRFIA. The ratio of the value (T) to the value (C) was calculated as the signal ratio (Y). A calibration curve was fitted and expressed as Y versus the natural log of the AFB1 concentration (CAFB1) [AFB1-free sample extracts spiked with AFB1 standard solutions of 0.1, 0.3, 1.5, 6.0, 9.0, and 12.0 µg·kg (CAFB1) :Y  was evaluated for repeatability. The TRFIA method was validated by comparing the results of TRFIA and HPLC methods.v/v) was vortexed for 30 s, and the extract was homogenized for 2 min. Subsequently, the solution was centrifuged at 8000 g for 2 min. The supernatant (1.0 mL) was diluted with 3.0 mL buffer before measurement using TRFIA. After mild vortexing, 150 μL solution was dropped into the penicillin bottle. The IC strip was inserted into the penicillin bottle and incubated at 37 °C for 6 min. The components of soybean sauce were more complex, and included salts, different amino acids, and pigments. Therefore, appropriate dilution ratio and diluted buffers were needed to reduce matrix interference.A representative 10 mL sample placed in a 50 mL centrifuge tube and extracted with 20 mL of 70% methanol . The analytical column comprised an Alltima C18 . The mobile phase consisted of a mixture of HPLC grade methanol-water (55:45) eluted at a flow rate of 1.0 mL·minas 10 µL ,30.To establish the optimal reaction system, a boric acid buffer solution at different pH values of 7.0, 7.4, 7.8, 8.2, 8.7, and 9.0, was used, respectively. The prepared Eu-probe was dissolved in boric acid buffer and added to strips to detect the signal on T line using the portable TRFIA. The result showed that excessively high or low pH promoted conjugation coagulation and obliterated the color of T line under UV light. The optimal pH was 8.2. The result was close to the AFB1-antibody isoelectric point, as reported previously ,28.−1 AFB1 standard solution were evaluated for antibody digestion during the coupling of antibody and Eu-nanospheres. The result showed that at 30 ng, the antibodies showed better CVs on T line, along with better stability and sensibility.The Eu-probe was prepared with different mass ratios of the antibody and Eu-nanospheres  in boric acid buffer. First, the lowest stabilizing concentration of antibody prevented coagulation. Following conjugation, the supernatant was collected for protein quantification for determination of protein absorbance. The results suggested that the absorbance increased at an antibody level exceeding 30 ng and the Eu-nanospheres conjugated with antibody were less than 30 ng. Further, the antibody concentration less than 30 ng showed low fluorescent intensity on T line at the same dilution. The blank solution and 0.5 µg·kg2a-BSA and rabbit anti-mouse IgG were prepared with serial dilutions of 0.1 to 1.0 mg·mL−1. The optimal concentrations were similarly screened as a checkerboard titration in an ELISA. Inhibitory rates were recorded to determine theoptimum concentrations. It was found that 0.8 µL·cm−1 AFB2a-BSA (0.25 mg·mL−1) and 0.7 µL·cm−1 rabbit anti-mouse IgG (0.5 mg·mL−1) were optimized to dispense on the T line and C line, respectively.The IC strip is a single-step test based on a competitive immunoassay format . The conThe sample pad was used to reduce matrix interference. The solutions containing 1.0% BSA, 2.5% sucrose, 2.5% Tween 20, and 0.02% NaN3 in 0.01 mol/L PBS (pH 7.4),were used to block the sample pad, which guaranteed a clear redline on the test line under the UV light for negative samples .Based on the competitive theory, the extract solutions or AFB1 standard solutions were pipetted into the penicillin bottle. After mild vortexing, the IC strip was inserted into the penicillin bottle and incubated at 37 °C for 6 min.In the negative samples (fortified AFB1-free), the Eu-probe flowed laterally along the strip toward T line. The Eu-probe was captured by the immobilized AFB2a-BSA on T line. Excess Eu-probe was continuously captured by the rabbit anti-mouse IgG on the C line, as seen in 2a-BSA on the T line, but reached the C line. The surplus Eu-probe flow was laterally captured by the T line as indicated by a faint red color , as seen in In the positive sample, the AFB1 toxins conjugated with Eu-probe, the compound did not interact with the immobilized AFB2 > 0.99) , yielded good correlation (R > 0.99) . The con−1 for the soybean sauce matrices, LOQ was 0.3 µg·kg−1, at the wider dynamic ranges of 0.3–10.0 µg·kg−1, respectively. The proposed method showed a lower LOD compared with the ELISA, gold particlesstrip and the reported previously using fluorescent microsphere probe-based IC strip more than 3 times [For the LOD, the mean value of 5 repeated experiments was recorded in 20 blank soybean bean samples. Based on the sample standard curve, the AFB1 concentration was further calculated and defined as the LOD. The LOD was 0.1 µg·kg 3 times ,31. More−1, the soybean bean sample was diluted using 20% methanol.In the recovery experiment, different soybean sauce samples were used in the inter batch repeated 5 times. Results showed an average recovery of 111.0%, ranging between 87.2% and 114.3%, with mean CVs of 6.0% (3.4%–8.6%), as shown in To test the repeatability, random soybean sauce samples were determined by HPLC before use. As shown in The 70% methanol solutions were used to extract the AFB1 in samples. Previous reports  showed t−1, and the AFB1 concentration ranged from 0.31 to 12.5 µg·kg−1. Only 3 samples exceeded AFB1 concentration over 5.0 µg·kg−1. In order to verify the accuracy of TRFIA, we randomly selected 10 samples containing the 3 positive samples for accuracy. Therefore, as shown in A total of 53 soybean sauce samples were analyzed by TRFIA, and 34 of these samples (64.2% of 53 samples) were contaminated by AFB1. The average content was 0.81 µg·kg−1, considerable linear range of 0.3–10.0 µg·kg−1, and recovery of 87.2%–114.3%. The CVs ranged from 3.4% to 8.6%. Furthermore, the intra-batch and inter-batch comparison revealed excellent repeatability and reproducibility. A high concordance of TRFIA and HPLC methods was recorded in soybean sauce samples. The TRFIA method can be widely utilized in rapid on-site analysis of AFB1 in soybean sauce samples.A TRFIA was developed for rapid and quantitative detection of AFB1 in soybean sauce samples. Nanospheres enclosing fluorescent europium before conjugation with anti-AFB1 antibody via EDC dramatically enhanced the sensitivity. The TRFIA enabled the detection of AFB1 in soybean within 10 min, with an LOD of 0.1 µg·kg


The gene encodes a multimodular xylanase that consists of a typical hydrophobic signal sequence, a catalytic domain of glycoside hydrolase (GH) family 11, a glycine-rich linker, and a family 1 carbohydrate binding module (CBM1). Deduced Xyn11B shares the highest identity of 74% with a putative xylanase from Podospora anserina S mat+. Recombinant Xyn11B was successfully expressed in Pichia pastoris and purified to electrophoretic homogeneity. Xyn11B had a high specific activity of 382.0 U mg−1 towards beechwood xylan and showed optimal activity at pH 6.0 and 50°C. Distinct from most reported acidic fungal xylanases, Xyn11B was alkaline-tolerant, retaining 30.7% of the maximal activity at pH 9.0. The Km and Vmax values for beechwood xylan were 2.2 mg mL−1 and 462.8 μmol min−1 mg−1, respectively. The enzyme exhibited a wider substrate specificity and produced a mixture of xylooligosaccharides. All these favorable enzymatic properties make Xyn11B attractive for potential applications in various industries.An endo-1,4- Efficieesterase . Among tGH.html) .Microbial xylanases have been applied in many industries, including animal feed, bread-making, paper and pulp, waste treatment, and brewing . Various Humicola is a well-known nonpathogenic and nontoxic one, which produces a wide variety of hemicellulases and cellulases [ Humicola insolens Y1 has been reported to be an excellent producer of xylanolytic enzymes, including three thermophilic GH 10 xylanases, one GH 11 xylanase (Xyn11A), and two bifunctional GH 43 xylosidase/arabinosidases [xyn11B) from H. insolens Y1 was reported. This enzyme had distinct properties from Xyn11A and showed high activity at neutral to alkaline pHs.The genusllulases –12. Therosidases , 14. In Humicola insolens Y1 CGMCC 4573  was the donor strain [ Pichia expression manual (Invitrogen).r strain . The plaβ-d-xylopyranoside (pNPX), barley β-glucan, and carboxymethyl cellulose-sodium (CMC-Na) were purchased from Sigma. Soluble and insoluble wheat arabinoxylan were obtained from Megazyme (Ireland). The DNA purification kit, Genome Walking kit, and LA Taq DNA polymerase were purchased from TaKaRa (Japan). T4 DNA ligase and restriction endonucleases were obtained from New England Biolabs (UK). All other chemicals were of analytical grade and commercially available.The substrates beechwood xylan, 4-nitrophenyl  xyn11B, was identified from the genome sequence of H. insolens Y1 (whole genome sequencing in progress). The PCR product was ligated into the pGEM-T Easy vector and transformed into Escherichia coli cells for sequencing. The total RNA was extracted from the mycelia after 3 days' growth on wheat bran medium [ xyn11B, the first-strand cDNA was synthesized using a ReverTra Ace kit . The cDNA sequence encoding mature Xyn11B without the signal peptide-coding sequence was amplified using two expression primer sets . The PCR products with appropriate size were ligated into the pGEM-T Easy vector for sequencing.The full-length GH 11 xylanase gene,n medium  by usinghttp://linux1.softberry.com/berry.phtml). The signal peptide was predicted by the SignalP 4.0 server [ Chaetomium thermophilum (PDB: 1XNK) and Trichoderma longibrachiatum (PDB: 2JIC) as the templates.Vector NTI 10.0 was used for DNA assembly and sequence analysis. Introns, exons, and transcription initiation sites were predicted using the online software FGENESH  and loaded onto a HiTrap Q Sepharose XL FPLC column  equilibrated with McIlvaine buffer (pH 9.0). Proteins were eluted using a linear gradient of NaCl (0-1.0 M) in the same buffer. Fractions with enzyme activities were collected and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described by Laemmli [β-N-acetylglucosaminidase H (Endo H) at 37°C for 1 h according to the manufacturer's instructions (New England Biolabs).To purify recombinant Xyn11B, the cell-free culture supernatant was collected by centrifugation at 12,000 ×g for 10 min at 4°C, followed by concentration with a Vivaflow 50 ultrafiltration membrane of 5 kDa molecular weight cut-off . The crude enzyme solution was dialyzed against 20 mM citric acid-Na Laemmli . The pro Laemmli , using aμL of 1% (w/v) beechwood xylan in 100 mM sodium citrate-phosphate buffer (pH 6.0) and 100 μL of appropriately diluted enzyme. After incubation at 40°C for 10 min, the reaction was terminated by adding 1.5 mL of DNS reagent, boiled for 5 min, and cooled to room temperature. The absorbance was measured at 540 nm. Each assay and its control were done in triplicate. One unit of xylanase activity was defined as the amount of enzyme that released 1 μmol of reducing sugar from the substrate equivalent to xylose per minute under the assay conditions.Xylanase activity was assayed using 3,5-dinitrosalicylic acid (DNS) method . The staIn order to study the biochemical characteristics of purified Xyn11B, buffers of a range of pHs  were used. The optimal pH of Xyn11B for enzyme activity was determined at 40°C for 10 min in buffers mentioned above. Similarly, optimum temperature was determined by assaying the enzyme at different temperatures (20–70°C) and optimum pH for 10 min. For pH-stability assay, the enzyme was preincubated in buffers with a pH range of 4.0–10.0 for 1 h at 37°C, and then the residual activities were examined under standard conditions . To estimate the thermal stability, Xyn11B was incubated in McIlvaine buffer (pH 6.0) at 30°C, 40°C, or 50°C for 5, 10, 15, 20, 30, and 60 min without substrate, and the residual enzyme activity was measured at standard conditions as described above.2, LiCl, CoCl2, CrCl3, NiSO4, CuSO4, MgSO4, FeCl3, MnSO4, ZnSO4, Pb(CH3COO)2, AgNO3, HgCl2, EDTA, SDS, or β-mercaptoethanol at 40°C for 10 min. Reactions without addition of any chemicals were used as controls.To investigate the effects of different metal ions and chemical reagents on Xyn11B activity, the enzymatic activities were measured in McIlvaine buffer (pH 6.0) containing 5 mM of NaCl, KCl, CaClβ-glucan, or CMC-Na.Substrate specificity of Xyn11B was estimated in the standard assay system containing 1% (w/v) of beechwood xylan, soluble or insoluble wheat arabinoxylan, barley Km) and the maximum velocity (Vmax) of  Xyn11B were calculated by Lineweaver-Burk plot [The Michaelis-Menten constant (urk plot . Each exμL of 100 mM citric acid-Na2HPO4 (pH 6.0) at 37°C for 12 h. After hydrolysis, the enzyme was removed from the reaction using the Nanosep Centrifugal 3 K Device . The hydrolysis products were analyzed by high-performance anion-exchange chromatography (HPAEC) with a model 2500 system from Dionex. Xylose, xylobiose, xylotriose, and xylotetraose were used as standards.Hydrolysis of beechwood xylan and soluble wheat arabinoxylan by 50 U of Xyn11B was performed in 500  xyn11B, from H. insolens Y1 has been deposited in the GenBank database under accession number KM275236.The nucleotide sequence of the xylanase gene, xyn11B, 995 bp in length, was identified in the genome sequence of H. insolens Y1. The cDNA of xyn11B contains 876 bp and encodes 291 amino acids. The deduced Xyn11B contains a putative N-terminal signal peptide at residues 1–19, a typical catalytic domain of GH 11 (residues 20–222), a glycine-rich linker at residues 223–255 (18/32 glycine residues), and a family 1 carbohydrate binding module (CBM1) at residues 256–291. The molecular weight and pI value of mature Xyn11B were estimated to be 29.1 kDa and 8.7, respectively.One xylanase coding gene, Podospora anserina S mat+ (XP_001903201.1), followed by the functionally characterized GH 11 xylanase XynC81 from Achaetomium sp. Xz-8 . The putative tertiary structure of Xyn11B catalytic domain displays the classical β-jelly-roll architecture, and two catalytic residues (Glu118 and Glu209) and consensus sequence PSIXG (X corresponds to a nonconserved residue) were identified. Solvent-exposed amino acid analysis revealed a high frequency of positively charged residues on the surface. In addition, Xyn11B contains two potential N-glycosylation sites (Asn93 and Asn255).Multiple sequence alignment of Xyn11B with available protein sequences from GenBank  indicate P. pastoris. After methanol induction for 72 h, the transformant with highest xylanase activity showed the activity of 101.5 U mL−1 in the culture supernatant. Recombinant Xyn11B was purified to electrophoretic homogeneity by a single-step anion-exchange chromatography . After deglycosylation with Endo H, the purified recombinant Xyn11B migrated as a single band of ~29.0 kDa, which was in agreement with the calculated molecular weight. Purified enzyme was identified by size-exclusion chromatography as monomer of 32.0 kDa without carbohydrate residue.The gene fragment coding for mature Xyn11B was cloned into vector pPIC9 and successfully expressed intography . The spePurified recombinant Xyn11B had a pH optimum of 6.0 at 40°C . It show2+ were strong inhibitors for Xyn11B activity. On the other hand, the activity of Xyn11B was significantly enhanced in the presence of β-mercaptoethanol.The effect of different metal ions or chemical reagents on the activity of purified Xyn11B was determined . Additioβ-glucan or CMC-Na.Xyn11B showed maximum activity on soluble wheat arabinoxylan (defined as 100%), moderate activity on birchwood xylan (80.5%) and beechwood xylan (76.2%), and weak activity on insoluble wheat arabinoxylan (7.0%). No activity was detected in the presence of barley  Km and Vmax values were 2.2 mg mL−1 and 462.8 μmol min−1 mg−1, respectively.Using beechwood xylan as the substrate, theThe products of beechwood xylan and soluble wheat arabinoxylan by Xyn11B hydrolysis were analyzed by HPAEC. The main products of beechwood xylan were 2.3% xylose, 25.1% xylobiose, 52.2% xylotriose, and 20.4 xylotetraose. The composition of the hydrolysis products from soluble wheat arabinoxylan was 50.5% xylose and 49.5% xylobiose. H. insolens Y1 was identified, cloned, and successfully expressed in P. pastoris. Xyn11B is a multimodular enzyme that contains four regions: an N-terminal leader sequence, a GH 11 catalytic domain, a glycine-rich linker, and a C-terminal CBM1. To our knowledge, about 25% of functionally characterized GH 11 xylanases carry one CBM; among them are the most characterized fungal GH 11 xylanases bearing a CBM1 module, which assists the catalytic domain to bind to insoluble and crystalline polysaccharides [In this study, a new GH 11 xylanase fromcharides , 23. Whe Aspergillus kawachii and XynA from Penicillium sp. 40 have optimal pHs at 2.0 [ Bispora sp. MEY-1 has an optimal pH at 2.6 [ H. insolens Y1 have optimal pHs at 6.0 and 7.0, respectively, and are both alkali-tolerant, exhibiting 50.6% and 30.7% of the maximum activities at pH 9.0, respectively. From the same strain, another three GH 10 xylanases were also found to be alkali-tolerant [ H. insolens Y1 xylanases with higher activities at alkaline pHs requires further study. In addition, Xyn11B had maximal activities at 50°C, lower than Xyn11A (60°C). Its weak thermostability (only stable at 30°C and below) is similar to other GH 11 xylanases from mesophilic fungi, such as PfXynC from P. funiculosum [ Penicillium sp. F63 [ Fusarium graminearum [Most fungal GH 11 xylanases are acidic and have pH optima below 5.5 , and soms at 2.0 , 26, andH at 2.6 . There aH at 2.6 . Xyn11B H at 2.6  from H. tolerant . The meciculosum , XYN11F6 sp. F63 , and Xylminearum .2+, a metal ion that interacts with Trp and oxidizes the indole ring [β-mercaptoethanol, which counteracts the oxidation effects of the S–S linkage between Cys residues [Like most GH 11 xylanases, Xyn11B activity was completely inhibited by Hgole ring , and wasresidues . Interesresidues , 31.Xyn11B had higher activity on soluble wheat arabinoxylan than beechwood xylan and birchwood xylan. Based on the HAPEC analysis, the hydrolysis products of wheat arabinoxylan compared to beechwood xylan were different. The hydrolysis products from soluble wheat arabinoxylan were equal amounts of xylose and xylobiose. However, the beechwood xylan products were a mixture of xylose, xylobiose, xylotriose, and xylotetraose. In the viewpoint of xylan composition, beechwood xylan and birchwood xylan have similar compositions (89% xylose and 1% arabinose), but soluble wheat arabinoxylan contains 38% arabinose and is more complex in side chain structure , 15. Xyn H. insolens Y1 are both alkali-tolerant but have different temperature optima  and thermostability. Xyn11B has complex hydrolysis products from various xylan substrates. These superior properties make Xyn11B a good candidate for the xylooligosaccharide production.In summary, Xyn11A and Xyn11B produced by


Here, we review the current knowledge on the glycosyltransferase and transpeptidase activities of PG synthases. We provide new data showing that the bifunctional PBP1A and PBP1B from Escherichia coli are active upon reconstitution into the membrane environment of proteoliposomes, and that these enzymes also exhibit DD-carboxypeptidase activity in certain conditions. Both novel features are relevant for their functioning within the cell. We also review recent data on the impact of protein–protein interactions and other factors on the activities of PBPs. As an example, we demonstrate a synergistic effect of multiple protein–protein interactions on the glycosyltransferase activity of PBP1B, by its cognate lipoprotein activator LpoB and the essential cell division protein FtsN.Peptidoglycan (PG) is an essential component in the cell wall of nearly all bacteria, forming a continuous, mesh-like structure, called the sacculus, around the cytoplasmic membrane to protect the cell from bursting by its turgor. Although PG synthases, the penicillin-binding proteins (PBPs), have been studied for 70 years, useful  This property has been used to obtain lipid II with a radiolabelled GlcNAc by a simple exchange reaction using purified lipid II and [14C]-GlcNAc in the presence of MurG .Radiolabelled UDP-Glclipid II . While p(c)S. aureus, the MurT/GatD complex amidates the d-Glu residue . I. Id-aminssay (d) . In thisssay (d) . However(c)α-helices followed by a proline/glutamine-rich unstructured region and a globular C-terminal SPOR domain which binds to PG but is not essential GlcNAc-labelled lipid II and dansylated lipid II were prepared as published GlcNAc-labelled lipid II (15 000 dpm), 0.01% EtOH, 5 µM MgCl2, 100 mM NaCl in a final volume of 540 µl. Samples were incubated at 37°C in a thermal microfuge tube shaker at 800 r.p.m. Aliquots of the reaction mix (95 µl) were taken at 0, 5, 10, 20 and 30 min. Un-reacted lipid II was immediately extracted by addition of 200 µl of a 1 : 1 mixture of butanol and 6 M pyridine–acetate, pH 4.2 [g for 2 min. The butanol phase (approx. 100 µl) was collected in a scintillation vial, 5 ml Ecoscint A liquid scintillation cocktail  was added and the radioactivity was measured using a HIDEX 300SL β-particle scintillation detector. Where indicated, samples included 0.2 mg ml−1 moenomycin  or 0.1 mg ml−1 ampicillin  to inhibit GTase and TPase, respectively.The escribed , except escribed  whereby , pH 4.2 . Samples(e)2, 150 mM NaCl, 0.05% Triton X-100, pH 7.5). Samples were incubated at room temperature for 10 min before addition of 0.2% w/v formaldehyde  and further incubation at 37°C for 10 min. Excess cross-linker was blocked by addition of 100 mM Tris/HCl, pH 7.5. Samples were applied to 100 µl of washed and equilibrated Ni-NTA superflow beads  and incubated overnight at 4°C, with mixing. The beads were then washed with 6 × 1.5 ml wash buffer  and boiled in SDS–PAGE loading buffer. Beads were then removed by centrifugation and samples analysed by SDS–PAGE. Gels were stained with Coomassie brilliant blue .Proteins were mixed at appropriate concentrations , 2 µM) in 200 µl of binding buffer (10 mM HEPES/NaOH, 10 mM MgCl


We explore here the potential of the Immunoscore® to predict the need for 5-FU adjuvant chemotherapy in Stage II and in Stage III CC.No biomarker has been validated so far to predict the need for adjuvant chemotherapy in Stage II colon cancer (CC), and current guidelines, e.g. NCCN, only rely on the identification of high-risk features such as MSI, pathological tumor characteristics or expected chemotherapy efficacy/safety ratio in each individual patient. In contrast, adjuvant chemotherapy is recommended for all Stage III colon cancer (CC). The Immunoscore® was calculated as previously described on a retrospective cohort of 456 colorectal cancer patients from Hôpital Européen Georges Pompidou, who underwent primary tumor resection between 1990 and 2003. For the analysis of chemotherapy benefit on disease-free survival, patients were grouped in 3 subcohorts based on their IS®: IS® 0-1 (worst prognosis), IS® 2-3 (intermediate prognosis) and IS® 4 (best prognosis).The IS® 0-1, IS® 2-3 and IS® 4 represented 11.6%, 35.2% and 53.2% in the Stage II population, and 18.6%, 36.4% and 45% of patients in the Stage III population. Disease-free survival of IS® 2-3 patients was improved by administration of 5-FU chemotherapy (Stage III: HR=2.69 (1.02-7.11) pV = 0.038, and Stage II: HR=1.47 (0.35-6.18) pV not reaching significance). IS® 4 patients had the best and IS® 0-1 patients the worst outcome in both Stage II and Stage III populations. There was no statistically significant benefit of chemotherapy on disease-free survival for IS® 4 or IS® 0-1 patients in both Stage II and Stage III populations.Stage II and Stage III cases represent resp. 327 (71.7%) and 129 (28.3%) of the cohort. 5-FU adjuvant chemotherapy was administered to 13.8% and 60.5% of Stage II and Stage III CC resp. IS®, which has been developed to be an easy-to-use routine practice biomarker, may represent a valid biomarker to precisely identify stage II and stage III CC patients most likely to benefit from 5-FU chemotherapy. These results, based on a single center retrospective cohort, warrant further validation on a larger, clinical trial, cohort.IS


A wider application of siRNA- and miRNA- based therapeutics is restricted by the currently available delivery systems. We have designed a new type of small molecule carrier (SMoC) system for siRNA modeled to interact with cell surface proteoglycans. This bifurcated SMoC has similar affinity for the model proteoglycan heparin to an equivalent polyarginine peptide and exhibits significant mRNA knockdown of protein levels comparable to lipofectamine and the previously reported linear SMoC. Small interfering RNAs (siRNA), which are capable of catalytically silencing specific genes via the RNA interference (RNAi) pathway, are attractive candidates for therapeutics for a wide range of diseases including genetic disorders and cancer. The direct application of unmodified siRNA to the target tissue has shown to be successful in clinical trials, for example an siRNA against a mutated form of keratin 6a to treat the skin condition pachyonychia congenita  in vivo as well as aiding cell targeting and internalization is a popular strategy for increasing delivery efficiency To be systemically effective, siRNA must be formulated in such a way as to overcome these delivery barriers. siRNA encapsulated in nanoparticles which protect the siRNA from degradation and improve its half-life in vivo, and form complexes via electrostatic interactions which neutralize the charge of the oligonucleotide and allow interaction with cell surface lipids and proteoglycans. As well as shielding the siRNA charge, these cationic materials have been found to promote cell entry of siRNA as they are effective at crossing cell membranes. Although the exact mechanism of cellular entry adopted by these polymers and peptides remains unclear, it is suggested that binding to cell surface proteoglycans such as heparan sulfate and chondroitin sulfate via their glycosaminoglycan (GAG) chains may be an initial internalization step, activating Rac1 and triggering subsequent rearrangement of the F-actin cytoskeleton leading to macropinocytosis initiation Cationic polymers such as polyethyleneimine π-cation interactions with the biphenyl backbone to promote electrostatic binding to siRNA and to anionic cell surface proteoglycans. The most successful SMoC compound for siRNA transfection described thus far is 4G-SMoC-SSPy 1,  class of compounds, which are small molecule mimics of penetratin, show potential as siRNA delivery agents 1H and 13C NMR spectra were recorded on either a Bruker AMX-300 or a Bruker AMX-500 spectrometer. Chemical shifts are reported as ppm relative to TMS internal standard. Mass spectra were recorded on either a Fisons VG70-SE spectrometer  or an Agilent 6100 Series LC-mass spectrometer  using either a C-18 or C-4 column. Microwave reactions were carried out using a CEM Discover microwave. All compounds were purified to ≥ 95% as measured by LC-MS. Proteoglycan binding was recorded using an Agilent 1200 series HPLC system equipped with a diode array detector and HiTrap heparin agarose column. A complete list of 1H NMRs for compounds used in this study is given in the supplementary information (Table S1).Starting materials were either commercially available or synthesized according to methods reported in the literature. 3 and 4 were initially built in MOE  using the ‘Molecular Builder’ with an extended conformation. All the guanidine groups are in +1 charge, reflecting the protonated state of the groups. Then, the initial structures were subjected to energy minimization using MMDD94X force field. The energy minimized structures of 3 and 4 were then carried through for dynamics simulations. All molecular dynamics simulations were conducted in a cube of water of depth of 10 Å using ‘Simulations-Dynamics’ in MOE. The protocol used in dynamics simulations is as follows: algorithm – Nosé-Poincaré-Andersen Hamiltonian equations of motion (NPA); temperature – constant at 300°K; mode – equilibration for 100 ps, forcefield – MMFF94X, cutoff at 8 Å. Conformations were collected in trajectory files every 0.5 ps, providing 200 conformers for each run for each molecule. The molecular dynamics simulations were also run in YASARA software Molecular modeling and dynamics were carried using Molecular Operating Environment (MOE) package (version 2011.10). The framework structures 3 or 4 were measured for each conformation. A histogram summarizing the data was calculated and plotted using Microsoft Excel.Data analysis, such as distance measurement, was carried out also in MOE using the graphics interface. The pairwise distances between the carbon atoms of the guanidine group from different phenyl rings in each molecule Crystal structure information for heparin (PDB ID: 1HPN) and chondroitin sulfate (PDB ID: 1C4S) was obtained from the PDB and imported into DS Visualizer  and the distance between sulfate residues measured between sulfur atoms.2dppf.CH2Cl2  and triethylamine  and the solution stirred for 2 min. 3,5-Diiodobenzoic acid, 5  was added, and the reaction heated in a microwave reactor for 10 min at 75 °C. The reaction mixture was cooled, filtered, and 1 m HCl added (50 mL). The product was extracted with dichloromethane (3 × 25 mL), and the combined organic extracts were washed with brine (50 mL), dried over MgSO4, filtered, and concentrated under vacuum. The mixture was purified by flash column chromatography using a gradient of 0–50% ethyl acetate in cyclohexane and the title product  obtained as a colorless oil.To a degassed solution of potassium carbonyl)amino)-ethoxy)phenyl)trifluoroborate 1H-NMR  δ: 3.27 , 3.48–3.74 , 4.11 , 4.97–5.10 , 6.68–7.11 , 7.23–7.32 , 7.66–8.29 .13C-NMR  δ: 40.81 (CH2), 41.13 (CH2), 66.87 (Cbz CH2), 68.22 (CH2), 113.43, 123.04, 124.90, 128.20 (Cbz ArC), 128.51 (Cbz ArC), 135.03, 136.44, 138.37, 145.06, 152.02, 156.77 (C = O).m/z): [M − H]− calcd for C59H57N4O14, 1045.3871; found, 1045.3804.HRMS-ES  in dichloromethane (20 mL) was added 30% hydrogen bromide in acetic acid (5 mL) dropwise and the mixture stirred at room temperature for 3 h. The reaction was diluted with dichloromethane (20 mL) and the product extracted with water (3 × 20 mL). The aqueous extracts were carefully dried under vacuum for several hours. The crude amine was then dissolved in methanol (20 mL), and N,N′-Di-Boc-1H-pyrazole-1-carboxamidine  and DIEA  were added. The reaction was stirred at room temperature for 24 h under nitrogen. The solvent was removed under reduced pressure and the residue redissolved in ethyl acetate (50 mL) and 1 m HCl added (50 mL). The layers were separated, and the aqueous layer washed with ethyl acetate (3 × 20 mL). The combined organic extracts were washed with brine (50 mL), dried over MgSO4, filtered, and concentrated under vacuum. The product was purified by flash column chromatography using a 0–25% ethyl acetate gradient in cyclohexane. The product was obtained as an off-white solid .To a solution of 3,5-biscarbonyl)amino)ethoxy)phenyl)-benzoic acid 1H-NMR  δ: 1.42–1.51 , 3.55 , 3.87 , 3.93 , 4.23 , 6.99 , 7.03 , 7.13 , 7.98 , 8.28 , 8.66 , 8.81 , 11.45 .13C-NMR  δ: 28.06 (Boc-CH3), 28.17 (Boc-CH3), 28.36 (Boc-CH3), 28.39 (Boc-CH3), 53.49 (CH2), 67.43 (CH2), 79.06 (Boc CCH3), 79.35 (Boc CCH3), 82.80 (Boc CCH3), 83.20 (Boc CCH3), 113.31, 123.34, 124.77, 129.78, 135.12, 138.33, 145.16, 152.11, 153.01, 156.43, 163.54, 169.33 (COOH).m/z): [M+ H]+ calcd for C71H107N12O22, 1479.7423; found, 1479.7423.HRMS-ES  was added and the mixture stirred for 4 h at room temperature. The solvent was removed under vacuum, and the residue washed three times with ether. The product was dissolved in water and lyophilized to form the tetra-TFA salt as a white solid .The tetra-Boc compound  was dissolved in DCM (2 mL), and TFA/1H NMR  δ 8.17 , 7.86 , 7.33–7.27 , 7.20 , 7.10 , 4.35–4.29 , 4.02–3.97 , 3.73 , 3.23–3.18 .13C NMR  δ 171.06, 157.88, 157.21, 151.90, 144.44, 138.55, 135.89, 135.33, 130.64, 130.37, 126.24, 123.69, 114.53, 71.96, 67.55, 42.15, 41.61. Also visible peaks for trifluoroacetate, 163.98, 163.74, 163.51, 163.27, 119.86, 117.93, 116.00, 114.06.m/z): [M+H]+ calcd for C31H43N12O6, 679.3429; found, 679.3428.HRMS-ES .2, 3, 2′, 3′-tetra(2-guanidino-ethyloxy)-4-cyanobiphenyl, N, N'-bis(tert-butoxycarbonyl)guanidino]-ethyloxy)-4-cyanobiphenyl 72O, washed three times with Et2O, dissolved in water, and lyophilized to give the title product  as a white powder.To a solution of 2, 3, 2′, 3′-tetra δ 7.50 , 7.20 , 7.15 , 6.91 , 4.38 , 4.24 , 4.00 , 3.92 , 3.68 , 3.28 .m/z (ES+) 584.3186, calculated C25H38N13O4+ 584.3170.HRMS 2CH2CO-RRRR-NH2, 9 (R4SSPy) or PySSCH2CH2CO-RRRRRRRR-NH2, 10, (R8SSPy),  in the molar ratios of 1/1, 1/5, 1/10, 1/20 and 1/50. The final siRNA concentration was 17 μm in 100 μL of water (1.7 nmol) in each case. The complexes were incubated for 30 min at room temperature. The samples were diluted with 2× RNA loading dye  containing ethidium bromide  and analyzed by gel electrophoresis on a 1% agarose gel prepared using 10× MOPS buffer , loading 25 μL of sample into each well. The gel was run at 5V/cm till the blue dye had migrated 2/3 of the gel. The gels were analyzed by UV illumination and the ethidium bromide bands quantified using the ImageJ software . The EtBr intensities were plotted, a dose–response curve fitted and EC50 values calculated using the Origin software .GAPDH siRNA  was mixed in RNAse-free water with varying concentrations of either SMoC, PySSCHm l-glutamine (Invitrogen) at 37 °C and 5% CO2. 4T1 cells were purchased from Caliper Life Sciences . MCF-7 cells were obtained from the European Collection of Animal Cell Cultures, Porton Down, UK.IMR-90 (ATCC# CCL-186) cells were obtained from LGC Standards, UK, at population doubling (PD) 12. Cells were cultured in Dulbecco's modified Eagles Medium (DMEM)  supplemented with 10% Fetal Bovine Serum (Invitrogen) and 2 m4 4T1 (murine) and MCF-7 (human) breast cancer cells were seeded in 35 mm diameter glass-bottomed fluorodishes . These were incubated overnight to encourage adherence and optimal subconfluent cell spreading for imaging. 4T1 cells were incubated with fresh RPMI-1640 Medium with l-glutamine and sodium bicarbonate  supplemented with 10% (v/v) fetal calf serum (FCS) ; MCF-7 cells were incubated with fresh DMEM containing 0.365 g/L l-glutamine, 4.5 g/L glucose , supplemented with 10% (v/v) FCS (Sigma-Aldrich), 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C in a humidified 5% CO2 incubator.Cell preparation: 1 × 102 was performed using image J to quantify the mean intracellular signal intensity compared to the background level of fluorescence. A one-way anova with Tukey's multiple comparison test was used to assess statistical significance. Appropriate controls were used throughot.Confocal: Cells were observed using an inverted Olympus Fluoview 1000 confocal laser-scanning microscope to determine intracellular localization of the oligonucleotide. Fluorescence confocal images were obtained using a 60× NA:1.35 oil immersion objective lens . Original image files were analyzed using Image J software. Analysis of the labeled siRNA ± 4G-BfSMoC-COOH, μm stock of Alexa Fluor® 555-labeled, double-stranded RNA oligomer in 100 mm KOAc, 30 mm HEPES-KOH, pH 7.4, and 2 mm MgOAc  was determined through confocal fluorescence microscopy. Lipofectamine® RNAiMAX (Invitrogen). The oligonucleotide was diluted to a final concentration of 1 μm either alone or in combination with 10 μm 4G-BfSMoC-COOH, 2. The initial dilutions were in phosphate buffered saline (PBS) . The oligonucleotide/4G-BfSMoC-COOH, 2 mixture was allowed to complex for 15 min at room temperature. Final dilutions were made in the appropriate cell medium. Either cell line was subsequently incubated with media , oligonucleotide, 4G-BfSMoC-COOH, 2 , or oligonucleotide/4G-BfSMoC-COOH, 2 for 24 h. Lipofectamine controls were transfected according to manufacturer's instructions to a final concentration of 3.75 μL/mL + oligonucleotide. Cells were washed twice with PBS and treated with phenol-red-free media prior to confocal imaging.The intracellular localization of Alexa Fluor® Red Fluorescent oligonucleotide (Invitrogen) supplied as a 20 mcdc7 siRNA , 100 nm Accell Non-targeting siRNA 1 negative control siRNA (Thermo Scientific), 8 μL/mL Lipofectamine LTX reagent (Invitrogen), 0.5 mm SMoC. SMoC/siRNA, and Lipofectamine/siRNA complexes were made by mixing equal volumes of the required solutions and incubating for 30 min at room temperature.Solutions were made up in Opti-MEM® (Invitrogen); 100 n5 cells/well in 1 mL DMEM containing 10% FCS and 2 mm glutamine and incubated at 37 °C under 5% CO2 for 24 h. The media was removed and replaced with 1.6 mL Opti-MEM® (Invitrogen). Four hundred microliters of SMoC/siRNA or Lipofectamine/siRNA complexes was added and the cells incubated for a further 72 h. The final concentration of SMoC was 50 μm and based on the dose–response analysis reported previously Cells were plated in 6-well plates at a density of 5 × 10Taq Mix and SYBR Green Reaction Mix from the Supersript III Platinum SYBR Green One-Step qRT-PCR kit (Invitrogen) according with the manufacturer's protocol. PCR conditions were 50 °C for 3 min, 95 °C for 10 min (one cycle), 95 °C for 15 seconds, 47 °C for 30 seconds, 60 °C for 30 seconds (45 cycles), 95 °C for 15 seconds, 60 °C for 15 seconds, ramp to 95 °C over 20 min (one cycle). Relative quantification data were obtained using the comparative Ct method with Realplex software according to the manufacturer's protocol . cdc7 RNA was measured using the following primers: forward primer: 5′-AAC TTG CAG GTG TTA AAA AAG-3′: reverse primer: 5′-TGA AAG TGC CTT CTC CAA T-3′. The following primers were used to measure GAPDH: forward primer: 5′-TCA ACT ACA TGG TTT ACA TGT TC-3′: reverse primer: 5′-GAT CTC GCT CCT GGA AGA T-3′. The signal obtained from the cdc7 primers was normalized using the signal from the GAPDH primers.RNA was extracted from the cell pellets using the PureLink RNA Mini kit (Invitrogen) using the protocol provided by the manufacturer. RNA was quantified using a Nanodrop spectrophotometer, and 40 ng of RNA was mixed with Superscript III RT/Platinum 9 and R8SSPy, 10 were applied to a heparin agarose column  (1 mL) and eluted with a gradient of sodium chloride from 500 to 1000 mm. The absorbance at 214 nm (CONH) was recorded and exported to Origin 9.0. The signals were normalized and replotted as shown in the Figure. HIVtat, 11, (sequence YGRKKRRQRRR) was obtained from Sigma.The test SMoCs and the marker oligoarginine peptides R4SSPy, t-test. A value of P < 0.05 was considered significant.Groups were compared using an unpaired two-sample Student's 1  4G-BfSMoC-COOH, 2 resulted in 4.9-fold (P < 0.01) increased uptake of the oligonucleotide-fluorophore in the 4T1 cells; and a 2.6-fold (P < 0.01) increase in the MCF7 cell line compared to siRNA alone. There was undetectable ‘background’ uptake of fluorescently labeled siRNA alone in the 4T1 cell line  and 3.3-fold (P < 0.01) increased uptake compared to siRNA alone in 4T1 and MCF-7 cell lines, respectively. In both cases, there was significant nuclear uptake at 24 h  was applied to the column. Polyarginine peptides R4SSPy, 9 and R8SSPy, 10 were applied as markers. Selected overlaid traces are shown in Figure 12 a 4G-SMoC marker compound bearing a 1-CN group, 8, and 6G-SMoC, 142 shows similar affinity to the linear 3G-SMoC-SSPy, 13 and somewhat less than the 4G-SMoCSSPy, 1 perhaps due to the carboxylic acid group which is negatively charged at physiological pH. Affinity to heparin was insensitive to the inclusion of the disulfide pyridyl group  liposome formulation of the polycationic lipid 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) and the neutral lipid dioleoyl phosphatidylethanolamine (DOPE) in membrane-filtered water. The only UV active component is expected to be DOSPA with a single weak amide UV absorbance expected at 214 nm. However, using our standard conditions and monitoring at 214 nm, we observed only a peak at the solvent front even with 10 μg of Lipofectamine injected (data not shown).To determine the relative proteoglycan binding of SMoCs, we utilized heparin bound to agarose beads and monitored the retention time when a gradient of sodium chloride 500–1000 m000 mm wain vivo as well as achieve efficient and targeted cellular internalization would be a major advancement for the therapeutic use of siRNA. Multicomponent siRNA delivery nanoparticles may consist of layers of different materials, such as cationic polymers for siRNA complexation, PEG to increase serum stability, and targeting ligands to confer cell specificity. An efficient siRNA transfection vector should tightly package the siRNA, bind to the cell surface so as to induce macropinocytosis, and finally escape from the endosome into the cytoplasm in order for the siRNA to act on the RNAi machinery and silence the target gene. In this study, we have examined the ability of the SMoC class of transporter to achieve the earlier stages of this process – namely siRNA complexation and cell surface proteoglycan binding.The development of a delivery vector that is able to complex and protect siRNA 1 was also active as a siRNA delivery vehicle. It was demonstrated that SMoCs were capable of packaging siRNA via electrostatic complexation through the guanidine side chains, thus neutralizing the siRNA negative charges. We also postulated that the biphenyl ring system of 4G-SMoC-SSPy formed π-cation interactions with the guanidine groups, therefore shielding the positive charges during membrane penetration, as predicted for penetratin. In the present study, we develop the structure of the original SMoC to produce a siRNA delivery agent that is able to bind sulfate groups on adjacent cell surface GAGs, is simpler to synthesize and contains a chemical ‘handle’ for potential conjugation.We previously showed that the protein transduction compound 4G-SMoC-SSPy, 2 described has been molecularly designed to explore binding to the common GAGs heparan sulfate and chondroitin sulfate, which have been shown to interact with cell-penetrating peptides as a precursor to internalization. In a model system utilizing the more highly sulfated proteoglycan heparin, we observed that the SMoCs bound with similar affinity to the reference tetra and octa-arginine 10, 11, and HIV-1 tat peptides. 4G-BfSMoC-COOH, 2 did not show increased binding over linear SMoCs despite the clear indication from the modeling studies that longer distances might be beneficial for binding and the molecular dynamics studies indicated that the larger structures were able to adopt more extended conformations. We should be cautious, however, in that cellular binding may differ from our model heparin system. Several studies have suggested that the binding of GAG by CPPs plays a role in cell penetration, with subsequent receptor clustering leading to reorganization of the actin cytoskeleton as a precursor to macropinocytosis 1 reagent or to Lipofectamine®; however, it does demonstrate that the disulfide pyridyl group, and therefore potential reaction with cell surface thiols, is not required for successful siRNA transfection. Further optimization of this system is in progress.The new 4G-BfSMoC-COOH,


A total of 41 B. mallei field strains, 36 B. pseudomallei field strains, and 1 B. thailandensis field strain from different geographic regions were tested and correctly identified. Testing of 55 non-Burkholderia bacterial species revealed 100% specificity of the assay. The minimum detection limit was 1 pg DNA or 160 GE for B. mallei and 130 GE for B. pseudomallei, respectively.Species specificity was tested with a panel of 13 B. mallei and B. pseudomallei/B. thailandensis.This assay enables the clear distinction between The online version of this article (doi:10.1186/s13028-015-0104-4) contains supplementary material, which is available to authorized users. Burkholderia mallei, B. pseudomallei and B. thailandensis being genetically closely related Gram negative bacteria, they display significant differences in pathogenicity and habitat. B. mallei, a facultative intracellular, non-motile, equine pathogen, is the causative agent of glanders, a highly contagious and frequently fatal zoonotic disease of the upper respiratory tract and lungs  of B. mallei (ATCC 23344) was utilized to design a simple conventional duplex PCR assay with fluorescent labelled primers enabling the distinction between B. mallei and B. pseudomallei/B. thailandensis. Bacterial strains were obtained from the strain collection of the National and OIE Reference Laboratory for Glanders at the Friedrich-Loeffler-Institute in Jena, Germany  glycerol. All other bacteria were grown on standard media and appropriate atmospheric conditions.Based on the results from a previous study , a frameB. mallei and an ATTO565 labelled 325 bp DNA fragment for B. pseudomallei/B. thailandensis, respectively. Labelled primers were obtained from Microsynth , the unlabelled primer from Jena Bioscience . PCR was conducted in a 20 μL reaction containing 0.3 μM of the primers , 1 × 5-Prime HotMasterMix , 2.5% DMSO and 10 ng template . The PCR was performed in a Mastercycler pro S™  under the following conditions: initial denaturation at 95°C for 1 min; 40 cycles at 95°C for 10 s, 63°C for 15 s, 70°C for 30 s, and the final extension at 70°C for 5 min. 13.3 μL PCR reaction mixed with 2.7 μL 6 × Loading Dye  were analysed by electrophoresis on a 1.25% agarose gel (wt/vol) at 9 V/cm for 40 min. Images were captured after an exposure period of 30 s for each LED/filter set using the G-Box EF2 Gel Documentation System : Blue-LED/Filt525 and Green-LED/Filt605 for the visualisation of 6-FAM and ATTO565 labelled PCR products, respectively. For optional ethidium bromide imaging (302 nm UV illuminator/FiltUV), the gel was stained after capturing the 6-FAM/ATTO565 images. Fragment sizes (326/327 bp) and correct labelling (6-FAM/ATTO565) of the amplicons were confirmed by means of capillary electrophoresis using a Genetic Analyzer 3130 with a G5 filter set . Species specificity was tested with a panel of 13 Burkholderia type strains. Additionally, a total of 41 B. mallei field strains from equines, 36 B. pseudomallei field strains from human and environmental origin, and one B. thailandensis field strain, all from different geographic regions were tested and correctly identified . All DNA samples were quantified using a NanoDrop 1000 spectrophotometer . The duplex polymerase chain reaction (PCR) was designed using the forward primer MBF04 (5′- CGTCAAGCGGGTGAACCA -3′), the 6-FAM labelled reverse primer MBR04-FAM (5′-6-FAM-GTCGTCCTCGCTCTTTCGC -3′), and the ATTO565 labelled reverse primer MBR10-ATTO565 (5′-ATTO565-GTCCTCGCTCTTCTTCGCG-3′). Primers were designed with the Genious software package (Ver. 6.1), to generate a specific 6-FAM labelled 326 bp DNA fragment for CR [fliC . DespiteB. mallei and B. pseudomallei and other Burkholderia species in the past few years [B. mallei and B. pseudomallei/B. thailandensis, respectively. These closely related species can cause considerable problems during the identification process in the laboratory as colony characteristics and routine biochemical tests are not sufficiently discriminative for species identification. The benefit of this assay is not only the unambiguous identification of B. mallei and the closely related species B. pseudomallei and B. thailandensis by fluorescence image capturing but also the possibility of detecting the B. mallei/pseudomallei/thailandensis complex on a standard ethidium bromide stained agarose gel.Fluorescent primers are widely used in real time PCR technology and several highly sophisticated and elegant PCR assays have been developed for the identification and differentiation of ew years . This st


To compare the long-term efficacy of ranibizumab versus bevacizumab for myopic choroidal neovascularization (CNV).This was a retrospective, multicenter, comparative, non-randomized study of 64 consecutive patients with myopic CNV treated with ranibizumab (22 patients) or bevacizumab (42 patients). Best-corrected visual acuity (BCVA) and central foveal thickness (CFT) on optical coherence tomography were evaluated before and after treatment. All the patients were followed for at least 12 months.P < 0.05 compared with baseline BCVA in each group). CFT decreased by 20.21%, 19.58%, and 22.43% from the baseline 304 ± 76 μm at 3, 6, and 12 months after treatment in the former group, and by 15.20%, 15.67%, and 15.56% from the baseline 297 ± 62 μm in the latter group . BCVA improvement and CFT reduction did not statistically differ when compared at the same periods from treatment between 2 groups. Neither ocular nor systemic safety problems appeared during follow up.BCVA  improved from 0.63 ± 0.30 to 0.43 ± 0.27, 0.41 ± 0.37, 0.40 ± 0.39, 0.39 ± 0.43, and 0.39 ± 0.42 at 1, 2, 3, 6, and 12 months after treatment in the ranibizumab group, and from 0.67 ± 0.28 to 0.52 ± 0.31, 0.49 ± 0.31, 0.47 ± 0.31, 0.42 ± 0.32, and 0.46 ± 0.43 in the bevacizumab group (all This study showed a similar functional and anatomical improvement after treatment of ranibizumab and bevacizumab for myopic CNV over a 12-month follow-up period. Myopic choroidal neovascularization (CNV) is the most common macular complication of pathologic myopia, with reported incidence ranging from 4–11% in highly myopic eyes . Myopic The advent of anti-vascular endothelial growth factor (anti-VEGF) agents has changed the paradigm of various retinal diseases. Several reports have demonstrated the effectiveness of anti-VEGF agents including ranibizumab and bevacizumab for myopic CNV -13. HoweOne hundred twenty two eyes of 110 patients who were diagnosed with myopic CNV from 2007 to 2009 at 3 tertiary medical centers  were retrospectively chart-reviewed in 2010, and 66 eyes of 64 patients satisfying inclusion and exclusion criteria were included in the study. The study protocol was approved by the Institutional Review Boards of the 3 hospitals and adhered to the tenets of the Declaration of Helsinki.Inclusion criteria were as follows: (1) axial length more than 26 mm or spherical equivalent less than -6.0 Dsph ; (2) pathologic myopia classified as M2 or more according to the criteria of Avila et al. ; (3) myoA thorough ophthalmic examination was performed in all patients. BCVA was measured using a Snellen chart and auto-refraction. Diagnosis of myopic CNV was confirmed when fluorescein angiography  revealed increased leakage over foveal area in the late phase compared with the early phase and the existence of neovascular lesion or subretinal fluid was observed with optical coherence tomography (OCT). The location of myopic CNV, which was classified into subfoveal, juxtafoveal, or extrafoveal one, was determined using both FA and fundus photography . Three OCT modalities were used to evaluate central foveal thickness (CFT): Stratus OCT , Cirrus OCT , and Spectralis OCT . The same OCT modality was always used in each patient to measure CFT so that the error arising from changing OCT modalities could be minimized.After explaining the patients the pros and cons of two anti-VEGF agents sufficiently, one anti-VEGF agent between bevacizumab  and ranibizumab  was selected by the patients. A volume of 0.05 mL (1.25 mg bevacizumab or 0.5 mg ranibizumab) was injected into the vitreous cavity using a 30-gauge needle after topical anesthesia was induced by 1 drop of 0.5% proparacaine hydrochloride  and povidone-iodine was applied inside the cul-de-sac.All the patients followed 1 + PRN (Pro Re Nata) protocol. They were followed at 4-week intervals after the first injection. At visits, BCVA, thorough ophthalmic examination, FA, and OCT were performed. When intraretinal or subretinal fluid remained on OCT or fluorescein leakage was persistent on FA, an additional intravitreal injection of anti-VEGF agent was performed to the first injection. When the lesion subsided totally, the follow-up period was lengthened from 4 weeks to 2–3 months without treatment. All the patients were followed for at least 12 months from the first visit.The primary outcome measures were changes in BCVA from baseline to 1, 2, 3, 6, and 12 months after treatment and the decreased rate (%) of the CFT from baseline to 3, 6, and 12 months after treatment in the bevacizumab and ranibizumab groups. Snellen visual acuity was converted to the logarithm of the minimum angle of resolution (logMAR). The secondary outcome measures were as follows: (1) the proportions of patients who gained or lost 2 or more lines of Snellen visual acuity at 6 and 12 months after treatment compared with baseline BCVA, (2) the proportions of patients in whom CFT was increased or decreased by 10% or more at 6 and 12 months in comparison with baseline CFT, (3) the rates at which FA revealed leakage at the late phase at 3, 6, and 12 months after treatment, (4) recurrence rates during 12 months after treatment, and (5) any complications associated with intravitreal injection over the follow-up period.P values < 0.05 were considered significant.We used independent t-test, paired t-test, Pearson’s chi-square test, Mann–Whitney test, and Wilcoxon signed rank test to analyze demographic data and outcome measures. Statistical analyses were performed using SPSS for Windows . Demographic data for the ranibizumab and bevacizumab groups are summarized in Table P = 0.27). In the ranibizumab group, logMAR BCVA improved from 0.63 ± 0.30 to 0.43 ± 0.27, 0.41 ± 0.37, 0.40 ± 0.39, 0.39 ± 0.43, and 0.39 ± 0.42 at 1, 2, 3, 6, and 12 months post-treatment, respectively, and statistically significant differences were observed between pretreatment and all post-treatment BCVA values . In the bevacizumab group, BCVA improved from 0.67 ± 0.28 to 0.52 ± 0.31, 0.49 ± 0.31, 0.47 ± 0.31, 0.42 ± 0.32, and 0.46 ± 0.43 at 1, 2, 3, 6, and 12 months post-treatment, respectively, and statistically significant differences were also observed between pretreatment and all post-treatment BCVA values . When BCVA values of the same period were compared between the 2 groups, no statistically significant differences were observed  . In the ranibizumab group, CFT decreased by 20.21%, 19.58%, and 22.43% at 3, 6, and 12 months post-treatment, respectively, and statistically significant differences were observed between pretreatment and all post-treatment CFT values . In the bevacizumab group, CFT decreased by 15.20%, 15.67%, and 15.56% at 3, 6, and 12 months post-treatment, respectively, and statistically significant differences were observed between pretreatment and all post-treatment CFT values . When the rates of decreased CFT at the same period were compared between the 2 groups, no statistically significant differences were observed . When the absolute CFT values at the same period were compared, there were also no statistically significant differences between the 2 groups  . Only 1 eye in each group experienced loss of 2 or more lines of BCVA despite the intravitreal injections (P = 0.99). At 12-month follow up, BCVA improved by 2 or more lines in 17 of 23 eyes (74%) and 27 of 43 eyes (63%) in each group (P = 0.43) and worsened in 1 of 23 eyes and 2 of 43 eyes in the groups (P = 0.99). At 6-month follow up, CFT decreased by 10% or more in 13 of 17 eyes (77%) and 24 of 32 eyes (75%) in each group (P = 0.91) and increased by 10% or more in only 1 eye in the bevacizumab group (P = 0.99). At 12-month follow up, CFT decreased by 10% or more in 14 of 17 eyes (82%) and 22 of 32 eyes (69%) in each group (P = 0.50) and increased by 10% or more in 2 of 32 eyes (6%) in the bevacizumab group (P = 0.54).The proportions of patients who gained or lost 2 or more lines of Snellen BCVA and in whom CFT decreased or increased by 10% or more at 6 and 12 months after treatment are summarized in Table P = 0.74), 1 of 22 eyes and 3 of 34 eyes at 6-month follow up (P = 1.00), and 1 of 19 eyes and 4 of 33 eyes at 12-month follow up (P = 0.64). Myopic CNV recurred in 1 of 23 eyes in the ranibizumab group and 5 of 43 eyes in the bevacizumab group during 12-month follow up . The former recurred at 9 months after treatment, and an additional intravitreal ranibizumab injection was performed. The latter all recurred at 12 months after treatment, and additional bevacizumab injections were performed. No severe complications such as endophthalmitis or increased intraocular pressure occurred in either group over the follow-up period.FA revealed leakage at the late phase in 4 of 22 eyes in the ranibizumab group and 8 of 31 eyes in the bevacizumab group at 3-month follow up  -22. In mBoth ranibizumab and bevacizumab are humanized murine monoclonal antibodies against VEGF, but there are some differences between them. The former consists of a 49-kD Fab fragment of antibody; the latter is a 149-kD whole antibody. Ranibizumab may have faster retinal penetration in comparison with bevacizumab due to its smaller molecular size, which would aid in approaching the lesion ,24. RaniIn our study, we demonstrated that the two agents led to similar functional and anatomical visual improvement in the 12-month follow-up period when injected as needed after the first intravitreal injection. LogMAR BCVA improved markedly at 1 month after the first injection in both groups, and it was maintained similarly over the follow-up period (Figure To date, four clinical studies have compared treatment effectiveness after intravitreal injection of ranibizumab and bevacizumab in myopic CNV -17. GharOur study has a few drawbacks. As a retrospective study, it has intrinsic limitations regarding bias control. The patients selected which drugs are injected under sufficient explanation, which would be a selection bias. However, any statistically significant differences were not found in demographic data between two groups. We calculated retinal thickness using several OCT modalities, making comparisons of retinal thickness between patients are less meaningful. However, only one OCT modality was applied to each patient during the follow-up period, which made comparisons of CFT between pre- and post-treatment period reasonable.In conclusion, we showed that in a retrospective study, ranibizumab and bevacizumab had similar functional and anatomical effectiveness for myopic CNV over a 12-month follow-up period. Given that this conflicts with previous results from Iacono et al. , some adCNV: Choroidal neovascularization; PDT: Photodynamic therapy; VIP: Verteporfin in photodynamic; anti-VEGF: Anti-vascular endothelial growth factor; BCVA: Best-corrected visual acuity; FA: Fluorescein angiography; OCT: Optical coherence tomography; CFT: Central foveal thickness; logMAR: Logarithm of the minimum angle of resolution; AMD: Age-related macular degeneration; CATT: Comparison of age-related macular degeneration treatment trial.The authors declare that they have no competing of interests.CDM the statistical analyses, acquisition of data and preparation of the first draft of the manuscript. KTW study concept and design, interpretation of data, drafting/revising the manuscript. WSJ, HJW, PKH, YHG, and CH Review and approval of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2415/14/69/prepub