In addition, the HA partially transformed to α-tricalcium phosphate (α-Ca3(PO4)2, α-TCP) and tetracalcium phosphate (Ca4(PO4)2O, TTCP), when the pellet samples were sintered at 1300 °C and 1400 °C, respectively, for 4 h. The maximum density and Vickers Hardness (HV) of sintered pellet samples were 2.85 g/cm3 ) and 407, which appeared at 1200 °C and 900 °C, respectively.In order to obtain the properties of the sintered as-dried calcium phosphate with [Ca]/[P] = 1.50, the characteristics of sintered pellets have been investigated using X-ray diffraction (XRD), inductively coupled plasma-mass spectrometry (ICP-MS), Fourier-transform infrared (FT-IR) spectra, Vickers hardness indentation and scanning electron microscopy (SEM). When the pellet samples were sintered between 700 °C and 1200 °C for 4 h, the hydroxyapatite (Ca The objects of the present study are: (i) to evaluate the phase stability of as-dried calcium phosphate powders with [Ca]/[P] = 1.50 and self accompanying NaThe element composition of as-dried calcium phosphate powders determined by ICP-MS analysis is listed in 10(PO4)6(OH)2, HA) (JCPDS Card No. 9-432) as the major phases with the rehenanite  as the secondary phase and β-TCP (JCPDS Card No. 86-1585) as the minor phase. The peak positions (2θ) at 31.90, 32.26, 32.98 and 53.24 correspond to the (211), (112), (300) and (004) reflections of HA, respectively. However, the split of (211) and (112) reflections is not complete due to the fact that HA is not fully crystallized from the pellet sample of as-dried calcium phosphate powders when sintered at 600 °C for 4 h.The XRD pattern of the pellet sample of the as-dried calcium phosphate powders with [Ca]/[P] = 1.50 was sintered at 600 °C for 4 h as shown in et al. /[P] = 1.50 sintered at 1300 °C and 1400 °C for 4 h are shown in When the sintering temperature is between 1300 °C and 1400 °C, the HA partially decomposes and transforms into α-TCP, TTCP according to the following reaction :et al. /[P] = 1.50 were sintered between 600 °C and 1200 °C for 4 h, the results of XRD patterns show HA as the major phase. Ishikawa et al.  have poiWang and Chaki  have poi2O at temperatures greater than 1300 °C. Moreover, due to the fact that TTCP is metastable, the synthesis of phase-pure TTCP requires either a rapid quench or the absence of moisture to prevent decomposition into HA and lime in the temperature range of 1000–1200 °C /[P] = 1.50 sintered at 600 °C for 4 h. However, the sintering sample has sufficient strength to measure the density in water. The density increases from 2.77 g/cm3 to 2.90 g/cm3 (91.77% T.D.) when the sintering temperature was increased from 800 °C to 1100 °C. When sintering at 1200 °C, the maximum density of 2.85 g/cm3 (90.18% T.D.) was obtained. Then, the density decreased to 2.05 g/cm3 (64.87% T.D.) when sintering at 1400 °C for 4 h because HA partially decomposed into α-TCP and TTCP. In addition, the density of 2.85 g/cm3 is only 90.33% of T.D., i.e., it is still lower than the theoretical density (3.21 g/cm3) of NaCaPO4 [The density of pellet samples of as-dried calcium phosphate powders with [Ca]/[P] = 1.50 sintered at various temperatures for 4 h is shown in  NaCaPO4 .3 with rising a sintering temperature from 600 °C to 1200 °C. This result was due to the NaCaPO4 increasing with increasing temperature. Moreover, the reaction of dehydroxylation also accompanied the OHA formation with NaCaPO4, as the sintering agent OHA has a good sinterability and, therefore, leads to an increased density. On the other hand, according to In the present study, the lower density of pellet samples was obtained when sintered at 600 °C for 4 h due to loss water of HA by dehydroxylation . The denIt may be concluded that during sintering of HA, grain growth was accompanied by the creation of many vacancies in the apatite crystallites, and that the sintered product is OHA ,41. The On the other hand, the density decreased rapidly when the pellet samples were sintered at 1300 and 1400 °C due to the partial HA decomposition into α-TCP and TTCP . The theThe variation of the hardness of sintered pellets with sintering temperatures was not similar to that of density, indicating that the hardness was not only controlled by the bonding among the particles in the sintered compact, but also controlled by other factors such as grain size and secondary phases. The present result of hardness did not agree with the result of Wang and Chaki . On the i.e., from 1000 to 1400 °C) could not be due to density effect but rather could be caused by the grain growth of HA.In the present work, it was found that the hardness of sintered pellets decreased with increasing temperature beyond the point at which maximum hardness was obtained. The decreases in hardness observed for pellet samples of as-dried calcium phosphate powders with [Ca]/[P] = 1.50 sintered above 900 °C (4 (indicated by arrows) precipitated between the grain boundaries of HA, and pores were also observed.Considering 4·2H2O, DCPD). This result also reveals that in order to obtain the HA it is not necessary for the calcium phosphate to have the exact value of [Ca]/[P] = 1.67. With calcium phosphate powders with values of [Ca]/[P] smaller than 1.67, the HA structure is also obtained.As mentioned above, the effects on the calcium phosphate pellets with [Ca]/[P] = 1.5 sintered between 700 and 1400 °C lead to a HA which possesses a good strength and hardness. In the present contribution, HA was obtained from the calcium phosphate powders with [Ca]/[P] = 1.5 prepared by hydrolysis of hydrogen phosphate dehydrate  and CaCO3 . DCPD and CaCO3 were dissolved in a deionized water and ethanol solution at a volume ratio of 1:5. The concentrations of DCPD and CaCO3 were 0.5 M. The ratio of [Ca]/[P] was designed as 1.5 for the synthesis process. The 0.5 M solution was 500 mL of 2.5 M NaOH solution , with CaCO3 added. Both mixtures were blended in an agitator with a speed of 200 revolutions per minute (rpm). When hydrolysis at 75 °C for 1 h attained the pH value of 11.0, then the reaction was terminated by cooling in ice water. The aggregates were filtered and then rinsed in deionized water five times. Subsequently, the product of calcium phosphate was dried at 60 °C for 12 h. Then, the as-dried calcium phosphate powders were obtained. In order to prevent the occurrence of serious agglomeration, the as-dried powders must be thoroughly. The as-dried powders were put in a laboratory ball mill using Al2O3 balls for 6 h. Then, the ground powders were put through an 80-mesh sieve.The starting materials were hydrogen phosphate dehydrate  was used for these experiments and dissolved in 50% concentrated The particle size distribution of as-dried calcium phosphate powders was measured by using an ultrafine particle analyzer with a 6328 Å He-Ne laser as a detecting source .−1 to 4000 cm−1. A spectral resolution of 4 cm−1 was chosen, and the composite spectrum for each sample was represented by the average of 64 scans, normalized to the spectrum of the blank KBr pellets.The chemical behavior and molecular bonding structure of the as-dried calcium phosphate powders sintering at various temperatures for 4 h were evaluated using a Fourier-transform infrared spectroscope . Each sintered pellet sample was reground to powders. Each sample was mixed with KBr (sample: KBr = 1:99 in mass ratio) and was pressed into 200 mg pellets, 13 mm in diameter, and the infrared adsorption spectra were measured in a frequency range of 400 cm2O3 powders from #100 to #1200 with H2O. Subsequently, polished by diamond paste for X-ray diffraction (XRD), density measurement and hardness test.The surface of sintered pellet samples was ground with Alθ) of 1°/min from 20 to 55°. Then, the XRD patterns were obtained for the polished bulk surface of the sintered pellets.Identification of the crystalline phases in the sintered pellets was carried out by XRD analysis  with Cu Kα radiation and Ni filter, operated at 40 kV, 30 mA, and a scanning rate  of the sintered pellet was measured by the Archimedes method using a sample size of 4.0 × 4.0 × 8.5 mmwa is the weight of a dried sample in air (g), wb is the weight of a sample in deionized water (g) and s is a specific gravity of a deionized water (g/cm3). When the sample weight in deionized water, the suspension wire of copper with diameter 0.12 mm may be twisted around the test specimen.where The hardness was measured by a Vickers indentation hardness tester . During Vickers indentation, the load applied and holding time on the polished surface of sintered pellet were 1.96 N and 30 s, respectively. In each sample, 10 indentations were measured. For scanning electron microscopy (SEM) observation, the polished sintered pellets were etched with dilute acetic acid. The operated conditions of gold-coating were 110 V, 10 mA and 2 min for voltage, current and time, respectively, to obtain the required film thickness for observation. The microstructure was observed by SEM .4 appeared as the major and second phases, respectively, with β-TCP as the minor phase. When the pellet samples were sintering at 1300 °C and 1400 °C, the HA partially decomposited into α-TCP and TTCP. The result of the FTIR showed that the dehydroxylation of HA occurred when sintered between 600 °C and 1200 °C. The density of sintering pellets increased from 2.38 to 2.85 g/cm3 (72.32 to 90.18% T.D.) with rising temperatures from 600 to 1200 °C due to the NaCaPO4 and oxyhydroxyapatite (OHA) formed with the temperature increase and the enhancement of densification. Whereas the density abruptly decreased to 2.05 g/cm3 (64.98% T.D.) when sintered at 1400 °C for 4 h due to partial decomposition of HA into α-TCP and TTCP. In addition, the maximum HV hardness of sintered pellets appeared at 900 °C, then decreased with rising sintering temperatures. When the pellet was sintered at 800 °C for 24 h, polygonization HA grains of a size of about 250–500 nm was observed. When sintering at 1000 °C for 4 h, HA grains of a size of about 0.6–2.0 μm were obtained.The properties of sintering as-dried calcium phosphate powders with [Ca]/[P] = 1.50 have been investigated using ICP-MS, XRD, FTIR, Vickers hardness test and SEM. The as-dried calcium phosphate powders have a single phase of HA when sintered between 600 and 1200 °C, whereby HA and NaCaPO


Saccharomyces cerevisiae to develop a strategy for domain mutagenesis aimed at introducing and in vivo recombining random mutations in defined segments of DNA. Mutagenic Organized Recombination Process by Homologous IN vivo Grouping (MORPHING) is a one-pot random mutagenic method for short protein regions that harnesses the in vivo recombination apparatus of yeast. Using this approach, libraries can be prepared with different mutational loads in DNA segments of less than 30 amino acids so that they can be assembled into the remaining unaltered DNA regions in vivo with high fidelity. As a proof of concept, we present two eukaryotic-ligninolytic enzyme case studies: i) the enhancement of the oxidative stability of a H2O2-sensitive versatile peroxidase by independent evolution of three distinct protein segments ; and ii) the heterologous functional expression of an unspecific peroxygenase by exclusive evolution of its native 43-residue signal sequence.Approaches that depend on directed evolution require reliable methods to generate DNA diversity so that mutant libraries can focus on specific target regions. We took advantage of the high frequency of homologous DNA recombination in  In the past two decades directed evolution strategies have had a huge impact on protein engineering and synthetic biology n possible permutations , it is still far from being fully explored Although the majority of the vast protein sequence space is probably non-functional, with 20Escherichia coli is by far the most common host in directed evolution experiments of prokaryotic proteins. However, broad differences with eukaryotic cells  preclude the use of this bacteria to engineer eukaryotic enzymes which mostly end up in misfolding and inclusion bodies formation. Alternatively, Saccharomyces cerevisiae is the model organism of choice for in vitro evolution of eukaryotic genes, permitting the development of comprehensive synthetic biology and metabolic engineering studies, particularly when dealing with the production of fuels and chemicals S. cerevisiae permits to simply shuffle foreign genes creating multiple crossover events, to repair linearized vectors for in vivo cloning or to promote the molecular evolution of multigenic phenotypes S. cerevisiae. Mutagenic Organized Recombination Process by Homologous IN vivo Grouping (MORPHING) randomly introduces mutations in specific protein segments using overlapping areas to favor in vivo splicing and recombination in yeast. The versatility of this method was evaluated in two case studies of ligninolytic oxidoreductases. First, we used MORPHING to enhance the oxidative stability of a versatile peroxidase (VP) from the basidiomycete Pleurotus eryngii. VP has three different catalytic sites for the oxidation of low-, medium- and high-redox potential compounds, which makes the enzyme extremely fragile in the presence of catalytic concentrations of H2O2Agrocybe aegerita, a heme-thiolate peroxidase with special catalytic capacity that includes oxygen transfer reactions Here, we present a simple, rapid and reliable random domain mutagenesis/recombination method for short fragments that is based on the physiological properties of Pleurotus eryngii (the R4 mutant) was used as the parental type in the construction of the library. This R4 mutant was engineered for secretion by 4 rounds of directed evolution, resulting in expression levels of 22 mg/L upo1 gene (clone C1A-2) from A. aegeritaS. cerevisiae . ABTS ), Taq polymerase, and the S. cerevisiae transformation kit were purchased from Sigma-Aldrich . The iProof High Fidelity DNA polymerase was purchased from Bio-Rad (USA). The Zymoprep Yeast Plasmid Miniprep Kit and Zymoclean Gel DNA Recovery Kit were obtained from Zymo Research , while NBD  was purchased from TCI America (USA). The Escherichia coli XL2-Blue competent cells and the GeneMorph II Kit (mutazyme II polymerase) were from Stratagene , and the uracil independent and ampicillin resistance pJRoC30 shuttle vector was obtained from the California Institute of Technology . The protease-deficient S. cerevisiae strain BJ5465 (α ura3-52 trp1 leu2Δ1 his3Δ200 pep4::HIS3 prb1Δ1.6R can1 GAL) was obtained from LGCPromochem , the NucleoSpin Plasmid kit was purchased from Macherey-Nagel (Germany), and the restriction enzymes BamHI and XhoI from New England Biolabs . All chemicals were of reagent-grade purity.VP from ddH2O to 650 mL. Flask expression medium contained 720 mL YP, 67 mL 1 M KH2PO4 buffer (pH 6.0), 111 mL 20% (w/v) D-galactose, 25 g/L ethanol, 500 or 300 mg/L bovine hemoglobin , 1 mM CaCl2 for VP or 2 mM MgSO4 for UPO, 1 mL 25 g/L chloramphenicol and ddH2O to 1000 mL. Microplate expression medium contained 720 mL YP, 67 mL 1 M KH2PO4 buffer (pH 6.0), 111 mL 20% (w/v) D-galactose, 100 or 50 mg/L bovine hemoglobin , 2 mM MgSO4 for UPO, 1 ml 25 g/L chloramphenicol and ddH2O to 1000 mL. YPD solution contained 1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) sterile D-glucose and 25 µg/mL chloramphenicol. SC drop-out plates contained 0.67% (w/v) sterile yeast nitrogen base, 1.92 g/L (w/v) sterile yeast synthetic drop-out medium supplement without uracil, 2% (w/v) bacto agar, 2% (w/v) sterile D-glucose and 25 µg/mL chloramphenicol. Luria-Bertani (LB) medium was prepared with 1% (w/v) peptone, 0.5% (w/v) yeast extract, 1% (w/v) NaCl and 100 µg/mL ampicillin.Minimal medium (SC) contained 0.67% (w/v) sterile yeast nitrogen base, 1.92 g/L sterile yeast synthetic drop-out medium supplement without uracil, 2% (w/v) sterile D-raffinose and 25 µg/mL chloramphenicol. YP medium contained 10 g yeast extract, 20 g peptone and All the PCR products generated were cleaned, concentrated and loaded onto a low melting-point preparative agarose gel and purified using the Zymoclean Gel DNA Recovery Kit (Zymo Research). The PCR products were cloned by replacing the corresponding parental gene in pJRoC30. To remove the parental gene, the plasmid was linearized with BamHI and XhoI. The pJRoC30-R4 variant was used as a template to construct MORPHING libraries of VP, while the pJRoC30-12C12 was used as the template to construct MORPHING libraries of UPO signal peptide.in vivo cloning in yeast. The targeted regions were subjected to random mutagenesis while the remaining segments were amplified by high-fidelity polymerases . The distal His environment region was selected to tune mutational loads. Small mutant libraries of around 500 clones were screened to optimize the mutagenic conditions. A similar protocol was followed for UPO MORPHING but in this case the UPO gene was split into two segments, one containing the signal peptide and the other the mature protein.For VP MORPHING, the whole gene was fragmented into three different segments in individual PCR reactions. Each segment contained homologous overhangs of ∼50 bp that overlapped one another to promote Figure S1), 0.3 mM dNTPs (0.075 mM each), 3% (v/v) dimethylsulfoxide (DMSO), 1.5 mM MgCl2, increasing concentrations of MnCl2  and 0.05 U/µL Taq polymerase. Error prone-PCRs were carried out on a gradient thermocycler  using the following conditions: 95°C for 2 min (1 cycle); 94°C for 45 s, 56°C for 45 s, 74°C for 45 s (28 cycles); and 74°C for 10 min (1 cycle). For the proximal His environment and Met environment, the final DNA template concentration was 0.92 ng/µL and the MnCl2 concentration was set at 0.2 mM and 0.01 mM, independently for both targeted regions.i) Mutagenic PCR of targeted regions: Reaction mixtures were prepared in a final volume of 50 µL containing the following: DNA template (0.1 ng/µL or 0.92 ng/µL), 90 nM Forward primer, 90 nM Reverse primer (different forward (F) and reverse (R) primers were used to amplify each region; Figure S1), 0.8 mM dNTPs (0.2 mM each), 3% (v/v) dimethylsulfoxide (DMSO), and 0.02 U/µL iProof polymerase. High fidelity PCRs were performed on a gradient thermocycler using the following conditions: 98°C for 30 s (1 cycle); 98°C for 10 s, 45 or 50°C for 30 s, 72°C for 1 min (28 cycles); and 72°C for 10 min (1 cycle).ii) High-fidelity PCR in non-mutated segments: Reaction mixtures were prepared in a final volume of 50 µL containing: DNA template (0.2 ng/µL), 0.5 µM Forward primer, 0.5 µM Reverse, (different forward (F) and reverse (R) primers were used to amplify each region; in vivo and recombined by transformation into S. cerevisiae cells using the Yeast Transformation Kit. The DNA transformation mixture contained the linearized plasmid (200 ng) mixed with the targeted region, as well as the segments upstream and downstream of those regions (400 ng per segment). Transformed cells were plated on SC drop-out plates and incubated for 3 days at 30°C. Subsequently, the mutant libraries were subjected to the HTP-protocol for oxidative stability, as described below.iii) Reassembly of the whole gene: The whole gene was reassembled Table S1), 0.3 mM dNTPs (0.075 mM each), 3% (v/v) dimethylsulfoxide (DMSO), 1.5 mM MgCl2, 0.1 mM MnCl2 and 0.05 U/µL Taq polymerase. Error-prone PCR was carried out on a gradient thermocycler using the following parameters: 95°C for 2 min (1 cycle); 94°C for 45 s, 50°C for 45 s, 74°C for 30 s (28 cycles); and 74°C for 10 min (1 cycle).i) Mutagenic PCR in signal peptide: Reaction mixtures were prepared in a final volume of 50 µL containing the following: DNA template (0.92 ng/µL), 90 nM RMLN F primer, 90 nM MORPH SP R primer (Table S1), 1 mM dNTPs (0.25 mM each), 3% (v/v) dimethylsulfoxide (DMSO), and 0.02 U/µL iProof polymerase. High fidelity PCR was carried out on the gradient thermocycler using the following conditions: 98°C for 30 s (1 cycle); 98°C for 10 s, 55°C for 25 s, 72°C for 45 s (28 cycles); and 72°C for 10 min (1 cycle).ii) High-fidelity PCR of non-mutated segment: Reaction mixtures were prepared in a final volume of 50 µL containing DNA template (0.2 ng/µL), 0.5 µM MORPH SP D primer, 0.5 µM RMLC R primer (in vivo and recombined by transformation into S. cerevisiae cells using the Yeast Transformation Kit. The DNA transformation mixture contained the linearized plasmid (100 ng) mixed with the targeted region, as well as the mature protein (200 ng per segment). Transformed cells were plated on SC drop-out plates and incubated for 3 days at 30°C. Subsequently, the mutant libraries were subjected to the HTP-protocol to assess activity as described below.iii) Reassembly of the whole gene: The whole gene was reassembled In vivo Assembly of Mutant libraries (IvAM) The whole UPO gene including its signal peptide was subjected to one round of directed evolution by Table S1), 0.3 mM dNTPs (0.075 mM each), 3% (v/v) dimethylsulfoxide (DMSO), 1.5 mM MgCl2, 0.01 mM MnCl2 and 0.05 U/µL Taq polymerase. Error-prone PCR was carried out under the following conditions: 95°C for 2 min (1 cycle); 94°C for 45 s, 53°C for 45 s, 74°C for 3 min (28 cycles); and 74°C for 10 min (1 cycle).Reaction mixtures were prepared in a final volume of 50 µL containing: DNA template (0.1 ng/µL), 90 nM RMLN F primer, 90 nM RMLC R primer (Table S1), 0.8 mM dNTPs (0.2 mM each), 3% (v/v) dimethylsulfoxide (DMSO), and 0.05 U/µL Mutazyme II. Error-prone PCR was carried out using the following parameters: 95°C for 2 min (1 cycle); 94°C for 45 s, 53°C for 45 s, 74°C for 3 min (28 cycles); and 74°C for 10 min (1 cycle).Reaction mixtures were prepared in a final volume of 50 µL containing: DNA template (56 ng/µL), 0.37 µM RMLN F primer, 0.37 µM RMLC R primer (Taq polymerase library and Mutazyme library were added in equimolar concentrations (200 ng each) to the linearized vector (100 ng). Transformed cells were plated on SC drop-out plates and incubated for 3 days at 30°C. Thereafter, the mutant libraries were subjected to the HTP-protocol to assay activity as described below.Table S1), 1 mM dNTPs (0.25 mM each), 3% (v/v) dimethylsulfoxide (DMSO), and 0.02 U/µL iProof polymerase. High fidelity PCR1 was carried out on the gradient thermocycler under the following conditions: 98°C for 30 s (1 cycle); 98°C for 10 s, 47°C for 25 s, 72°C for 15 s (28 cycles); and 72°C for 10 min (1 cycle).Reaction mixture was prepared in a final volume of 50 µL containing: DNA template (0.2 ng/µL), 0.5 µM Forward primer RMLN F, 0.5 µM Reverse primer SP* R , 0.5 µM Forward primer SP* F, 0.5 µM Reverse primer RMLC R (Table S1), 1 mM dNTPs (0.25 mM each), 3% (v/v) dimethylsulfoxide (DMSO), and 0.02 U/µL iProof polymerase. High fidelity PCR was carried out on the gradient thermocycler under the following conditions: 98°C for 30 s (1 cycle); 98°C for 10 s, 52°C for 25 s, 72°C for 40 s (28 cycles); and 72°C for 15 min (1 cycle). PCR fragments corresponding to the SP* and the native upo (200 ng each) were recombined together with the linearized vector (100 ng) by IVOE the native Table S1)), 0.8 mM dNTPs (0.2 mM each), 3% (v/v) dimethylsulfoxide (DMSO), and 0.02 U/µL iProof polymerase. High fidelity PCRs were carried out on the gradient thermocycler under the following conditions: 98°C for 30 s (1 cycle); 98°C for 10 s, 53°C for 30 s, 72°C for 1 min (28 cycles); and 72°C for 10 min (1 cycle). The whole gene was reassembled in vivo and recombined by transformation into S. cerevisiae cells using the Yeast Transformation Kit. The DNA transformation mixture was composed of the linearized plasmid (200 ng) mixed with the mutated fragments (400 ng per fragment). Transformed cells were plated on SC drop-out plates and incubated for 3 days at 30°C. Thereafter, the mutant libraries were subjected to the HTP-protocol described below.Reaction mixtures were prepared in a final volume of 50 µL containing: DNA template (0.2 ng/µL), 0.2 µM Forward primer, 0.2 µM Reverse primer . After 48 h, 160 µL of expression medium was added to each well and the plates were incubated for a further 24 h. The plates (master plates) were centrifuged for 15 min at 3000 rpm and 4°C  and the master plates were duplicated with the help of a robot  by transferring 20 µL of supernatant into two replica plates: the initial activity plate (IA plate) and the residual activity plate (RA plate). Next, 180 µL of stability buffer  was added to the IA plates and 180 µL of incubation solution (Buffer A containing 0.3 mM H2O2) was added to the RA plates using a Multidrop robot . Both plates were briefly stirred and incubated at room temperature for 60 min, such that the activity assessed in the RA plates was reduced by 2/3rds with respect to the initial activity of the parental type. The supernatants (20 µL) were transferred from both RA and IA plates to new plates to measure the residual and initial activity values by adding ABTS in specific buffers: 180 µL of 100 mM sodium tartrate buffer [pH 3.5] containing 2 mM ABTS and 0.1 mM H2O2 to estimate of residual activity; and 180 µL of 100 mM sodium tartrate buffer [pH 3.5] containing 2 mM ABTS and 0.13 mM H2O2 to estimate the initial activity. The plates were stirred briefly and the absorption at 418 nm  was recorded  on a plate reader . The plates were then incubated at room temperature until a green color developed and the absorption was measured again (t1). The relative activities were calculated from the difference between the absorption value after incubation and that of the initial measurement normalized to the parental type in the corresponding plate (Δt1-t0). Oxidative stability values were calculated as the ratio between residual activity and the initial activity values (RA/IA). To rule out false positives, two consecutive re-screenings were carried out. Moreover, a third re-screening was performed to determine the increase in the apparent half-life of each selected variant  relative to the parental R4 in different molar ratios [H2O2]/[VP in supernatants].Individual clones were selected and cultured in sterile 96-well plates  containing 50 µL per well of SC minimal medium. In each plate, column number 6 was inoculated with the parental R4 mutant as an internal standard, and well-H1  was inoculated with untransformed First re-screening: Aliquots of 5 µL of the best clones were removed from the master plates and used to inoculate 50 µL of minimal medium in new 96-well plates. Columns 1 and 12, and rows A and H, were not used to prevent the appearance of false positives. After incubating for 24 h at 30°C, 225 rpm, and 80% relative humidity, 5 µL was transferred to the adjacent wells and incubated for a further 24 h. Finally, 160 µL of expression medium was added and the plates were incubated for another 24 h. Accordingly, each mutant was grown in 4 wells. The parental types were subjected to the same procedure  and the plates were assessed using the same protocols as those used for the screening described above.E. coli cells  and plated on LB/amp plates. Single colonies were picked and used to inoculate 5 mL LB/amp media, and they were grown overnight at 37°C and 225 rpm. The plasmids were then extracted  and S. cerevisiae was transformed with plasmids from the best mutants as well as with the parental type. Five colonies for each mutant were selected and re-screened as described above.Second re-screening: An aliquot from the wells with the best clones in the first re-screening was inoculated in 3 mL of YPD and incubated at 30°C and 225 rpm for 24 h, recovering the plasmids from these cultures (Zymoprep Yeast Plasmid Miniprep Kit). As the product of the zymoprep was very impure and the concentration of DNA extracted very low, the zymoprep mixtures containing shuttle vectors were transformed into super-competent t1/2 H2O2): A single colony from the S. cerevisiae clone containing the parental R4, the new mutants and untransformed yeast were picked from a SC drop-out plate (SC supplemented with uracil for untransformed cells), used to inoculate 5 mL of minimal medium, and incubated for 48 h at 30°C and 225 rpm . An aliquot of cells was removed and used to inoculate a final volume of 5 mL of minimal medium in a 50 mL falcon tube , and they were incubated until two growth phases had been completed . Thereafter, 9 mL of expression medium (500 mg/L bovine hemoglobin) was inoculated with 1 mL of this preculture in a 100 mL flask (OD600 = 0.1). After incubating for ∼48 h at 30°C and 225 rpm , the cells were separated by centrifugation for 15 min at 3000 rpm and 4°C , and the supernatants were collected and stored at 4°C. The protein concentration was estimated from supernatants using the Bio-Rad protein assay kit, . VP apparent concentration was calculated as the difference between the total protein content of yeast expressing VP and that in its absence (from non-transformed yeast cells -lacking VP gene-). The parental R4 and the mutants  were incubated at room temperature in 20 mM sodium tartrate buffer [pH 5.0] in the presence or absence of different concentrations of H2O2 . Aliquots (20 µL) were removed at different times and the residual/initial activities were measured in a final volume of 200 µL containing 100 mM sodium tartrate buffer [pH 3.5], 2 mM ABTS and 0.13 mM or 0.16 mM H2O2 . The residual activity was determined relative to the corresponding mutant incubated in the absence of H2O2, taking into account the final concentration of H2O2 in each activity assay. Each point represents the mean of three independent experiments performed in triplicate. Increment in apparent half-lives (Δt1/2vs H2O2) were calculated for each mutant at given molar ratio VP (supernatants) : H2O2.Third re-screening  were centrifuged  for 15 min at 3000 rpm and 4°C, and the supernatants (20 µL) were transferred from the master plate to two replica plates by a robot , adding the reaction mixture (180 µL) together with ABTS or NBD to each replica plate. Both colorimetric assays were used for properly detecting secretion levels improvements regardless of the substrate used . The reaction mixture with ABTS contained 100 mM sodium citrate/phosphate buffer [pH 4.4], 0.3 mM ABTS and 2 mM H2O2, while the reaction mixture with NBD contained 100 mM potassium phosphate buffer [pH 7.0], 1 mM NBD, 15% (v/v) acetonitrile and 1 mM H2O2. Plates were stirred briefly and the initial absorptions at 418 nm  and 425 nm  were recorded on the plate reader . The plates were then incubated at room temperature until a green (ABTS) or yellow (NBD) color developed, and the absorption was measured again. The values were normalized against the parental type in the corresponding plate. To rule out false positives, two re-screenings were carried out as described above for VP.Individual clones were selected and inoculated in sterile 96-well plates  containing 50 µL per well of SC minimal medium. In each plate, column number 6 was inoculated with the corresponding parental type, and one well (H1-control) was inoculated with untransformed 2O2 was added to the samples to measure activities. The thermostability values were calculated as the ratio between the residual activity at different temperature points and the initial activity at room temperature. The T50 value was determined as the transition midpoint of the inactivation curve of the protein as a function of temperature, which in our case was defined as the temperature at which the enzyme lost 50% of its initial activity after a 10 min incubation.Appropriate dilutions of the supernatants were prepared such that aliquots (20 µL) produced a linear response in kinetic mode. A gradient profile was constructed using a thermocycler  for the selected mutants and the parental type, using 50 µL for each point in a gradient scale ranging from 30 to 80°C. After a 10 min incubation, samples were removed and chilled on ice for 10 min. Thereafter, 20 µL samples were removed and incubated for 5 min at room temperature. Finally, 180 µL of 100 mM sodium tartrate buffer [pH 3.5], 2 mM ABTS and 0.1 mM HPlasmids containing the VP/UPO variants were sequenced on an ABI 3730 DNA Analyzer-Applied Biosystems Automatic Sequencer at the Secugen . The following primers were designed using Fast-PCR software :5′-GTTCCATCATCGCGTTCG-3′); 5F-reverse (5′-GGATTCCTTTCTTCTTGG-3′) and RMLC R (Table S1). For UPO: RMLN F; upo1 sec direct (5′-GAAGGCGACGCCAGTATGACC-3′); upo1 sec reverse (5′-GGTCATACTGGCGTCGCCTTC-3′) and RMLC R (Table S1).for VP: RMLN F; 3R-direct  was used to generate a model to map the new mutations found with the help of the PyMOL Molecular Visualization System (Schrödinger). A homology model was generated by carrying out a structural alignment in PyMOL with the following crystal structures (PDB IDs are indicated): 3FJW, native VP from P. eryngii; 1IYN, recombinant chloroplastic ascorbate peroxidase (ApX) from N. tabacum expressed in E. coli; 3M5Q, native manganese peroxidase (MnP) isozyme 1 from P. chrysosporium; 1H3J, native peroxidase from C. cinerea (CiP); and 1W4W, recombinant horseradish peroxidase C1A from horseradish (HRP) expressed in E. coli. IDENTITY and SIMILARITY percentages were obtained using SEQUENCE SIMILARITY AND IDENTITY Software (http://imed.med.ucm.es/Tools/sias.html).The crystal structure of VPL2 from S. cerevisiae. In a single step, this approach allows us to assemble delimited randomly mutagenized regions with the remaining, unaltered fragments of a gene. Unlike most evolution methods focused in restricted areas, mutations are randomly generated and they do not depend on the engineering of a set of spiked/degenerate synthetic oligonucleotides. By MORPHING, small segments are targeted and subjected to error-prone PCR with defined mutational frequencies, while the remaining portions of the gene are amplified using high-fidelity polymerases  between segments is directly proportional to the number of segments assembled, allowing several regions to be studied alone or in a combinatorial manner. Depending on the distance between mutations, crossover events can occur between the different mutations in the target fragment(s), mediated by the S. cerevisiae recombination machinery, fostering enrichment. The success of this method is facilitated by the high fidelity DNA splicing of fragments through the small overhangs with overlapping sequences of ∼50 bp that flank each segment. These overhangs ensure the in vivo reconstitution of the whole gene with random mutations only in the segments specifically targeted. Under these rules, up to six recombination events between fragments can be created without significantly affecting transformation efficiencies .MORPHING is a method of generating DNA diversity based on the high frequency of homologous recombination of ymerases Figure 12O2 due to a mechanism-based phenomenon known as suicide inactivation that is common to all peroxidases 2+ binding site), and two access channels, the dense production and traffic of free radicals jeopardizes the enzyme's stability and function, . The first region subjected to MORPHING was the distal His environment (L28-G57) that contains the H2O2 binding site within a helix that is highly conserved in all high-redox potential peroxidases. The second target region was the proximal His environment (L149-A174) located on the opposite side to the distal His, in the surroundings of the heme domain. The third region was the Met environment (I199-L268), containing three of the five putative oxidizable Met in the VP-R4 variant. Several mutational loads were assayed for each region to construct independent mutant libraries and then explore them for oxidative stability. Mutational loads were adjusted by modifying the PCR conditions in order to introduce 1 to 5 mutations per segment (including the crossover areas). The frequencies of mutation were estimated from different landscapes of mutant libraries (500 clones each), calculating the number of clones with <10% of the parental enzyme activity, and they were further verified by DNA sequencing of a random sample of mutants including active and non-active variants  and Tv of 35%  was observed in the libraries under study (i.e. Ts/Tv ratio∼1.8). Mutational frequencies of 0.5 nucleotide changes/100 bp were obtained with Ts G→A when [MnCl2]≤0.1 mM, regardless of the DNA template concentration. In segments as short as 30 residues long , we obtained high mutational loads at [MnCl2]>0.1 mM (frequencies of ∼3 mutations/100 bp). Among the high mutational variants, the 1E11  and 2G4  mutants incorporated 5 mutations each that inactivated the protein due to the highly conserved nature of this region  were identified. We found two beneficial mutations at the L28-G57 segment. The 3G10 variant showed a 2.1-fold improvement in stability vs H2O2, with a significant Δt1/2vs H2O2 of 18 min with respect to the parental type and a 5.5°C increase in the T50 , it conferred a 1.3-fold improvement in stability and it was associated with a Δt1/2vs H2O2 of ∼10 min with respect to the parental type . Situated at the heme entrance, Pro141 is a highly conserved residue in all fungal peroxidases. However, the P141A mutation resulted in a 1.4-fold improvement in oxidative stability, producing a Δt1/2vs H2O2 of ∼5 min with respect to the parental type and no change in thermostability (t1/2vs H2O2 to 45 min (data not shown). This mutant was the departure point to engineer an oxidative stable VP variant by successive rounds of in vitro evolution .After the initial screening and three consecutive re-screenings, several mutants with an improved increment in apparent half-life . Similarly, Met152 in the proximal His environment was not mutated.No mutants were discovered in the I199-L268 segment (the Met environment), suggesting that the three oxidizable methionines in this area are not involved in the oxidative stabilization of VP. This result was corroborated by subjecting Met262 and Met265 to combinatorial saturation mutagenesis and screening. No improved variants were identified in this way and ∼95% of the clones were inactive in this mutagenic landscape, indicating that these residues are very sensitive and do not tolerate changes  is a new, potentially ligninolytic, peroxidase that has attracted much research attention due to its versatility and applicability in a variety of synthetic processes S. cerevisiae (unpublished data). To further enhance the secretion of this protein, we used MORPHING to independently evolve the 43 amino acid UPO signal peptide in order to enrich the signal leader in beneficial mutations without altering the biochemical properties of the enzyme. To reliably compare the two directed evolution strategies, we exposed the signal peptide alone and the entire UPO gene  to one round of directed evolution in two parallel experiments. Mutational rates for the leader library and the full-gene library were 0.5 and 0.1 nucleotide changes/100 bp, respectively. Both libraries were screened with the help of an ad-hoc dual-colorimetric assay to estimate the enzymes peroxidase (with ABTS) and peroxygenase (with NBD) activities. The mutagenic landscape generated by MORPHING revealed an increased tolerance to mutations in the signal peptide than in the whole UPO gene. Indeed, we found that when random mutagenesis of the leader and the whole UPO gene was compared, 30% and 49% of clones had <10% of parental enzyme activity on ABTS as a substrate, respectively . Each of these mutations individually enhanced secretion by ∼20% with respect to the parental type. Although MORPHING was useful to unmask these beneficial mutations for secretion, the eukaryotic machinery of S. cerevisiae was unable to join these positions by homologous recombination due to their proximity. Therefore, we constructed a signal peptide containing the full set of these mutations by conventional site-directed mutagenesis looking for a synergic effect in secretion levels. The evolved signal peptide also included the beneficial mutation GCCA21DGAC (discovered in the earlier stages of evolution). Finally, the signal peptide of native UPO was replaced by the evolved signal peptide F12Y-A14V-R15G-A21D  could be enhanced in ectively Figure 7S. cerevisiae secretory pathway.This second case study demonstrates that the use of our focused mutagenesis method to direct the evolution of signal leaders is a suitable approach to promote heterologous functional expression of complex eukaryotic genes in yeast. Targeting mutational loads to leader sequences is a simple means of detecting mutations that are beneficial for secretion and that can be subsequently combined in a single signal peptide to generate potential synergies along the one-pot approach for the construction of focused mutant libraries of eukaryotic genes in S. cerevisiae. In general terms, MORPHING allows the researcher to focus exclusively on the random introduction of mutations and their recombination in restricted region/s, while protecting critical domains from mutagenesis. The selection of the target regions to be evolved is as important as the mutational loads chosen for each mutant library, which can be easily varied to enrich the target segments in beneficial mutations.The random domain mutagenesis/recombination method presented here is a reliable 2O2 inhibition in VP that could be applied to other high redox-potential fungal peroxidases, it also effectively decoupled secretion and catalytic activity for functional UPO expression. This approach can also be used to explore other complex problems, such as to alter substrate specificity or enantio-selectivity by subjecting several segments of the same gene to random mutagenesis, promoting their in vivo assembly in one transformation step. Indeed, this strategy is currently being used by our group to evolve a fungal aryl alcohol oxidase for the selective oxidation of different alcohols. Apart from structure-function relationship studies, MORPHING can be also useful when structural information is absent, e.g., for the evolution of leader peptides for secretion, for the modification of promoters, or in the evolution of unknown regions of biochemical relevance that have been revealed by conventional directed evolution.The two case studies presented here validate the versatility of our method by tackling two distinct problems. While MORPHING proved useful to explore limited targeted regions, allowing us to identify several structural determinants of HAdditional advantages of this method include the reduction of the sequence space to be explored , the conservation of certain catalytic properties while improving other traits, and the discovery of new structural/catalytic determinants that can be further optimized using focused saturation mutagenesis. The combination of MORPHING with classical directed evolution and semi-rational approaches, or with neutral genetic drift, may lead to the development of new adaptive pathways to engineer more robust eukaryotic enzymes in yeast Figure S1VP MORPHING. Three different regions of VP were targeted for random mutagenesis and recombination . The VP gene is shown in blue, the α-factor prepro-leader to promote secretion in yeast in red and the shuttle vector in green. The areas of crossover between the fragments are represented by crosses. The overlapping areas between segments were created by superimposing PCR reactions in defined regions .(TIF)Click here for additional data file.Figure S2Selected areas of VP subjected to MORPHING: proximal His environment (red), Met environment (yellow), and distal His environment (blue). The heme domain is depicted in stick mode and CPK colors. The amino acids delimiting mutated regions and the most relevant residues are highlighted .(TIF)Click here for additional data file.Figure S3Combinatorial saturation mutagenesis landscapes at positions 262 and 265 of VP. Clone activity is plotted in descending order. The solid horizontal line indicates the activity of the parental type in the assay.(TIF)Click here for additional data file.Table S1Oligos used for VP and UPO MORPHING. The lengths of the PCR products and the overlapping areas are shown. NNS and SNN indicate NN(G/C) and (G/C)NN codons for the saturation mutagenesis libraries. CSM, combinatorial saturation mutagenesis; UPOsp, UPO signal peptide; IvAM, In vivoAssembly of Mutant libraries for the directed evolution of the whole UPO gene; epPCR, error-prone PCR; HF-PCR, high-fidelity PCR.(TIF)Click here for additional data file.


To the Editor: Tick-borne rickettsioses are zoonoses caused by intracellular bacteria belonging to the spotted fever group rickettsiae  were included in a prospective study. The mean (± SD) interval between onset of lesions and consultation was 7 ± 1.8 days. Underlying conditions were present in 13 (33%) patients: diabetes , hypertension , chronic renal failure , cervical cancer , bronchial cancer , and tobacco consumption . Fever and generalized maculopapular rash  were found for 38 (97.4%) patients, including 5 (12.8%) with purpuric rash. One (2.5%) patient had 2 eschars on the back. Eschars were frontal (1 [2.5%] patient), cervical (3 [7%]), axillary (4 [10.2%]), on the back (6 [15%]), on the abdomen (2 [5%)], on the thorax (1 [2.5%]), inguinal (6 [15%]), on the leg (8 [20.5%]), on the arm (1 [2.5%]), on the buttock (2 [5%]), on the breast (1 [2.5%]), on the nipple (1 [2.5%]), on the penis (1 [2.5%]), and on the scrotum (2 [5%]). Conjunctivitis was reported for 24 (61.5%) patients and myalgia for 34 (87.8%). Lymphadenopathy was found near the eschar for 8 (20.5%) patients. Antimicrobial drug therapy (doxycycline 200 mg 1×/d) for 3.5 ± 1.4 days was empirically prescribed for all case-patients before diagnoses were confirmed.A dry sterile swab  sample was collected from the inoculation eschar of each patient by the same person (N.M.). Two patients had 2 swab samples collected from the same eschar. Only 1 swab sample was collected from the patient with 2 eschars. Scabs were removed from eschars before swabbing. The swabs, while being rotated vigorously, were directed to the base of the eschar at a 50°–60° angle for 5–6 times. For 4 patients, an eschar biopsy sample was collected under sterile conditions. Swabs were then placed back in their tubes and stored at –20°C before transportation to Unité des Rickettsies, Marseille, France.,t) value of the β-actin gene for all swab samples was 24.9 (± 2.7). Of 41 swab samples, 26 were positive for rickettsial DNA (63.4%) by qPCR targeting the RC0338 gene . Specific Rickettsia conorii conorii qPCR, targeting the putative acetyltransferase gene , significantly lower than that for R. conorii conorii–negative samples . Of 4 patients for whom swab and skin biopsy samples were available, 3 had positive results.In the laboratory, each sample was placed in 2 mL of culture medium. DNA was extracted from 200 µL of solution of eschar swab or skin biopsy samples by using the QIAamp DNA Mini Kit  according to the manufacturer’s instructions, with a final elution volume of 100 µL. We used quantitative real-time PCR (qPCR) to determine quality of DNA extraction and level of housekeeping gene coding for β-actin (www.graphpad.com/prism/Prism.htm) to p<0.5.Opinions of health care providers and patients were evaluated by using standard questionnaires . Most het values of β-actin, results in low rickettsial load, explaining the false-negative results. This test can be used at the bedside or in an outpatient clinic and could be useful for epidemiologic and clinical studies. Because qPCR results can be obtained <4 hours after sampling, this technique might be useful for point-of-care diagnosis.Swabbing an eschar is a rapid and simple technique that can be easily performed without risk for the side effects associated with biopsy sampling. Insufficient material taken during swabbing, evidenced by high C


Reduced muscle mass is a hallmark of metabolic diseases like diabetes and cancer. The mammalian (mechanistic) target of rapamycin complex 1/S6 kinase 1 (mTORC1/S6K1) pathway is critical to the regulation of muscle protein synthesis and mass but its mechanism of action is not completely understood.Using L6 myotubes, we characterized the regulation of programmed cell death 4 (PDCD4), a recently described substrate of S6K1. The abundance, but not Ser67 phosphorylation, of PDCD4 was sensitive to amino acid and serum deprivation: values in starved cells were 4.5X of control (P < 0.001). Refeeding had opposite effects. Growth factors, compared to amino acids, appeared more critical in regulating PDCD4 abundance. Furthermore, inhibition of mTORC1 or the proteasome prevented the refeeding-associated decrease in PDCD4 abundance. Amino acid and serum deprivation significantly increased PDCD4 binding to eIF4A (P < 0.05); this was reversed during refeeding. PDCD4 depletion by RNA interference had no significant effect on phenylalanine incorporation into myotube mixed proteins in control cells but further suppressed (30%) this measure in nutrient-deprived cells (P < 0.0005). This was not observed in myoblasts. In starved myotubes, PDCD4 depletion further reduced the association of eIF4G with eIF4E.Our data suggest that in myotubes, PDCD4 abundance is sensitive to nutritional manipulation in an mTORC1 and proteasome depended manner. Furthermore, the role of PDCD4 in regulating protein synthesis appears dependent on the developmental state of the cell. The mammalian (mechanistic) target of rapamycin complex 1/ribosomal protein S6 kinase 1 (mTORC1/S6K1) signalling is a critical regulator of skeletal muscle mass and metabolism, and mechanisms that regulate it are studied as possible targets for the treatment/prevention of loss of muscle mass in diverse muscle atrophying conditions . HoweverProgrammed cell death 4 (PDCD4),  , H731 99, and inMuch of what is known about PDCD4 is from cancer studies where PDCD4 is proposed to function as a cell cycle inhibitor/tumor suppressor. Loss of this protein is associated with invasion, progression or increased aggression of numerous, but not all , cancersIn the present work, using L6 myotubes, our specific objectives were to: 1) examine the requirement for mTORC1/S6K1 and the ubiquitin proteolytic system in regulating PDCD4; 2) examine the contribution of amino acids vs. growth factors in mediating the effect of nutrition on PDCD4; and 3) determine whether nutritional status affects the interaction of PDCD4 with components of eIF4F. Because others have suggested that signalling pathways that regulate protein metabolism may be regulated differently in myotubes versus myoblasts  and becaGiven the identification of PDCD4 as a substrate of mTORC1/S6K1 signalling, and the fact this kinase pathway is regulated by nutrients, we examined the effect of nutrient deprivation on the regulation PDCD4 in L6 myotubes. Neither 12 h of serum and amino acid deprivation nor refeeding in a complete medium had any significant effect on PDCD4 Ser67 (the residue targeted by S6K1) phosphorylation . Amino acid-free medium (RPMI 1640) was obtained from US Biological . PDCD4 siRNA oligonucleotides, phosphatase and protease inhibitor cocktails were purchased from Sigma-Aldrich . Alpha Modification of Eagle’s Medium (AMEM) was obtained from Wisent .Fetal Bovine Serum (FBS), Horse Serum (HS), Lipofectamine RNAiAntibodies to eIF4A, eIF4G, phosphorylated (Thr389) S6K1, and horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit and anti-mouse) were purchased from Cell Signaling Technology . Antibody against PDCD4 was from Cell Signaling Technology or Santa Cruz Biotechnology . Antibodies against phosphorylated  PDCD4 were from Sigma-Aldrich or Aviva Systems Biology .L6 myoblasts were cultured in 12-well plates in growth medium  until they reached ~80% confluency. Cells were then shifted into differentiation medium (DM: AMEM as above except that 2% HS replaced 10% FBS). Experiments were carried out on day 4–5 of differentiation. For starvation experiments, myotubes were grown in differentiation medium or starved in amino-acid free, serum-free medium (amino acid-free RPMI 1640) for 12 h. They were then refed in DM for 1 or 3 h. To examine the roles of amino acids and growth factors in regulating PDCD4 abundance, in some experiments refeeding was done in incubation media of varied composition. To examine the requirement for mTORC1 or the ubiquitin dependent proteolytic system on the regulation of PDCD4, additional refeeding experiments were carried out in the presence of inhibitors of these pathways  or equivalent volumes of DMSO. At the end of the experiments, cells were harvested in a lysis buffer  sodium dodecyl sulphate (SDS), 1 mM DTT, supplemented with protease (10 μl/mL) and phosphatase (10 μl/mL) inhibitor cocktails).Max (Life Technologies) as previously described [Myotubes on day 4 of differentiation were transfected with 30 nM siRNA oligonucleotides designed against PDCD4 or with a proprietary scrambled oligonucleotide ), using Lipofectamine RNAiescribed . We usedescribed ,40.Proteins were resolved on 7.5, 10, or 15% SDS-PAGE (depending on the antigens being studied), transferred onto polyvinylidene difluoride (PVDF) membranes, which were then immunoblotted for the indicated antigens, as previously described . ImmunobTo immunoprecipitate eIF4A or PDCD4, myotubes were cultured in 10-cm plates. Following appropriate treatments, cells were rinsed in ice-cold PBS and then lysed in 500 μl of ice-cold lysis buffer (40 mm HEPES (pH 7.5), 120 mM NaCl, 1 mM EDTA, 10 mM pyrophosphate, 10 mM glycerol 2 phosphate, 0.5 mM orthovanadate) supplemented with 0.03% CHAPS, 1 mM DTT, 0.5 mM NaV, 1 mM benzamidine, 6.25 mM N-ethyl-maleimide (NEM) and protease inhibitor cocktail (10 μL/mL). One hundred micrograms of myotube proteins were combined with either anti eIF4A or anti-PDCD4 (Santa Cruz biotechnology) antibodies and the mix rotated overnight at 4°C. The following day and in order to precipitate the antigen-antibody complex, 50 μL of re-suspended BioMag protein G–bound beads  were added to each of the immunoprecipitation tubes and the suspension rocked gently at 4°C for 1 h. The beads were collected on a magnetic stand (Life Technologies) and washed 3 times with 0.1 M sodium phosphate buffer. After the last wash, beads were re-suspended in 1X SDS-PAGE sample buffer and boiled at 95°C for 2 minutes. Following a brief centrifugation, eluates were collected, separated on 10% SDS-PAGE, and blotted for PDCD4 and eIF4A.Data are presented as means ± SEM. Treatment means were compared using a one-way analysis of variance (ANOVA) and differences among individual means assessed using the Bonferroni multiple comparison test or, as in Figures The authors declare that they have no competing interest.OAJA conceived, designed, conducted experiments, wrote and reviewed the manuscript; DK, NI and NM conducted experiments and reviewed the manuscript. All authors read and approved the final version of the manuscript.PDCD4 phosphorylation (Ser457) in L6 myotubes.Click here for fileMyofibrillar protein synthesis in L6 myotubes.Click here for file


The enzymatic conjugation of (DOTA)1-decalysine, (DOTA)3-decalysine or (DOTA)5-decalysine to the antibody heavy chain (via Gln295/297) gave rise to immunoconjugates containing two, six or ten DOTA moieties respectively. Radiolabeling of the immunoconjugates with 177Lu yielded specific activities of approximately 70 MBq/mg, 400 MBq/mg and 700 MBq/mg with increasing numbers of DOTA chelates. Biodistribution experiments in SKOV3ip human ovarian cancer cell xenografts demonstrated a high and specific accumulation of radioactivity at the tumor site for all antibody derivatives with a maximal tumor accumulation of 43.6±4.3% ID/g at 24 h for chCE7agl-[(DOTA)-decalysine]2, 30.6±12.0% ID/g at 24 h for chCE7agl-[(DOTA)3-decalysine]2 and 49.9±3.1% ID/g at 48 h for chCE7agl-[(DOTA)5-decalysine)]2. The rapid elimination from the blood of chCE7agl-[(DOTA)-decalysine]2 (1.0±0.1% ID/g at 24 h) is associated with a high liver accumulation (23.2±4.6% ID/g at 24 h). This behavior changed depending on the numbers of DOTA moieties coupled to the decalysine peptide with a slower blood clearance (5.1±1.0 (DOTA)3 versus 11.7±1.4% ID/g (DOTA)5, p<0.005 at 24 h) and lower radioactivity levels in the liver (21.4±3.4 (DOTA)3 versus 5.8±0.7 (DOTA)5, p<0.005 at 24 h). We conclude that the site-specific and stoichiometric uniform conjugation of the highly DOTA-substituted decalysine ((DOTA)5-decalysine) to an anti-tumor antibody leads to the formation of immunoconjugates with high specific activity and excellent in vivo behavior and is a valuable option for radioimmunotherapy and potentially antibody-drug conjugates (ADCs).Site-specific enzymatic reactions with microbial transglutaminase (mTGase) lead to a homogenous species of immunoconjugates with a defined ligand/antibody ratio. In the present study, we have investigated the influence of different numbers of 1,4,7,10-tetraazacyclododecane-N-N′-N′′-N′′′-tetraacetic acid (DOTA) chelats coupled to a decalysine backbone on the  One of the remaining challenges of immunoconjugation is product homogeneity with regard to site-specificity and stoichiometry of antibody modification et al. used an approach to attach a high number of chelating agents to the anticardiac myosin antibody R11D10 et al. designed a dendrimer incorporating up to 128 chelating agents (DOTA) and conjugated it to the anti-EGFR antibody hMAb425 et al. have designed a tris-tetraazamacrocycle comprising three chelating moieties in order to increase the specific activity of a RIC 64Cu. In an in vivo biodistribution study in tumor-bearing nude mice, both RICs showed similar tumor uptake. However, it is noteworthy that adverse liver uptake was threefold higher for the tris-tetraazamacrocycle RIC than for the mono-tetraazamacrocycle RIC. Furthermore, in the examples mentioned above, the exact sites of antibody modification were not known.To overcome this dilemma Slinkin We have recently reported a novel strategy using an enzyme of the transglutaminase family  for site-specific and stoichiometric uniform antibody modification with various lysine-comprising or lysine-mimicking substrates including transition metal chelators in vitro and in vivo characteristics of an antibody. We employed the anti-L1-CAM antibody chCE7agl (N297Q mutation), as it has shown excellent tumor-targeting capacity In this study, we make use of constructs comprising a decapeptide of lysine and different numbers of DOTA moieties attached to the lysine side chains. The oligolysine backbone was primarily chosen in order to permit a convergent synthesis with different numbers of DOTA moieties Streptomyces mobaraensis) was purchased from Zedira . All chemicals were purchased from Sigma-Aldrich or Fluka, Buchs, Switzerland, unless otherwise stated. All cell culture media and additives were purchased from BioConcept . 177Lu chloride was purchased from ITM (Isotopen Technologien München) AG . 2-(1H-benzo-triazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU) was purchased from Novabiochem . The peptides were purified and analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) on a Chromolith SemiPrep 100 × 10 mm column , and characterized by LC/MS using an Exactive (Thermo Scientific) mass spectrometer. The composition of the immunoconjugates was determined via LC/MS analysis on a Waters LCT Premier mass spectrometer. Samples were chromatographed on an Uptisphere BP1 column  heated to 40°C, using a linear gradient starting from 20% A, 75% B and 5% C  and changing to 50% A, 45% B and 5% C in 25 min at a flow rate of 0.5 mL/min. The eluent was ionized using an electrospray source (ESI+). Data were collected with MassLynx V4.1  and deconvolution was performed using MaxEnt1. Before LC/MS analysis, immunoconjugates (10 µg in 10 µL) were mixed with guanidine buffer  and reacted with 50 mM DTT at 70°C for 30 min to reduce disulfide bonds and thereby separate the heavy and light chains of the antibody.The chCE7agl antibody was expressed and purified following a previously published protocol In vivo studies were performed in female athymic immunodeficient nude mice  in compliance with Swiss laws on animal protection.SKOV3ip1 cells were maintained in Dulbecco’s modified Eagle medium  supplemented with 10% fetal calf serum (FCS), 2 mM of glutamine, 100 units/mL of penicillin, 100 µg/mL of streptomycin, and 0.25 µg/mL of fungizone. ε side chain of specific lysines, Fmoc-Lys(Mtt)-OH was used in the positions to be modified. The Nε Mtt protecting groups were removed using 1% trifluoroacetic acid (TFA) in dichloromethane (DCM), tris-tBu-DOTA  was coupled to obtain DOTA-conjugated peptides.Compounds were assembled on a 0.05 mmol Tenta Gel R Ram resin  with a degree of substitution of 0.19 mmol/g. Fmoc L-lysine derivatives  with the following protecting groups were employed: Fmoc-Lys(Mtt)-OH, Fmoc-Lys(Boc)-OH, and Boc-Lys(Boc)-OH. The peptides were obtained by solid phase peptide synthesis (SPPS) in a fully automated peptide synthesizer (ABI 433A Applied Biosystems) using fluorenylmethyloxycarbonyl (Fmoc) chemistry. HBTU was used as the coupling agent. In order to attach DOTA to the NDeglycosylation of mAb chCE7 was performed as previously described 177Lu solution (5 µL with 70 MBq) in 225 µL ammonium acetate buffer  at 37°C for 2.5 h. After incubation, 25 µL of a 50 mM EDTA solution was added to give a final concentration of 5 mM EDTA and stirred for 5 min to complex free lutetium. The radioimmunoconjugates were analyzed and purified using size exclusion chromatography (SEC) on a Superose-12 Fast Protein Liquid Chromatography (FPLC) column  and eluted with PBS buffer containing 1 M NaCl at a flow rate of 0.5 mL/min. Fractions of 500 µL were collected and the major peak fractions were pooled, after which specific activities were determined. For the animal study the radiolabeling procedure was scaled up and 500–1000 µg antibody derivatives were labeled with 500–700 MBq 177Lu in a volume of 900 µL ammonium acetate buffer.For small scale radiolabeling, 50 µg of the immunoconjugate was reacted with 177Lu-labeled derivatives were assayed with cell-binding tests using increasing numbers of SKOV3ip1 cells as described before 6) in a total volume of 550 µL PBS on a shaking platform at 37°C for 2 h. Non-specific binding was determined by parallel incubations in the presence of 10 µg of the unconjugated antibody. Human plasma stability studies were performed according to a previously published protocol The immunoreactivities of the Animal studies were conducted in compliance with the Swiss laws on animal protection. All experiments were approved by the Tierversuchskommission der Kantone BS-BL-AG, Switzerland, and permitted by the local government . All efforts were made to minimize suffering. Housing and animal husbandry was conducted according to local law on animal protection.nu mice, 5 weeks old, with a weight of 16–19 g  were injected in the peritoneum (i.p.) with 5 x 106 SKOV3ip1 human ovarian carcinoma cells. The cells were a gift from Prof. P. Altevogt  and the identity of the cell line was determined by STR profiling . After three weeks, groups of 15–16 animals were injected i.p. with 1.2 MBq of chCE7agl-[(DOTA)-decalysine]2, chCE7agl-[(DOTA)3-decalysine]2 or 0.9 MBq of chCE7agl-[(DOTA)5-decalysine]2 (400 µl PBS containing 12.4 µg protein). At 24, 48, 72, and 120 h post injection, groups of 3–4 mice for each derivative were sacrificed. Tumors and major organs were weighed and measured together with an aliquot of the injected solution in a gamma counter. Results are expressed as % injected dose per gram (%ID/g). Statistical analysis of data was performed using the Student’s t test .Female CD1-foxn1n-decalysine]2 and chCE7agl (5 µg/µL) were dialyzed against deionized water, after which 5 µL of each sample was loaded 4 cm from the cathode into an applicator mask slot. IEF protein marker mixture from Serva, pI 3.5–10.7  or Bio-Rad, pI 4.45–9.6  was used to determine the pI of the derivatives. Focusing was performed with a Multiphor II electrophoresis unit . After prefocusing at 1 W constant power for 10 min and focusing  the gel was fixed in 30% trichloroacetic acid for 20 min. After drying, proteins were stained with a standard Coomassie stain solution.Isoelectric focusing (IEF) was conducted on an IsoGel IEF agarose plate  from Lonza  according to manufacturers instruction. Samples of chCE7agl-2, respectively  and the immunoreactivity as determined by cell binding assays was between 39–81% n 39–81% . Fischer in vivo .et al. observed large differences in biodistribution among anti-CEA F(ab’)2 fragments conjugated to chelating polymers with various charges in vivo behavior. Therefore, we determined the isoelectric point of chCE7agl and the different chCE7agl-[(DOTA)n-decalysine]2 conjugates on an IsoGel IEF agarose plate (pH 3–10). The pI of the unmodified chCE7agl antibody was ∼ 7.8. Two species of the mAb are visible  can produce changes in the biodistribution and kinetics of immunoconjugates. In general, an increase in net positive charge generally results in increased tissue retention and increased blood clearance  visible , lane 2.sible 3-decalysine]2 respectively. From this point the accumulated activity declines to 34.3±9.0 (48 h) and 17.6±7.8% ID/g (120 h) for chCE7agl-[(DOTA)-decalysine]2. The same trend applied for chCE7agl-[(DOTA)3-decalysine]2. For the two conjugates, the blood pool radioactivity dropped to 1.0±0.1 chCE7agl-[(DOTA)-decalysine]2 and 5.1±0.15% ID/g chCE7agl-[(DOTA)3-decalysine]2 at 24 h. At the same time high liver accumulations of over 20% ID/g were observed. We speculate that the high tumor accumulation of these two derivatives at 24 h was due to the injection route of the conjugates. Otherwise, more of the conjugates may be trapped in the liver. The situation was different for chCE7agl-[(DOTA)5-decalysine]2: this conjugate showed a maximal tumor uptake at 48 h p.i. with 49.9±3.1% ID/g  with a slow drop in uptake at 72 h  and 120 h p.i. . This higher tumor accumulation was associated with a higher blood pool concentration (11.8±1.4–2.0±1.1% ID/g 24–122 h) of the conjugate over the whole time period . Furthermore, the high tumor accumulation persisted in the same way as has been described for 177Lu-DOTA chCE7agl 5-decalysine]2 was only 5.8% ID/g at 24 h (p<0.005). Thus, our results reflect to a certain extent the pI-dependence of biodistribution of radioimmunoconjugates reported in the literature.The results of the time-dependent biodistribution data are summarized in 177Lu-chCE7agl-[(DOTA)-decalysine]2 revealed high tumor-to-blood (between 44.0±6.5 at 24 h to 87.8±33.0 at 120 h) and liver-to-blood (between 23.2±3.6 at 24 h to 36.4±10.6 at 120 h) ratios at all time points (in 177Lu-chCE7agl-[(DOTA)3-decalysine]2 and 177Lu-chCE7agl-[(DOTA)5-decalysine]2 showed similar tumor-to blood ratios (between 6.1±2.1/3.5±0.7 at 24 h to 15.9±2.7/17.8±2.3 at 120 h), while, 177Lu-chCE7agl-[(DOTA)5-decalysine]2 displayed significantly better tumor-to-liver ratios (between 7.1±1.0 at 24 h to 5.6±2.9 at 120 h) than the conjugates with lower amounts of DOTA ligands. This is somewhat contrary to the trend observed by Knogler et al. where the chCE7agl was chemically functionalized with 7–15 DOTA chelators (randomly attached to lysine side chains of the mAb) nu mice), while Kukis et al. observed a similar trend with a murine anti-lymphoma IgG2a mAb Lym-1 chemically functionalized with different numbers (an average of 2 to 11 chelators/mAb) of the macrocyclic chelating agent TETA  labeled with 67Cu. In these studies, higher number of chelators resulted in lower tumor uptake, faster blood clearance and higher liver accumulation The differences in blood clearance and tumor uptake of the three tested radioimmunoconjugates resulted in significantly different tumor-to-organ ratios. et al.177Lu-DOTA-chCE7agl with an average number of 4.2 DOTA/mAb  and a range of 1 to 9 ligands/antibody molecule. These mAbs revealed an excellent biodistribution with high and persistent tumor accumulation and low activity in off-target organs. This antibody was highly potent in the treatment of disseminated ovarian tumor nodules in vivo. The chemically modified mAbs described by Fischer et al. in vivo bioditribution of the chCE7agl-[(DOTA)5-decalysine]2 conjugate was very similar to the described chemically modified mAbs. Importantly, the enzymatic conjugations lead to immunoconjugates with a uniform and well-defined substitution only on the heavy chain. For a possible subsequent approval by the authorities, this can be advantageous.Recently, Fischer 177LuCl3. The advantage of the high specific activity was not counteracted by the simultaneous decrease of the immunoreactivity. In mice bearing human SKOV3ip1 tumor xenografts expressing the antigen L1CAM all immunoconjugates showed high tumor uptake at 24 h after intraperitoneal administration. However, high liver uptake and fast clearance from the blood pool was observed for the immunoconjugates with higher isoelectric points (pI >9.5). Therefore, our results provide evidence that ligand/mAb ratios per se are not the sole factor which influence target-to-organ ratios of (radio)immunoconjugates but factors such as pI and hydrophilicity/lipophilicity  are equally important. While this might not be surprising, the possibility of site-specific and stoichiometric functionalization of targeted mAb via our enzymatic method permits a systematic investigation of the influence of the individual factors in order to optimize the pharmacological properties of the radioimmunoconjugates.We have demonstrated that it is possible to use microbial transglutaminase to site-specifically modify the chimeric IgG1-type tumor-targeting anti-L1CAM antibody chCE7agl with various numbers of DOTA metal chelating systems attached to a polylysine backbone. This method allowed the production of homogenous immunoconjugate with a precise and predictable number of metal chelating agents per antibody. The site of modification was identified to be Gln295 and Gln297, which are located in a flexible loop region of the Fc-part of the antibody chCE7. Increasing numbers of DOTA moieties coupled to the decalysine substrate was accompanied by an increasing specific activity of the immunoconjugates when labeled with Supporting Information S1(DOC)Click here for additional data file.


Initially epithelial (E) in nature, the HCT-8 cells become rounded (R) after seven days of culture on soft substrate. R cells show a number of metastatic hallmarks  The weak adhesion of R cells with substrate is also consistent with the observation that R cells show a smaller projected area, than E cells on the same stiffness substrate  to 197.9±83.6 μm2 (n = 56) on a 20 kPa region) (2 (n = 56), 182.3±32.2 μm2 (n = 63), and 190.9±82.5 μm2 (n = 57), respectively , indicating constitutive rounded shape independent of the substrate stiffness. On 5 kPa, 10 kPa and 15 kPa regions, the shape factors of single R cells are 0.8±0.1, 0.8±0.2, and 0.9±0.2, respectively (2 on the 1 kPa region to 578.1±429.8 μm2 on the 20 kPa region (2 (n = 51), 276.0±104.8 μm2 (n = 62), and 442.7±367.7 μm2 (n = 55) on 5 kPa, 10 kPa and 15 kPa regions, respectively (2 (n = 46) on the 1 kPa region to 1421.7±845.7 μm2 (n = 31) on the 20 kPa region (2 (n = 62), 749.9±355.5 μm2 (n = 63), and 1218.6±773.5 μm2 (n = 59) on 5 kPa, 10 kPa and 15 kPa regions, respectively. Concurrently with increasing substrate stiffness, single MKF cells spread to a more irregular morphology, with their shape factor decreasing from 0.9±0.1 on the 1 kPa to 0.5±0.2 on the 20 kPa regions, respectively  . Across ectively . Also, tectively . After pectively . Their sectively . Across ectively . The spra region . As the ectively . On the 2, 3662.1±1105.3 μm2, 4249.5±919.5 μm2, 9736.5±4032.7 μm2 and 11748.7±2144.9 μm2, respectively , respectively, as well as those of single R cells displaying no cell-cell contacts  for prolonged culture times (months); again suggesting weak cell-cell adhesion among R cells. Taken together, these results suggest that during or after E-to-R transition, R cells acquire cell autonomy that is characterized by markedly reduced cell-cell and cell-substrate adhesive contacts.Furthermore, it is interesting to note that as HCT-8 E cells or MKF cells undergo homotypic cell-cell adhesion, their individual cell areas and cell shape factor become remarkably less substrate stiffness-dependent , the cell stiffness of HCT-8 E cells cultured on stiffness-gradient substrates is determined by indentation using silicon-nitride cantilevers with a spring constant of 148.14 pN/nm (with consistent cell indentation speed 0.1 μm/sec). Hertz theory see  was usedth day of culture on PA gel) and R cells were measured using a micro-fabricated bio-MEMS force sensor (2). The probe and the sensor are not functionalized. The sensor is manipulated with an x-y-z piezo stage. The flat probe is brought in contact with E-cell islands' lateral convex surface at the boundary. Each E-cell island consists of 100 s of cells with multiple cells stacking at the island periphery . Cell force is calculated from the change of gap and force-deformation calibration of the sensor spring. At a critical value of force, Fc, the cone suddenly detaches from probe (c is the maximum force on the force-displacement curves. We consider Fc as a measure of cell-probe adhesion. We measured Fc for 12 such cell clusters and obtained Fc  = 256.3±33.7 nN (n = 12). Similar experiments with R cells show negligible cell-probe adhesion with Fc  = 1.14±0.13 nN  with an objective 20× and a high-speed SPOT camera was used to record cell phenotypes and deformation behavior Human colon adenocarcinoma HCT-8 cells (ATCC No.: CCL-244) were cultured in RPMI 1640 (Gibco No.: 23400–062) supplemented with 2 grams of sodium bicarbonate per liter, giving final concentrations of 10% horse serum (Gibco No.: 26050–088), 1× antibiotic-antimycotic (Gibco No.: 15240–062) and 1 mM of sodium pyruvate (Gibco No.: 11360) 0 + δ, where D0 is the initial deflection of the sensor beam and δ is the additional deformation due to applied force F. Both F and D are in SI units, Newton and meter, respectively. Before measuring cell adhesion, the sensor was sterilized using Alcohol and DI water multiple times. During the experiment, the T-shaped sensor probe was allowed to contact the cell lateral membrane for 2 minutes and was then moved away horizontally. Due to cell adhesion, the sensor beams deform during retraction by δ, giving the force according to Eqn. (1). Note that the probe is non-functionalized by any extracellular matrix proteins and only has a coating of SiO2 on the surface. Therefore, non-specific adhesive interactions were formed between the cell and the SiO2-coated probe. The entire pulling process lasts 10–30 seconds.We characterized the non-specific adhesion strength of the HCT-8 cells using a novel Bio-MEMS force sensor −1 (Veeco) was used to characterize the stiffness of the HCT-8 cell monolayer. A conical tip approximation . α  = 35° is the half open-angle of cantilever tip. During experiments, the curves of force versus sample indentation were obtained and used to determine Elastic modulus distribution.Atomic force microscopy (Asylum) with silicon-nitride cantilever having a spring constant k = 148.14 pN×nmximation  for the 5 cells) were placed in flat bottom vials  and rotated in a gyratory water bath shaker  at 12 rpm at 37°C for 5, 10, 20, 40, 60, 80, 100 and 120 minutes, respectively. At the end of each time period, cells were diluted with 8 mL 0.9% saline and placed on ice to stop further cell aggregation. The number of single cells present at each time point was measured in the Coulter counter as described in The cells were harvested and individualized by trypsin/EDTA treatment followed by restoration in complete culture medium containing serum to neutralize residual trypsin. Since fibronectin was used for cell adhesion on PA gel substrates and there was no tissue present, trypsin/EDTA (not collagenase) were used to remove cells from culture substrates into single cell state. The cell suspensions were placed in 17×100 mm capped polypropylene tubes  and were rotated end over end at 7–8 revolutions per minute in a conventional Labquake shaker (Barnstead/Thermolyne Model No.: 41510) for 1 hour at 37°C to allow recovery of any surface cell adhesion molecules (CAMs) or other proteins. The recovery of CAMs following trypsinization was guaranteed by identifying the increase in cell aggregate number as incubation duration prolongs, as shown in Cultures were fixed with 4% paraformaldehyde at 37°C for 30 minutes followed by the 15-minute permeabilization in 0.1% Triton (×100) solution. Rhodamine phalloidin  was used as fluorescent conjugate to stain specifically F-actin filaments. Image-iT™ FX Signal enhancer  was used to block all non-specific binding and enhance the imaging quality. The actin structures were imaged using laser-scanning confocal microscopy  with appropriate fluorescent filters, and data were analyzed using Andor IQ software . Multiple images were combined using Amira  software .Image stacks processing was performed using ImageJ (NIH) and Photoshop CS3 (Adobe Inc.) software. Statistical data processing and analysis were performed using Office Excel (Microsoft), Origin Pro (OriginLab Corp.) and Matlab (the MathWorks) programs.


DMBT1), is an important component in innate immune defense. The first scavenger receptor cysteine rich domain (SRCR1) of gp340 has been shown to inhibit HIV-1 infection through binding to the N-terminal flank of the V3 loop of HIV-1 gp120.gp340, a member of scavenger receptor cysteine rich family encoded by Deleted in Malignant Brain Tumors 1 (Through homology modeling and docking analysis of SRCR1 to a gp120-CD4-X5 antibody complex, we identified three loop regions containing polar or acidic residues that directly interacted with gp120. To confirm the docking prediction, a series of over-lapping peptides covering the SRCR1 sequence were synthesized and analyzed by gp120-peptide binding assay. Five peptides coincide with three loop regions showed the relative high binding index. An alanine substitution scan revealed that Asp34, Asp35, Asn96 and Glu101 in two peptides with the highest binding index are the critical residues in SRCR1 interaction with gp120.We pinpointed the vital gp120-binding regions in SRCR1 and narrowed down the amino acids which play critical roles in contacting with gp120. DMBT1), a gene originally identified in brain tumors [streptococcus gordonii and streptococcus mutans, playing important roles in dental caries [E. coli, Lactobacillus casei, Helicobacter pylori, S. aureus, S. pneumoniae, and Haemophilus. Influenzae[Salmonella enterica in intestinal epithelial cells [Salivary agglutinin (SAG) and lung scavenger receptor glycoprotein gp340) are multi-splicing products of Deleted in Malignant Brain Tumors 1  was built based on the known structure of M2bp SRCR domain (PDB ID: 1BY2) using software MODELLER9v4 [The structure of SRCR1  was performed using ZDOCK3.0.1, a rigid-body protein-protein docking software [A complete set of 15-mer peptides, overlapping by 10 amino acids, was derived from SRCR1. These peptides were numbered according to the SRCR1 sequence, P1 standing for 1–15 residues, P2 for 5–20 and so on. To study the role of the vital amino acids for gp120 binding, a set of peptides was introduced alanine substitutions in corresponding residues. All peptides were synthesized and purified by high-performance liquid chromatography (HPLC) to >95% purity . The biotin tag was labeled at the N-terminus of the peptide.Recombinant gp120s were purchased from Immuno Diagnostics, Inc  or from the NIH AIDS Reagent Program . 96-well polyvinylchloride plates  were coated with 50 μl of 8ug/ml gp120 diluted in 50 mM bicarbonate buffer, pH 9.6, and incubated at 4°C for 16 hours. Unbound protein was removed by repeated washing with 20 mM Tris–HCl, pH7.4, containing 0.05% Tween-20 (washing buffer). Nonspecific sites were blocked with 200 μl 2% nonfat milk dissolved in washing buffer, at 37°C for 60 min. 50 μl of 30 μg/ml biotinylated peptides diluted in washing buffer were added and allowed to bind gp120 at 37°C for 60 min. Unbound peptides were removed by repeated washing. For competition studies, biotinylated P19 (40 μg/ml) was mixed with increasing concentrations of non-biotinylated P19, added to a gp120-coated plate (8ug/ml) and incubated at 37°C for 60 min. Unbound peptides were removed by repeated washing. Since P1 showed no binding to gp120 in the previous binding analyses, it was used as a control at the maximum concentration (640 μg/ml). The bound peptides were detected with AP-conjugated streptavidin  and PNPP liquid substrate , and measured at 405 nm.DMBT1 SRCR1 and M2bp SRCR shared high homology (50%) in primary sequence. The SRCR1 3D structure was organized around a curved four-stranded β-sheet cradling two α-helices , either individually or in combination. All the mutant peptides showed reduced binding with gp120  was shown to bind streptococcal and mediated bacterial aggregation . InteresWe performed docking analysis on gp120-SRCR1 interaction and made a number of predictions on the SRCR1 amino acid residues that may interact with gp120. Binding analysis using a series of synthetic peptides derived from SRCR1 demonstrated that three high binding loop regions of aa33-36, 73–76 and 94–97 on SRCR1 bound gp120. The relative binding indexes were consistent with the predictions of the docking model, suggesting that our model is appropriate for further analysis of SRCR1-gp120 interaction and that, by combining biochemical approaches, the model may facilitate the designing of new HIV-1 target drugs.The authors declare that they have no competing interests.YC and JHL carried out overall research work; YC and XLW carried out the peptide binding assay; YC, JHL, ZCH and ZWW conceived and designed the experiments; YC and ZWW drafted the manuscript. All authors have read and approved the final manuscript.


Thlaspi caerulescens is a natural selected heavy metal hyperaccumulator that can not only tolerate but also accumulate extremely high levels of heavy metals in the shoots. Thus, to identify the transportors involved in metal long-distance transportation is very important for understanding the mechanism of heavy metal accumulation in this hyperaccumulator.TcOPT3 of OPT family from T. caerulescens. TcOPT3 was pronouncedly expressed in aerial parts, including stem and leaf. Moreover, in situ hybridization analyses showed that TcOPT3 expressed in the plant vascular systems, especially in the pericycle cells that may be involved in the long-distance transportation. The expression of TcOPT3 was highly induced by iron (Fe) and zinc (Zn) deficiency, especially in the stem and leaf. Sub-cellular localization showed that TcOPT3 was a plasma membrane-localized protein. Furthermore, heterogonous expression of TcOPT3 by mutant yeast (Saccharomyces cerevisiae) complementation experiments demonstrated that TcOPT3 could transport Fe2+ and Zn2+. Moreover, expression of TcOPT3 in yeast increased metal  accumulation and resulted in an increased sensitivity to cadmium (Cd) and copper (Cu).We cloned and characterized a novel gene TcOPT3 might encode an Fe/Zn/Cd/Cu influx transporter with broad-substrate. This is the first report showing that TcOPT3 may be involved in metal long-distance transportation and contribute to the heavy metal hyperaccumulation.Our data demonstrated that  Thlaspi caerulescens, a Cd/Zn/Ni hyperaccumulator, has been used as a model plant to study the physiological and molecular mechanisms of heavy metal hyperaccumulation Metal hyperaccumulators can not only tolerate high concentration of heavy metals in the soils but also take them up actively and accumulate and distribute them to appropriate tissues at extreme high levels, thus make them very attractive for the remediation of heavy metal polluted soils The efficient metal loading and unloading ability in the vascular systems was regarded as the key step of hyperaccumulating process for hyperaccumulators Candida albicansSchizosaccharomyces pombeSaccharomyces cerevisiaeplanta. For example, YSLs mediate long-distance transport of specific Fe species ZmYS1 cloned from Zea mays is the founding member of YSL family, which functions in root Fe-phytosiderophore (Fe-PS) uptake from the soil HvYS1 encoding a YSL transporter is a specific transporter for iron (III)-PS in barley roots YSL genes cloned from T. caerulescens express at higher levels as compared with their Arabidopsis thaliana homologs with distinct patterns. TcYSL3 was further confirmed as a Fe/Ni-NA influx transporter T. caerulescens.The first OPT family is identified in the pathogenic yeast Arabidopsis, nine putative OPT orthologues (AtOPT1 to AtOPT9) have been identified AtOPTs have been proved to play a role in metal homeostasis and movements. Transcript of AtOPT2 is induced in root by iron and zinc deficiency, and that of AtOPT3 by iron, copper and manganese deficiency In Thlaspi caerulescens, named TcOPT3. We characterized its expression in different tissues, and conducted heterologous expression in yeast to investigate the role of TcOPT3 in metal uptake. We demonstrated that TcOPT3 was involved in Fe/Zn/Cd/Cu transportation and it should be a very important component in the metal long-distance transport system, and contribute to heavy metal hyperaccumulation in the shoots.In the present study, we isolated a member of the OPT family from a metal hyperaccumulator, AtOPT3, BjGT1 and ZmGT mRNA sequence. We isolated the full-length cDNA from Thlaspi caerulescens by RACE PCR, and designated it as TcOPT3 (Genebank accession no. HQ69984). The coding region of TcOPT3 was 2211 bp, and a corresponding 737 amino-acid sequence was predicted.Degenerated primers were designed from the most conserved regions according to the TcOPT3 exhibited 79% identity with its homologous AtOPT3, and the deduced protein TcOPT3 showed 95% identity with AtOPT3  was used as a control. The transcript level of TcOPT3 mRNA was 2-fold more in the leaf and stem tissues than that in the root  under the control of the cauliflower mosaic virus 35 S promoter and pm-rk (Plasma membrane marker) were co-expressed in the onion epidermis cells. The confocal microscopy observation showed that the green fluorescence and red pm-rk were both confined to the plasma membrane  and can TcOPT3 cDNA could restore the growth of yeast double mutants fet3fet4 (strain DEY1453) and zrt1zrt2 (strain ZHY3), which can not grow on the Fe- and Zn-limited medium respectively fet3fet4 and zrt1zrt2 (2+ or 50 µM Zn2+), suggesting that TcOPT3 can transport both Fe2+ and Zn2+.To study the function of TcOPT3 in Fe and Zn transport, we tested whether expression of a zrt1zrt2  when bot3, 50 µM ZnSO4, 50 µM CuSO4 or 20 µM CdSO4, respectively. Compared with those empty vector-expressing strains, the TcOPT3-expressing trains grew worse  cultured in the solutions containing 50 µM FeClew worse , meanwhiew worse .AtOPT3 in yeast can transport Cu, Mn and Fe S. cerevisiae opt2 mutants and contributed to Cd accumulation Thlaspi caerulescens, a Cd/Zn/Ni hyperaccumulator, ecotype of Ganges, from southern France, can accumulate 10 000 mg Cd kg−1 at shoot dry weight base TcOPT3 from T. caerulescens.The Oligopeptide transporters (OPTs) were initially characterized as small-peptide transporters. However, this has been challenged by their new functions in metal trafficking recently. The first report is from Wintz et al who found that heterologous expression of A. thalianaSaccharomyces cerevisiae rescued the growth of Fe-depleted mutant fet3fet4 , and found that altough the growth was negatively affected by Ni addtion , Nramp , MTP , and HMA  families in y metals , 9.TcOPT3 was preferentially expressed in the aerial parts (stem and leaf) than roots both under normal condition or Fe/Zn deficient conditions  Increased Zn2+ influx across the root-cell PM; (2) Reduced Zn sequestration in the root-cell vacuole; (3) Increased Zn transport into the xylem and via the xylem to the shoot; (4) Increased Zn2+ influx into leaf mesophyll cells; and (5) Zn and Cd stored primarily in leaf epidermal cells. In situ hybridization results showed that, TcOPT3 was highly expression in vascular cells both in roots and shoots  for two weeks at 14-h/25°C d and a 10-h/18°C night regime, a light intensity of 100 µmol photons m−2 s−1. Then the seedlings were transferred to modified Hoagland nutrient solution (2 mM Ca(NO3)2, 0.1 mM KH2PO4, 0.5 mM MgSO4, 0.1 mM KCl, 0.7 mM K2SO4, 0.1 mM Fe-EDTA, 10 µM H3BO3, 0.5 µM MnSO4, 10 µM ZnSO4, 0.2 µM CuSO4 and 0.01 µM (NH4)6Mo7O2, pH 5.5).Seeds of T. caerulescens roots or leaves by the Trizol reagent  and purified by DNase I (RNase Free) Kit . First strand cDNA of T. caerulescens synthesis was performed using M-MLV reverse transcriptase  and PCR-amplified with degenerated primers designed from conserved AtOPT3 sequence. One fragment was cloned into the pMD19-T Vector , sequenced and identified as AtOPT3 orthologs by BLAST using the NCBI database. RACE PCR (5′ and 3′) were performed in order to obtain full-length sequences from uncomplete cDNA fragments . The putative amino acid sequences were aligned with the program Clustal X Version 1.8 http://www.megasoftware.net) The predicted amino-acid sequence from TcOPT3 cDNA and other selected OPT protein sequences were aligned using the CLUSTAL W program, version 1.8 . The putative trans-membrane domains were predicted by the TMHMM  as described above. RT-PCR with oligo d(T)-anchor primer were performed with PrimeScript RT  reagent kit . For real-time RT-PCR, 2 µl of the diluted (1∶5) cDNA products were used as templates with 5 µl SYBR Pre mix , 0.5 µl primers (10 µM each) and 2.5 µl water. Calculation of the ΔCp values was performed as described Total RNA was isolated from roots, stems and leaves of one-month-old TcOPT3 was amplified by PCR  containing 4% paraformaldehyde overnight at 4°C, then dehydrated in ethanol series, and embedded in paraffin. A gene-specific fragment containing the 416 bp fragment cross 3′UTR of TcOPT3 cDNA fragment containing SmaI and SacI restriction sites  E.C. Hansen knock-out strain fet3fet4  fet3fet4 and zrt1zrt2 transformants were grown overnight in liquid SD-ura and supplemented with 10 µM FeCl3 or 50 µM ZnSO4, respectively. Yeast cells were recovered by centrifugation, resuspended in SD-ura (without added Fe or Zn) at an OD600 of 1, 0.1, 0.01 and 0.001. Then 10 µL of each culture was spotted on SD-ura plates; plates were incubated at 30°C for 2 d and then photographed.For complement test, 600 of 1. After washed by CaCl2 and centrifugation, resuspended in SD-ura with 50 µM FeCl3, 50 µM ZnSO4, 20 µM CdCl2, or 50 µM CuSO4 were grown for another 0, 6, 12, 24 hours. After measured the OD values and washed by CaCl2 to remove adsorbed transit metals in apoplast cells, aliquots of yeast cells were taken for measurement of metal accumulation.Wild-type yeast DY1455 was transformed with TcOPT3-pFL61, or with pFL61, which served as a negative control. DY1455 transformants were grown on liquid SD-ura medium overnight at an OD2 for 5 min twice to remove adsorbed transit metals from the yeast cell walls. Then cell sediments were dried at 70°C for 2 days, and digested in HNO3. Metal concentration in the digested solution was determined by inductively coupled plasma-atomic emission spectrometry .For metal accumulation, yeast cells were concentrated at 5,000 rpm for 5 min and washed by 0.5 mM CaClt-test.Data were statistically analyzed using analysis of variance (ANOVA) in Origin 8, and tested for significant (P≤0.05) treatment differences using Student’s Figure S1Growth of the wild-type (DY1455) and TcOPT3-transformed yeast cells under different metal supplies. Yeast cells were grown to an OD600 of 1.0, and then supplemented with 100 µM PbNO3 or 100 µM NiSO4 respectively. Data are the means ± SE per experiment (n = 3), P<0.05 by Student’s t-test.(EPS)Click here for additional data file.Figure S2Heavy metal accumulation of wild-type (DY1455) and TcOPT3-transformed yeast cells. Pb and Ni accumulation in yeast transformants. Metal accumulation was conducted in liquid SD media supplemented with 100 µM PbNO3 or 100 µM NiSO4 respectively. Data are the means ± SE per experiment (n = 3), P<0.05 by Student’s t-test.(EPS)Click here for additional data file.