Bos grunniens) domesticated under alpine hypoxia environments, is well adapted to survive and grow against severe hypoxia and cold temperatures for extended periods. In this study, a full coding sequence of the IGF2 gene of Tianzhu white yak was amplified by reverse transcription PCR and rapid-amplification of cDNA ends (RACE) for the first time. The cDNA sequence revealed an open reading frame of 450 nucleotides, encoding a protein with 179 amino acids. Its expression in different tissues was also studied by Real time PCR. Phylogenetic tree analysis indicated that yak IGF2 was similar to Bos taurus, and 3D structure showed high similarity with the human IGF2. The putative full CDS of yak IGF2 was amplified by PCR in five tissues, and cDNA sequence analysis showed high homology to bovine IGF2. Moreover the super secondary structure prediction showed a similar 3D structure with human IGF2. Its conservation in sequence and structure has facilitated research on IGF2 and its physiological function in yak.The IGF family is essential for normal embryonic and postnatal development and plays important roles in the immune system, myogenesis, bone metabolism and other physiological functions, which makes the study of its structure and biological characteristics important. Tianzhu white yak ( RACE teectively . The seqectively . The BLA2.1.2.N-terminal of the sequence was considered M (Met) and the chemical composition of the predicted protein is illustrated in The molecular analysis of the 179 amino acid sequence of yak IGF2 using the program PROTEAN [2.1.3.The amino acid sequence of yak IGF2 was aligned with that of eight mammalian species by ClustalW ,19 Figu. The disBos primigenius and Bos bubalis, 99% with Bos taurus, 96% with Ovis aries and Capra hircus, 92% with Cervus elaphus, 84% with Sus scrofa and Homo sapiens, respectively  [The 3D structure of yak IGF2 was predicted using homology structure modeling on a Swiss-model server . The 3D  sapiens . The 3D  sapiens . Yak IGF sapiens  (regionserminus) . The B a2.1.5.http://pymol.org) [vs. human IGF2 and human IGF1 were 95% and 80%, respectively. The overall RMSD (root mean square deviation) between yak IGF2 and human IGF2 structure was 0.09  when compared with human IGF2. Similarly, the superimposed yak IGF2 structure with human IGF1 had a Q-score of 0.45. The p-score was used to evaluate the significance of structure similarity. Superimposition of yak IGF2 with human IGF2 and IGF1 had p-scores of 10.8 and 1.8, respectively  . The ovemol.org)   and the expression of GAPDH as a housekeeping gene (endogenous control). The relative expression of yak IGF2 in the testis, muscle, spleen, kidney, ovary and brain were compared with that of liver. The expression level in testis was 1.35 fold (135%) higher than in liver, followed by muscle (53%), spleen (12%), kidney (1%), ovary (1%) and brain (0.6%) relative to liver (100%), respectively , which also indicate a higher similarity of yak to human . The p-to human .IGF2, followed by liver, muscle, spleen, kidney, ovary and brain. This result showed that the expression of yak IGF2 may be related to the reproductive axis and regulate the signaling pathway in reproduction to control IGF2 gene expression and to promote bone and muscle growth, and then increase the meat production. Although this paper provides detailed information about yak IGF2 sequences and its structure, more detailed studies are required to explore the role of yak IGF2 in gene regulation, function, response to muscle growth, and action at the molecular level. The yeast two-hybrid system will be used for further studies of yak IGF2 function. RNAi and conditional gene knockout technology will also be applied to study the muscle growth mechanism using yak as an animal model.Real time PCR data suggested that yak testis has the highest expression of 3.3.1.Bos Taurus, Sus scrofa and Ovis aries available from National Center for Biotechnology Information (NCBI) for initial screening. Bioinformatics software and website were used for the analysis of the biological sequence information. The related software and website were illuminated in IGF2 gene expression.The PCR primers and RACE primers were designed based on the conserved sequence homology from 3.2.Fresh liver, kidney, spleen, lung and testis tissues from adult male yaks were obtained immediately after slaughtering from the Slaughterhouse of Tianzhu County . Yak tissues were immediately stored at −80 °C.3.3.Total RNAs were extracted from yak liver tissues by using RNAiso Plus  following manufacturer’s instructions and was further used for cDNA synthesis. RNA was quantified by NanoDrop-8000  and RNA integrity was assessed on denaturing formaldehyde agarose gel (1%) electrophoresis . Two micrograms of total RNAs were subjected to reverse transcription to single-stranded cDNA using BioTeke Thermo RT Kit , the reverse transcription PCR reaction was 48 °C for 50 min, followed by 70 °C for 10 min. The total 20 μL cDNA synthesis reaction contained 2 μL total RNA, 1 μL Oligo dT or Random Primer (50 μM), dNTP Mixture (10 μM), Thermo M-MLV (200 U/μL), RNase Inhibitor (40 U/μL), 4 μL 5× first-strand buffer and 10 μL of ultrapure millipore water .3.4.IGF2 primers were designed based on conserved sequence homology from Bos taurus and Sus scrofa available from NCBI for initial screening. The special primers used for initial screening and obtaining partial IGF2 cDNA of Yak were Forward 5′-CTGGAAGCAGTCCACCCAG-3′ and Reverse 5′-TTCTGGTGTTGCTAAAAAGCC-3′. The PCR reaction mixture (30 μL volume) contained 2 μL of template cDNA, 1.5 μL (10 μM) of each primers set, 15 μL 2× Power Taq PCR MasterMix,  and 10 μL of ultrapure millipore water and was carried out by Bio-Rad PCR . The PCR amplification conditions were as follows; initial denaturation at 95 °C for 5 min followed by 35 cycles at 95 °C for 1 min, 52 °C for 1 min and 72 °C for 1 min. Final extension step was carriedout at 72 °C for 10 min. The products of PCR reactions were electrophoresed on an agarose gel (1.5%) using DNA Ladder DL2000 . The 544 bp fragment from the PCR reaction was eluted and purified using BioTeke Gel Extraction kit . Purified fragments were quantified and validated on agarose electrophoresis before sequencing. PCR fragments were sequenced by BGitech Company, Beijing, China.The 3.5.IGF2 using RACE kits . Yak total RNA was annealed with 5′- and 3′-end adaptor primers, and reversely transcribed respectively to respective 5′- and 3′-cDNA. The resulting single stranded 5′- and 3′-cDNA were then used as templates in PCR. For 5′ RACE, the 5′-end adaptor primer (forward primer 5′-CGCAGACAGGACGGTACAGGGATTTCA-3′) and IGF2 5R (reverse primer 5′-CTCACTTCTAATCGCTGGATGCCTTGGA-3′) were used. For 3′ RACE, IGF2 3F primer (forward primer 5′-TCCAAGGCATCCAGCGATTAGAAGTGAG-3′) and the 3′-end adaptor primer (reverse primer 5′-TCTGAAATCCCTGTACCGTCCTGTCTGC-3′) were used. The cycling program was set for five cycles of 94 °C, 5 min; 5 cycles of 94 °C 15 s, 70 °C 15 s, 72 °C 3 min; 5 cycles of 94 °C 15 s, 68 °C 15 s, 72 °C 3 min; 5 cycles of 94 °C 15 s, 65 °C 15 s, 72 °C 3 min; 25 cycles of 94 °C 15 s, 60 °C 15 s, 72 °C 3 min; 1 cycle of 72 °C, 5 min. The RACE PCR products were purified and cloned into pMD-18 vector  to confirm the sequences of the 5′- and 3′-end, respectively. The full-length cDNA PCR product was cut with the restriction enzymes, purified, and ligated with T4 DNA ligase into a pMD-18 vector. Competent E. coli DH5α cells were transformed with the plasmids and selected by means of antibiotic resistance. The sequence of the inserted full-length cDNA was confirmed with DNA sequencing.Rapid-amplification of cDNA ends (RACE-PCR) was used to identify and isolate the 5′- and 3′-end of 3.6.IGF2 gene was analyzed for nucleotide, protein translation, sequence alignment and comparisons with other mammalian species [http://www.cbs.dtu.dk/services/SignalP/) [et al. [The sequence of yak  species . The seq species  and the  species . DNA and species . Signal ignalP/) . BioinfoignalP/) . The met [et al.  was used [et al. .3.7.IGF2 mRNA sequence with the free energy minimization algorithm. RNA tertiary structures was characterized by secondary structural elements based on hydrogen bonds within the molecule that form several recognizable “domains” of secondary structure like stems, hairpin loops, bulges and internal loops. A poly(A) tail and poly(A) signal for the yak IGF2 mRNA was predicted based on the free minimization algorithm of CLCbio  [Bioinformatics Toolkit Quick 2D program  was usedGermany) . The pre3.8.The amino acid sequence of yak cIGF2 was used as template to identify homologous mammalian sequences in PSI-BLAST. Nine homologous sequences from different mammals were used for multiple sequence alignment by ClustalW on MEGA5.1 software . The out3.9.The 3D structure of yak IGF2 was predicted using a Swiss-model server base on homology structure model . The simThe antigenic properties of yak IGF2 were predicted according to the methods of Kolaskar and Tongaonkar . The ant3.10.cIGF2 was detected by quantitative Real time PCR. 200 ng total RNA was used for the synthesis of single-stranded DNA (cDNA) with BioTeke Thermo RT Kit . PCR was performed using 2 μL of cDNA in a 25 μL reaction volume with ABI7300  Real time System. SYBR Premix Ex Taq™ II  and specific primers were used in each reaction. The sense and antisense primers were designed with the Primer 5.0 based on the IGF2 cDNA sequences. Primer sequences used were as follows: sense: 5′-ATACCCCGTGGGCAAGTTC-3′, antisense: 5′-ACGACACTTTGGCTTTGGCTCACTTCTAATC-3′, product size 138 bp), GAPDH . The expression of IGF2 in the liver was taken as a reference sample  and the expression of GAPDH as a housekeeping gene (endogenous control). ABI7300 PCR system was used, and the 2-step standard procedure for amplifying the samples was applied. Stage 1: for denaturation, Reps 1, 95 °C for 30 s. Stage 2: PCR reaction, Reps 40, 95 °C for 5 s and 60 °C for 31 s. The final step is the dissociation stage. All PCR reactions were performed in triplicate. The results were calculated applying the 2−ΔΔCt method [The gene expressions level of t method .4.The putative full sequence of yak IGF2 was successfully cloned for the first time, and its sequence and predicted product structure was found to be very similar to human IGF2. As a specific species and a model animal living in the highland with low oxygen content, research of IGF will greatly facilitate resistance studies, and benefit yak breeding in similar areas. There were several binding sites in yak IGF2 that can accept different receptors, which play crucial roles in regulating skeletal muscle growth and differentiation, as well as maintaining homeostasis. The present work represents initial research into yak IGF2; future studies will be aimed at the clarifation of the functions of IGF2 and the mechanisms of bone and muscle growth.


It is known that Korean red ginseng (KRG) slows depletion of CD4 T cells in human immunodeficiency virus type 1-infected patients [To my knowledge, there is only one report on gross deletions in the  (gΔpol)  althoughpatients . We repopatients  in 10 HIpol gene  using 68 PBMC samples from these 10 patients. The pol gene was amplified using two primer sets, outer primers OBP1/ OBP2 (nt. 3733 to 3752/ 5297 to 5278) and inner primers OBP3/OBP4 (nt. 3837 to 3860/ 5049 to 5068). Nucleotide positions were based on NL4-3.This study consisted of 10 patients  infected with HIV-1 subtype B. All patients had consistently taken pure KRG powder for 42 ± 4 months  for up to 3 years, with at least five blood samples available. The average monthly dose was 99 ± 25 g . The daily dose was 5.4 g for men and 2.7 g for women. They were not treated with antiretroviral therapy. We amplified the pol gene in the 10 patients. Among the 277 amplicons, 25 were grossly deleted. There was no amplicon with a stop codon. Size of deletion was 660 ± 277 bp (49 to 1008). Seven patients exhibited gΔpol, ranging from 4.8% to 19.2%, with significant increases after KRG intake relative to baseline (12.3% vs. 1.7%) (p<0.05). Interestingly, 3 of 4 patients who took KRG <100 grams per month did not reveal any gΔpol, whereas all 6 patients who took KRG > 100 grams per month revealed gΔpol (>10% of amplicons). The proportion of gΔpol increased about 2-fold over the first 6 months on KRG and was statistically significantly higher after 6 months. Median time to the first detection of gΔpol was 22 months.We obtained 277 amplicons of the These findings show that occurrence of gΔpol is associated with KRG intake.


Differential thermal analysis (DTA) and infrared spectroscopy (IR) were used to study the chemical changes during the xerogel annealing. After the sol evaporation at 100 °C, the formed gel was annealed from 300 to 900 °C for 30 min under a rich O2 atmosphere, and the yielded product was analyzed by X-ray diffraction (XRD) to characterize the microstructural behavior and confirm the crystalline structure. The results showed that Lu2O3 nanopowders start to crystallize at 400 °C and that the crystallite size increases along with the annealing temperature. A transmission electron microscopy (TEM) study of samples annealed at 700 and 900 °C was carried out in order to analyze the microstructure, as well as the size, of crystallites. Finally, in regard to scintillating properties, Eu3+ dopant (5 mol%), Tb3+ codoped Lu2O3 exhibited a typical red emission at 611 nm (D°→7F2), furthermore, the effect of Tb3+ molar content  on the Eu3+ radioluminiscence was analyzed and it was found that the higher emission intensity corresponds to the lower Tb3+ content.Nanocrystalline Eu Further(Z = 71) , especiathe ions . On the length ~6 nm that etectors , and, moroscopic ,14. Anottons/keV , compare2O3:Eu3+ . Finallymoisture ,18. Furtt of GOS  and thatt of GOS . On the t of GOS ,22. Becat of GOS , many sot of GOS –26, solvt of GOS –31, moltt of GOS , and thet of GOS –37. Oxalt of GOS . Therefot of GOS , it is iopowders –43. Fina2O3:Eu3+ . In Y2O3u3+ ions ,46; furtu3+ ions , it has 3+,Tb3+ co-doped Lu2O3 scintillating nanopowders by a simple sol-gel process, starting with nitrates as precursors and without the use of urea or oxalic acid, and instead, diethyleneglycol (DEG) C4H10O3 as polymerization agent. It was studied the chemical changes during the sol gel process of the nanopowders by means of a Thermogravimetric (TGA), differential thermal analyses (DTA) and infrared spectroscopy (IR). The structural evolution during the annealing process was analyzed by X-ray diffraction (XRD), the morphology by means of TEM observations, and their scintillating properties under X-ray (30 keV) radiation as a function of the heat treatment temperature and the Tb3+ content.The aim of the current work is to synthesize for first time Eu2O3 xerogel 5 mol % Eu3+, 0.01 mol % Tb3+. As observed, the TGA curve shows three stages of weight loss. The first stage (I), from 100–175 °C, shows a weight loss of 9% and two endothermic events, at 120 and 170 °C. The first event can be attributed to the release of water of hydration and OH, and the second one to the decomposition of the organic matter, this event suggest the formation of lutetium hydrated species or even carbonate species to form precipitates with Lu3+ ions [u3+ ions . The sec2O3: Eu3+ 5 mol %, Tb3+ 0.01 mol% are depicted in −1 on the dried precursor sol, thermally treated at different temperatures, in order to determine the evolution of the decomposed products of the xerogel powders calcined up to the crystallization process. For the xerogel at 100 °C, bands observed at 3400 cm−1 (ν), 1650 cm−1 (δ) and 750 cm−1 (δ) can be ascribed to O-H stretching (ν) and deformation (δ) vibrations, due the presence of water and alcohol groups. Since the heating at 400 °C was not enough to remove these species, we have to conclude that they were structurally built into the host and not merely adsorbed on the surface. After a 600 °C heat treatment, these O-H vibrations were less intense than those observed at lower temperatures, exerting an influence on the powder’s microstructure, as demonstrated by XRD analysis. While the heating of the powders at much higher temperatures , the vibrations related with the O-H impurities [i.e., these vibrations are nearly missing at 900 °C. The absorption peak around 1380 cm−1 indicates the N-O stretching vibration [3 −, which remains present until 500 °C. The peak situated at 1530 cm−1 can be attributed to the asymmetrical stretching of C-O, while the absorption bands at 1090 cm−1 and 850 cm−1 are due to the symmetrical stretching of C-O and deformation vibrations of C-O in CO3 2−. These absorption peaks indicate the presence of carbonate groups. Bands of C-O-H and CH2- corresponding to bending vibrations appear around 1410 cm−1 and 1460 cm−1, respectively, arising from the decomposition of DEG [−1, characteristic of the C-C bond [3 − residues were adsorbed in the sample, which could be removed after 900 °C thermal treatment. Finally, the bands at 580 and 489 cm−1, observed from 400 °C, and attributed to the Lu-O stretching vibrations of cubic Lu2O3 (a Lu2O3 host lattice vibration) [The Fourier transformed infrared spectra of Lupurities  are almon of DEG , and oneC-C bond . The intbration) –56, indi2O3: Eu3+ 5 mol %, Tb3+ 0.01 mol% nanopowders annealed in air at temperatures ranging from 300 to 900 °C. At 300 °C, the xerogel exhibits an almost amorphous behavior; however, at 400 °C, the system possesses an aspect of an amorphous phase characterized by broad diffraction peaks, which also indicates that the crystallites are very small, lower than 5 nm.2O3 structure (JCPDS 431021) with a spatial group Iā3 (lattice parameter 10.391 Å). With an increments the annealing temperature, the diffraction peaks become narrower, which reflects an increase in the size of the Lu2O3 crystallites. β is the full-width radiation at half-maximum (FWHM) of the peak, and θ is the Bragg angle of the diffracted X-ray. The crystallite size ranges from 5.3 nm at 500 °C to 25.5 nm at 900 °C; these observations were confirmed by TEM observations for the 700 and 900 °C co-doped lutetia nanopowders.This result is in good agreement with the DTA observations, since the crystallization process began between 300 and 400 °C. As the annealing temperature reaches 500 °C, it becomes evident that the crystallization process has ended in a cubic Lu2O3: Eu3+ 5 mol %, Tb3+ 0.01 mol % scintillating nanopowders, annealed at 700 °C. As observed, the morphology of the powders is mainly angular; however, some of the faces are rounded and the particles are highly agglomerated. Bright field, diffraction pattern and dark field micrographs, as well as particle size distribution of the sample annealed at 900 °C are shown in 2O3: Eu3+ 5 mol%, Tb3+ 0.01 mol% nanopowders annealed at 700 and 900 °C, under X-ray excitation of 30 kV and 40 mA. The scintillating spectra of the sample display a group of emission lines situated in the 575- to 725-nm spectral region, corresponding the Eu3+ transitions from the excited 5D0 level to 7Fj  levels, with no evidence of any Tb3+ emission (545 nm). The maximum emission line at 611 nm, which matches well the spectral sensitivity range of CCD cameras, corresponds to the 5D0 → 7F2 electric dipole transition, and is a product of the Eu3+ ion located at C2 sites in the Lu2O3 host cubic lattice [3+ emission centers according to a recombination mechanism [5D0→7F0, 5D0→7F1, 5D0→7F3 and 5D0→7F4 transitions, respectively, and are in good agreement with the energy levels described elsewhere [3+ ion in the nanopowder sample. Furthermore, it has been demonstrated that until the complete elimination of residual OH contamination, the non-radiative multiphonon relaxation process can be stimulated with high-energy phonons introduced by this impurity [− impurity has been almost completely eliminated, which is a temperature considerably lower to the 1300 °C range that has been previously reported [ lattice , and itsechanism . The leslsewhere . The difimpurity . In the 3+ 5D0→7F2 (611 nm) emission as a function of the Tb3+ concentration for nanopowders annealed at 700 and 900 °C. In both cases, the lutetia co-doped at 0.01 mol % Tb3+ presents the highest light yield, and for reasons explained earlier, the emission of the sample heat-treated at 900 °C is higher than that of the 700 °C one. The enhancement of the Eu3+ emission in the nanopowders is due to a non-radiative energy transfer from Tb3+ to Eu3+, as Tb can absorb more X-ray radiation. At higher Tb3+ concentrations, a self-quenching mechanism [3+ emission. Similar results have been observed for Gd2O3 co-doped Eu, Tb nanopowders [echanism  exists, opowders .2O3:Eu3+, Tb3+ nanopowders were prepared by a simple sol-gel process method. 3)3•6H2O , europium nitrate Eu(NO3)3, terbium nitrate Tb(NO3)3 , acetic acid CH3COOH (Fermont 98%) used as a catalyst, and diethyleneglycol (DEG) C4H10O3  used as a polymerization agent. Initially, the lutetium nitrate was dissolved in an ethanol-deionized water solution (95-5 vol%) using a hot stirrer at 40 °C for 3 hours to obtain 25 mL of a 0.95 M Lu sol. Thereafter, the solution pH was adjusted by incorporating acetic acid (0.17 M), and the DEG was added (0.42 M). Finally, the co-doping elements, previously dissolved in ethanol, were incorporated the initial lutetium sol. The sol obtained was mixed for 2 hours to produce a transparent solution, stable for more than 3 months. Subsequently, the sol was dried at 100 °C for 24 hours, and the yielded gel was calcined at different temperatures for 1 hour to obtain the Lu2O3:Eu3+, Tb3+ scintillating nanopowders.Lu−1. The IR spectra of the samples were recorded in the range of 4000–400 cm−1 using Fourier transform infrared spectroscopy  and the KBr pelleting technique. The phase composition of the powders was identified by X-ray diffraction at room temperature on a powder diffractometer (Bruker D8Advance) using Cu Kα radiation (1.5418 Å). The morphology and particle size of the powders were observed with a transmission electronic microscope (JEOL 2200) operating at 200 keV. The scintillating properties were recorded employing an X-ray generator (PW-1830 Phillips) operating at 30 kV and 40 mA, using W Kα radiation (0.2086 Å) and a photon flux of 1012 ph s−1.Thermogravimetric (TGA) and differential thermal analyses (DTA) of dried gel were conducted using a SDT Q600 TA instrument; the studies were performed in air with a heating rate of 10 °C min2O3:Eu3+, Tb3+ nanopowders have been prepared by a simple sol-gel method, using lutetium, europium and terbium nitrate as precursors, and DEG as a polymerization agent. This nanopowder crystallizes into a cubic system from 400 °C, and the process is completed at 500 °C, resulting in particles composed of crystallites ranging in size from 5.3 to 25.5 nm. The increments in crystallite sizes depend on the heat treatment temperatures. The powder exhibits interesting scintillating properties, and at 611 nm presents a reddish emission corresponding with the Eu3+ 5D0→7F2 transition, which makes it a promising material for X-ray detection systems, since this emission matches the maximum efficiency of the CCD cameras. The light yield of the nanopowders was analyzed at two different annealing temperatures: 700 and 900 °C. It was established that at the sample heat treated at the higher temperature, presents an enhanced light output, presumably due to better crystallinity and to the complete removal of OH−. Finally, it was determined that with the co-doping of Tb3+ at a 0.01 mol% level, the light yield was enhanced compared to trials with higher Tb contents, presumably due to the effect of a self-quenching process.Lu


SBcas3.3, was identified by functional screening of a forest-soil metagenomic library on agar plates supplemented with AZCL-casein. Overproduction in Escherichia coli revealed that the enzyme is produced as a 770-amino-acid precursor which is processed to a mature protease of ~55 kDa. The latter was purified by affinity chromatography for characterization with the azocasein substrate. The enzyme proved to be an alkaline protease showing maximal activity between pH 9 and 10 and at 50°C. Treatment with the chelating agent ethylenediaminetetraacetic acid irreversibly denatured the protease, whose stability was found to depend strictly on calcium ions. The enzyme appeared relatively resistant to denaturing and reducing agents, and its activity was enhanced in the presence of 10 ml/l nonionic detergent . Moreover, SBcas3.3 displayed oxidant stability, a feature particularly sought in the detergent and bleaching industries. SBcas3.3 was activated by hydrogen peroxide at concentrations up to 10 g/l and it still retained 30% of activity in 50 g/l H2O2.A novel serine protease gene, The online version of this article (doi:10.1186/2193-1801-2-410) contains supplementary material, which is available to authorized users. Microorganisms produce various enzymes and antimicrobial agents potentially useful in agriculture, the chemical industry, and both human and veterinary medicine Daniel . Among tEscherichia coli, and screening the metagenomic libraries obtained for a desired activity on medium supplemented with a substrate of the type of enzyme sought , had been successfully screened for lipolytic (Biver and Vandenbol Desulfobacter postgatei 2ac9 (ZP_10168560.1).Two forest-soil metagenomic libraries were screened in Bacillus licheniformis. These enzymes are characterized by the active site residues  located in the motifs D-(T/S)-G, H-G-T-H, and G-T-S-M-A-X-P. Within this subfamily, the biochemically characterized protease to which SBcas3.3 is most similar  is the alkaline protease KP-43 isolated from Bacillus sp. strain KSM-KP43 . The proteolytic activity of each collected fraction was evaluated at several pH values (4–10) and temperatures (4-50°C) (data not shown). SBcas3.3 was found exclusively in the insoluble fraction, as an inactive protein migrating as a single band corresponding to a molecular mass of ~80 kDa, close to its theoretical molecular mass of 83 kDa  vector allowing overexpression in a Figure . The proa Figure . Sequenca Figure  suggestea Figure . The pro2+ concentration, since calcium promotes thermal stability of many subtilisin-like proteins  , one lacking the first 254 amino acids  and the other, the first 210  Figure a, and coThe mature form of the protease was thus purified by Ni-NTA affinity chromatography with calcium (5 mM) added at all steps. This form of the protein was also found exclusively in the insoluble fraction, whatever the growth conditions tested , with an optimum near 50°C  before estimating its activity in the presence of Ca2+ at optimal concentration. Addition of 5 or 10 mM EDTA completely abolished protease activity even at low temperature, and this inhibition was found to be irreversible, as no activity was recovered after CaCl2 repletion. This confirmed our preliminary results obtained with cellular extracts: SBcas3.3 is thus strictly dependent on Ca2+ for its stability.The calcium dependence of SBcas3.3 was quantified by measuring its relative activity in the presence of CaClTo estimate the kinetic parameters of SBcas3.3, the Folin-Ciocalteu method was used to perform initial velocity measurements at various casein concentrations. The maximal velocity (Vmax) and Michaelis affinity constant (Km) were then deduced from a Lineweaver-Burk plot. At its optimal temperature and pH, 1 mg enzyme released 34 μmol tyrosine per min and displayed a Km of 3.8 mg/ml casein.2+, thus complicating result interpretation. As SBcas3.3 appears related to oxidant-stable proteases, we also tested the effects of reducing agents and oxidants. Interestingly, SBcas3.3 was found to be moderately affected by 5–20 mM dithiothreitol and 2-mercaptoethanol and strongly resistant to high concentrations of hydrogen peroxide. Its activity actually increased when H2O2 was added at a concentration up to 10 g/l, and 30% activity was still measurable in 50 g/l H2O2 (1.47 M). SBcas3.3 thus displays characteristics of an oxidant-stable protease.The ability of the protease to degrade the azocasein substrate was next assayed in reaction mixtures containing various additives Figure . ProteolE. coli colonies expressing metagenomic DNA were first incubated at 37°C for 2 days and then for a few weeks at ~22°C. Screening at low temperature is sometimes used to search for cold-active enzymes  , notably because of oxidation of a conserved methionine residue adjacent to the catalytic serine , in contrast to other oxidant-stable proteases such as AprX from Virgibacillus sp. SK37 , these proteins share several features in addition to the fact that they are produced as large precursors with N-ter parts playing the role of intramolecular chaperones. Both SBcas3.3 and KP-43 have a preference for an alkaline pH (optimum: respectively 9–10 and 11–12) and for relatively high temperatures (optimum: respectively 50 and 60°C). Interestingly, SBcas3.3 is also highly resistant to the oxidizing agent hydrogen peroxide. Most subtilisin-like proteases are rapidly inactivated in the presence of 50 mM HFunctional screening of a forest-soil metagenomic library allowed the identification of a novel alkaline serine protease showing only low sequence identity to known proteases. The new enzyme is active over a relatively large pH range and displays high resistance to oxidizing agents, making it particularly attractive for various biotechnological applications.Sau3AI, and fragments ranging in size from 4 to 9 kb  and 9 to 20 kb (medium-insert library), recovered after separation in a 1% low-melting-point agarose gel, were inserted into the BamHI-linearized, dephosphorylated pHT01 vector (MoBiTec). The ligation products were introduced into electrocompetent DH10B Escherichia coli cells (Invitrogen). About 35,000 E. coli colonies from each library  were screened for protease activity by spreading the transformants on 2×YT (2× yeast extract and tryptone) medium  supplemented with 15 g/l agar (Oxoid), 50 μg/ml ampicillin, and 1 g/l AZCL-casein (Megazyme). This substrate releases a soluble blue dye upon hydrolysis. Colonies producing this activity were identified by a blue halo surrounding them. All plates were incubated at 37°C for 2 days and then transferred to room temperature (~22°C) for three weeks.Two forest-soil metagenomic libraries differing in average insert size (7 and 12 kb) were constructed (Biver and Vandenbol http://merops.sanger.ac.uk)  using the Sanger method. Sequence similarity searches were carried out with the NCBI BlastP program (standard protein blast) and by comparison with proteases of the MEROPS database , 5’-gggaattcCATATGGCGCGCGTATTAAAGGTT-3’ as forward primer, and 5’-cgcGGATCCTCATCAGTGGTGGTGGTGGTGGTGCGTTCGGCCGACGATCAC-3’ as reverse primer. The restriction sites  used for subcloning are italicized and the histidine tag sequence appears in bold. PCR products were cloned into the pET-30b(+) vector (Novagen) and the resulting plasmids introduced into E. coli Rosetta 2 (DE3) (Novagen) after checking the inserts by DNA sequencing. The proteolytic activity of transformants was evaluated by spotting 5 μl overnight culture  onto solid 2×YT medium (15 g/l agar) supplemented with 10 μM isopropyl β-D-1-thiogalactopyranoside (IPTG) (Fermentas), 1g/l AZCL-casein, and appropriate concentrations of antibiotics (30 μg/ml chloramphenicol and kanamycin). The bacteria were allowed to grow at 37°C for 16 h before transfer to incubators set at various temperatures .The entire coding sequence of the CATATGGGATTCAAGATCCTCGATCGTC-3’ (cas560) or 5’-gggaattcCATATGGCCATCAAGCGTCCAAGCA-3’ (cas516) as forward primer.Two N-terminally truncated mutants of SBcas3.3 were obtained similarly, using the same reverse primer as for amplification of the full-length protease and 5’-gggaattcFor protein size determination by SDS-PAGE, bacteria were grown for 16 h at 25 or 37°C in liquid Terrific Broth (TB) medium (Difco) containing 10 μM IPTG. Cells of 25-ml cultures were harvested and resuspended in 5 ml lysis buffer . After lysis at 22°C for 30 min and subsequent sonication on ice , the total extracts obtained were centrifuged  to separate soluble from insoluble proteins (the latter were next resuspended in 5 ml lysis buffer). Each fraction was mixed with an equal volume of Laemmli’s sample buffer 2× containing 100 ml/l 2-mercaptoethanol and heated at 95°C for 5 min before electrophoresis. Western blot analysis with anti-His tag antibodies was performed as previously described  for 16 h . Total cell extracts were then prepared as described above and diluted twice in 0.1 M buffer  supplemented with 2 g/l AZCL-casein. Incubation was performed on a wheel (2 rpm) at 4, 22, 29, 37, and 50°C until the appearance of the blue colour.To better evaluate the activity of SBcas3.3, cas516, and cas560, their ability to degrade the AZCL-casein substrate was also investigated on total extracts of 2. This was followed by pre-incubation at 4°C for 0 or 30 min, addition of 1 g/l AZCL-casein, and transfer to 4, 22, 29, 37, or 50°C.Preliminary information about the calcium requirements of the enzymes was obtained by diluting total cell extracts twice in 0.1 M Tris–HCl pH 9 supplemented with 0 or 10 mM CaClE. coli Rosetta cells transformed with the empty vector was included in all experiments. No activity was observed under any of the conditions tested.A negative control corresponding to extracts of E. coli Rosetta cells was diluted to OD600nm=0.005 in a 250-ml shake flask containing 50 ml TB medium and antibiotics (30 μg/ml chloramphenicol and kanamycin). The culture was grown at 37°C under shaking at 160 rpm until the OD600nm reached about 0.4. Overproduction was induced by addition of 10 μM IPTG followed by incubation at 25°C for 16 h. Cells were harvested by centrifugation and lysed at 22°C for 30 min in 20 mM Tris–HCl buffer (pH 8) containing 5 mM CaCl2, 0.3 M NaCl, 1 ml/l Triton X-100, and 5 mg/ml lysozyme. After sonication on ice , the lysate was centrifuged at 10,000 g for 20 min (4°C) to separate insoluble from soluble proteins. The soluble fraction, which did not contain SBcas3.3, was discarded and the insoluble fraction containing the active recombinant protease was resuspended in 20 mM Tris–HCl pH 8, 5 mM CaCl2, 0.5 M NaCl, 20 ml/l Triton X-100, 1.5 M urea to solubilize the protease. The lysate was then centrifuged again to collect the soluble fraction now containing the protease, which was next purified by Ni-NTA affinity chromatography (Qiagen). For this, the soluble fraction was first diluted to reduce the urea and Triton X-100 concentrations to 7.5 ml/l and 0.1875 M, respectively, and then incubated overnight at 4°C with 500 μl Ni-NTA agarose pre-equilibrated with binding buffer . The resin was recovered by centrifugation at 800 g for 5 min and washed once with binding buffer (flow-through 1) and twice with washing buffer  (flow-through 2) before elution with 20 mM Tris, 5 mM CaCl2,0.5 M NaCl, 175 mM imidazole, pH 7.5 (10 ml). The purified proteins were dialysed at 4°C against 20 mM Tris–HCl pH 7.5 and 5 mM CaCl2 and analysed by SDS-PAGE.To purify the recombinant protease, an overnight culture of transformed 2, 5 g/l azocasein) at various temperatures from 5 to 70°C. After pre-incubation of the reaction mix, the reaction was started by adding enzyme and stopped after 20 min with 300 μl of 100 g/l ice-cold trichloroacetic acid (TCA). The precipitated proteins were removed by centrifugation at 10,000 g for 10 min (4°C). To increase the sensitivity of the measurement, the supernatant (400 μl) was alkalinized by addition of an equal volume of 2 M NaOH. The mix was centrifuged at 10,000 g for 5 min (22°C) to remove any precipitate before estimation of the azo dye concentration at 440 nm. To determine the optimal pH of the enzyme, protease activities were measured at 50°C over a pH range of 5 to 12 in a mix containing 5 mM CaCl2, 5 g/l azocasein, and 50 mM buffer . The effects of several additives  on protease activity were evaluated by pre-incubating the enzyme with each additive (in 50 mM Tris–HCl pH 9 and 5 mM CaCl2) at 30°C for 30 min before incubation with azocasein (in 50 mM Tris–HCl pH 9 and 5 mM CaCl2) at 50°C for 20 min. As PMSF has a short half-life at alkaline pH, pre-incubation was done at pH 7.5 instead of 9. The effect of isopropanol was tested because this was the solvent used to prepare the stock solution of PMSF (200 mM) added to the mix. To evaluate the calcium requirements of the purified enzyme, SBcas3.3 was pre-incubated on ice for 30 min with various CaCl2 concentrations (0.5 to 20 mM) before measuring its azocaseinolytic activity at 50°C. To see if calcium is required for SBcas3.3 activity or for its stability, Ca2+ was removed from the enzyme by pre-incubation with 5 or 10 mM EDTA for 30 min at 4°C before calcium repletion (at 4°C) by dilution in 5 mM CaCl2 . The residual azocaseinolytic activity of SBcas3.3 was measured after 4 hours of calcium repletion and compared with that of the enzyme pre-incubated without EDTA.Protease activity was estimated by measuring the release of acid-soluble material from azocasein (Sigma-Aldrich). The optimal temperature was determined by incubating 10 μl purified enzyme with 290 μl reaction mix . TCA  was then added to stop the reactions (600 μl). The proteins were removed by centrifugation and the amino acids present in the supernatants quantified by the Folin-Ciocalteu method. For this, the supernatants (500 μl) were incubated at 37°C for 30 min with 1250 μl Na2CO3 0.5 M and 250 μl Folin-Ciocalteu reagent diluted 10× (Sigma-Aldrich) before absorbance measurement at 660 nm. Activities were determined by comparison with a standard curve of L-tyrosine (0–0.2 mM) and expressed in μmol tyrosine released per min per mg protein. A Lineweaver-Burk plot was used to determine Km and Vmax.To determine the kinetic parameters of SBcas3.3, 10 μl enzyme was incubated for 20 min at 50°C in 50 mM Tris–HCl pH 9, 5 mM CaClAll experiments were performed at least twice in triplicate, and the results of one representative experiment are shown. They are expressed as means ± standard error of the mean. Comparisons with the control condition were performed with Student’s t-test.The nucleotide sequence of the insert containing the gene encoding the proteolytic activity was deposited in the GenBank database under accession number KC433657.Caseinolytic activity ofE. coliRosetta cells overproducing the full-length protease SBcas3.3 or one of its truncated mutants, cas516 or cas560. (PDF 30 KB)Additional file 1:


People with velo-cardio-facial syndrome or 22q11 deletion syndrome (22q11DS) have behavioral, cognitive and psychiatric problems. Approximately 30% of affected individuals develop schizophrenia-like psychosis. Glutamate dysfunction is thought to play a crucial role in schizophrenia. However, it is unknown if and how the glutamate system is altered in 22q11DS. People with 22q11DS are vulnerable for haploinsufficiency of PRODH, a gene that codes for an enzyme converting proline into glutamate. Therefore, it can be hypothesized that glutamatergic abnormalities may be present in 22q11DS.1H-MRS) to quantify glutamate and other neurometabolites in the dorsolateral prefrontal cortex (DLPFC) and hippocampus of 22 adults with 22q11DS (22q11DS SCZ+) and without (22q11DS SCZ−) schizophrenia and 23 age-matched healthy controls. Also, plasma proline levels were determined in the 22q11DS group.We employed proton magnetic resonance spectroscopy (We found significantly increased concentrations of glutamate and myo-inositol in the hippocampal region of 22q11DS SCZ+ compared to 22q11DS SCZ−. There were no significant differences in levels of plasma proline between 22q11DS SCZ+ and 22q11DS SCZ−. There was no relationship between plasma proline and cerebral glutamate in 22q11DS.in vivo1H-MRS study in 22q11DS. Our results suggest vulnerability of the hippocampus in the psychopathology of 22q11DS SCZ+. Altered hippocampal glutamate and myo-inositol metabolism may partially explain the psychotic symptoms and cognitive impairments seen in this group of patients.This is the first  Velo-cadio-facial-syndrome or 22q11 deletion syndrome (22q11DS) is a genetic syndrome caused by a deletion on chromosome 22 which is accompanied by several somatic, behavioral, cognitive and psychiatric problems, and structural and functional brain abnormalities People with 22q11DS are vulnerable to haploinsufficiency of approximately 30 genes located on the deleted region of chromosome 22q11, including the proline dehydrogenase gene (PRODH) The involvement of glutamate in schizophrenia is particularly related to NMDA receptor hypofunction. Evidence for the role of NMDA receptor hypofunction in schizophrenia comes from pharmacological studies of phencyclidine (PCP) and ketamine. These NMDA receptor antagonists have shown to produce schizophrenia-like behaviors in rodents The brain areas associated with NMDA receptor hypofunction in schizophrenia include the prefrontal cortex and hippocampus 1H-MRS) is a feasible method for in vivo quantification of glutamate concentration and other brain metabolites that, if altered, may reflect abnormal neuro-developmental features 1H-MRS studies have been conducted. Although inconclusive, 1H-MRS findings also suggest abnormal glutamatergic neurotransmission Proton Magnetic Resonance Spectroscopy  and without schizophrenia (22q11DS SCZ−). We hypothesized altered glutamate concentrations in individuals with 22q11DS SCZ+ compared to healthy individuals and, in 22q11DS SCZ+ compared to 22q11DS SCZ−. Besides glutamate, we also analyzed other neurometabolites from 1H-MRS spectra including N-acetylaspartate, choline, myo-inositol and creatine which reflect the status of neuronal functioning and glial cells, possibly disturbed in 22q11DS.To date, the glutamatergic system in 22q11DS has not been investigated. People with 22q11DS have an increased prevalence of schizophrenia and similar neuroanatomical abnormalities. Hence, in this study we employed Furthermore, we assessed levels of plasma proline and plasma glutamine in the 22q11DS group. Increased proline has been reported in 22q11DS patients n = 12, age 29.25±8.24; 22q11DS SCZ− n = 10, age 28.50±8.47) and 23 healthy controls .We included 22 adults with 22q11DS (mean ± SD) , The Netherlands and was approved by the Medical Ethics Testing Committee/AMC. All participants were capable of giving written informed consent and did so, after receiving full information on the study.All individuals with 22q11DS were assessed by an experienced psychiatrist and a physician for people with an intellectual disabilities using available information from medical records and a semi-structured psychiatric interview. All diagnoses reported are DSM-IV diagnoses . The 22q11DS group was subdivided into 2 groups: those who were fulfilling DSM-IV criteria for schizophrenia (22q11DS SCZ+) all taking antipsychotic medication and having duration of illness >1 year  For assessment of intelligence quotient (IQ) we used the shortened Dutch version of the Wechsler Adult Intelligence Scale (WAIS-III–NL) consisting of 5 subtests: vocabulary, comprehension, similarities , block design, and object assembly (performance IQ) All healthy volunteers were seen by a physician. They were included in the study after screening for psychiatric disorders and medical conditions affecting the brain. None of the participants had a history of substance or alcohol abuse. Urine drug screening  was performed at study day and was negative in all subjects. Healthy participants were not using any medication at the time of testing.1H-MRS data acquisition took place at the Department of Radiology  using a 3 Tesla Intera MRI system  equipped with a 6 channel sense head coil. For estimation of metabolite concentrations, two single 8 ml voxels of interest positioned in the left dorsolateral prefrontal cortex (DLPFC) (2×2×2 cm) and left hippocampus (2×2×2 cm) were obtained for each subject  and a TR/TE of 9.8/4.5 milliseconds  were obtained.1H-MRS spectra were analyzed using the Linear Combination of Model spectra (LCModel) commercial spectral-fitting package plus phosphocreatine (Cr), glycerophosphocholine plus phosphocholine (choline), myo-inositol, N-acetylaspartate (NAA), NAA plus N-acetylaspartylglutamate (NAAG), glutamine and glutamate.plus glutamine (Glx). Glutamate and glutamine are closely related amino acids involved in intermediary metabolism, protein synthesis and neurotransmission. Metabolite concentrations are expressed in millimoles per liter.In addition, we analyzed the combination of glutamate Data were excluded from analysis if the voxel coordinates were not or incorrectly recorded. Spectral width  was always lower than 0.1 p.p.m. and signal to noise ratio (SNR) greater than 11 as estimated by LCModel. Cramer-Rao minimum variance bounds (SD) was lower than 50% for glutamine and lower than 15% for the other metabolites.Plasma proline and plasma glutamine concentrations of the 22q11DS group were assessed by automated ion exchange chromatography with post-column ninhydrin derivatization. Plasma amino-acid analyses were performed on a JEOL AminoTac  following a morning blood draw.P≤0.05 (2-tailed). Statistical analyses were performed with SPSS, release 16.0.2 for Windows .We used non-parametric Kruskal-Wallis H test to compare metabolite concentrations, age and IQ between the 3 groups  because the assumption of normal distribution was not met. Following, Post Hoc analyses were conducted with Mann-Whitney U tests. Correlation analyses were conducted with Spearman's rho test. Results are reported as significant when P = 0.45) and age .Patients and healthy controls did not differ with regard to sex . Also verbal IQ  and performance IQ  were significantly different between the groups. Post hoc analysis showed that HC compared to 22q11DS SCZ+ differed significantly for total IQ P = 0.001, verbal IQ P = 0.001 and performance IQ P = 0.001. HC compared to 22q11DS SCZ− differed significantly for total IQ P = 0.004, verbal IQ = P = 0.005 and performance IQ P = 0.01. 22q11DS SCZ+ compared to 22q11DS SCZ− differed significantly for total IQ P = 0.02 and verbal IQ P = 0.02 but not performance IQ P = 0.17.Patients had a lower total IQ than healthy controls  Glx (P = 0.03) and myo-inositol (P = 0.03). Post Hoc analysis indicated that these metabolite concentrations were significantly higher in 22q11DS SCZ+ compared to 22q11DS SCZ− patients . Hippocampal Glx was higher in 22q11DS SCZ+ compared to HC (P = 0.02). In the DLPFC there was a significant positive correlation between glutamine concentration and antipsychotic dosage (n = 10 ρ = 0.64 P = 0.05) and a trend towards a positive correlation between Glx and antipsychotic dosage (n = 10 ρ = 0.59 P = 0.07). There were no significant correlations between hippocampal metabolites and antipsychotic dosage.Metabolite concentrations for the DLPFC and hippocampal region are displayed in P = 0.53). The normal laboratory range for plasma proline was 77–343 µmol/l and for plasma glutamine 344–743 µmol/l.For the whole 22q11DS group, the mean±SD for plasma proline was n = 13, 354±128.88 µmol/l and for plasma glutamine n = 8, 540.62±68.14 µmol/l. The correlation between these variables was not significant (n = 8 ρ = 0.26 P = 0.56) or plasma glutamine . There was no significant correlation between plasma proline and plasma glutamine in any of the two 22q11DS groups.There were no significant differences between 22q11DS SCZ− and 22q11DS SCZ+ for plasma proline . Also, there was no significant correlation between proline and DLPFC glutamate for the 22q11DS SCZ− (n = 5 ρ = 0.30 P = 0.62) and 22q11DS SCZ+ group (n = 6 ρ = 0.37 P = 0.47).The correlation between DLPFC glutamate and plasma proline for the whole 22q11DS group was not significant (n = 11 ρ = 0.26 P = 0.56). There was no significant correlation between proline and hippocampal glutamate for the 22q11DS SCZ− (n = 6 ρ = 0.03 P = 0.96) and 22q11DS SCZ+ group (n = 4 ρ = 0.40 P = 0.80).The correlation between hippocampal glutamate and plasma proline for the whole 22q11DS group was not significant  have been shown to potentiate glutamate transmission in hippocampus and cerebral cortex In addition to increased hippocampal glutamate, we found higher concentrations of myo-inositol in 22q11DS SCZ+ compared to 22q11DS SCZ−. Increased concentrations of myo-inositol have previously been reported in mild cognitive impairment and Alzheimer disease The finding of increased glutamate and myo-inositol may be tightly related to each other in the psychopathology in 22q11DS SCZ+. Myo-inositol is primarily found in astrocytes vs. 22q11DS SCZ−) vs. healthy controls. In patients with chronic schizophrenia, 1H-MRS studies of the frontal cortex have shown increased We found no significant variation in neurometabolites concentration between the whole 22q11DS patient group and the healthy control group. This might be explained by group differences in the proportion of gray matter/white matter within the DLPFC and hippocampal voxels. Also, we found no evidence for altered glutamate in the DLPFC of 22q11DS patients  The strengths of this study include the evaluation of neuronal integrity in 22q11DS according to psychiatric status of 22q11DS SCZ− and 22q11DS SCZ+ and in comparison to age matched healthy controls. Also, all MRS spectra were carefully inspected and were included only if fulfilling the quality criteria of LCmodel.i.e., assessment of metabolite concentrations without addressing the impact of different tissue included in the voxel of interest) may increase the standard error of measurement and diminish the power to detect significant differences. The cubic shape of hippocampal voxel may have allowed for contamination signals from adjacent regions of the hippocampus. Moreover, the effect of medication can be a potentially confounding factor in 1H-MRS studies We have to acknowledge some limitations of the study; unfortunately at the time of the study we were not able to analyze plasma samples of proline and glutamine of healthy controls. Future studies with large sample sizes including healthy volunteers, should elucidate the relationship between plasma levels , cerebral metabolites and the vulnerability to schizophrenia. We did not determine the size of deleted region in each 22q11DS patient although the majority of patients have a typically deleted 3 Mb region. We did not to apply a correction for multiple comparisons because the possibility of inflating type II error 1H resonance spectrum. The use of higher field strengths and implemented spectroscopy analysis technique makes it possible to improve glutamate quantification. Discrepancies across earlier 1H-MRS studies that proposed to investigate glutamate in psychosis could have resulted from differences in brain regions of interest, patient population and stage of disease or issues of spectroscopy measurements.Due to similar chemical components glutamate and glutamine overlap significantly in the 1H-MRS studies with larger sample sizes including other prefrontal and temporal brain regions will help to clarify brain metabolism and integrity in 22q11DS.In conclusion, our findings suggest vulnerability of the hippocampus in the psychopathology of 22q11DS SCZ+. Although the generalizability of the results is restricted by the relatively small sample size, altered glutamate and myo-inositol metabolism may partially explain the psychotic symptoms and cognitive impairments seen in this group of patients. Future


Arabidopsis thaliana immunophilin ROF1 with phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-bisphosphate was identified using a phosphatidylinositol-phosphate affinity chromatography of cell suspension extracts, combined with a mass spectrometry (nano LC ESI-MS/MS) analysis. The first FK506 binding domain was shown sufficient to bind to both phosphatidylinositol-phosphate stereoisomers. GFP-tagged ROF1 under the control of a 35S promoter was localised in the cytoplasm and the cell periphery of Nicotiana tabacum leaf explants. Immunofluorescence microscopy of Arabidopsis thaliana root tips verified its cytoplasmic localization and membrane association and showed ROF1 localization in the elongation zone which was expanded to the meristematic zone in plants grown on high salt media. Endogenous ROF1 was shown to accumulate in response to high salt treatment in Arabidopsis thaliana young leaves as well as in seedlings germinated on high salt media (0.15 and 0.2 M NaCl) at both an mRNA and protein level. Plants over-expressing ROF1, (WSROF1OE), exhibited enhanced germination under salinity stress which was significantly reduced in the −rof1 knock out mutants and abolished in the double mutants of ROF1 and of its interacting homologue ROF2 (WS−/2−rof1). Our results show that ROF1 plays an important role in the osmotic/salt stress responses of germinating Arabidopsis thaliana seedlings and suggest its involvement in salinity stress responses through a phosphatidylinositol-phosphate related protein quality control pathway.A direct interaction of the  Absence of Fab1p, the only kinase that synthesizes PIP2 in yeast through a direct modification which converts phosphatidylinositol-3-phosphate [PI(3)P] to PIP2, results in enlarged vacuoles 2 is increased following hyperosmotic stress which also accelerates the PIP2 mediated fission of lysosomal subcompartments 2 have been implicated in osmotic stress responses 2 related response in A. thaliana seedlings or suspension cultures 2 contained in plants is increased in response to osmotic stress, however, our knowledge of the functional importance of PIP2 on plant osmotic stress responses is very limited. In addition, PIP2 presents a peculiarity compared to other PIPs. In contrast to the PI(3)P, phosphatidylinositol-4,5-bisphosphate [PIP2] and phosphatidylinositol-3,4,5-trisphosphate [PIP3], PIP2 recognizing domains are highly diverse; e.g. HEAT/ARM repeat containing ENTH domains 2 during osmotic stress responses in plants and a still unidentified PIP2 effector, the identification of novel PIP2 binding candidates from osmotically stressed A. thaliana extracts would provide useful information on both the functional importance of PIP2 in plants as well as for novel binding domains interacting with this phosphoinositide.Phosphatidylinositol phosphates (PIPs) are phospholipids with important regulatory properties in signaling and trafficking processes 2 and is involved in plant responses to high salinity and osmotic stress. Following its isolation using a phosphatidylinositol-phosphate (PIP) affinity chromatography its mode of interaction with PIP stereoisomers was investigated as well as the possible functional importance of this interaction in cellular processes during plant osmotic stress responses.In this work we show that ROF1, a plant immunophilin that belongs to the FK506-Binding Protein (FKBP) subfamily, directly interacts with PI(3)P and PIP2 binding candidates from A. thaliana cell suspension cultures. A T87 cell suspension culture treated with either 0 (C) or 0.4M NaCl for 5 mins was centrifuged, cell pellet was collected and homogenized and extracts were first incubated with a positively and a negatively charged ion exchange chromatography (Q and S Sepharose) in order to reduce sample complexity. Sequential washings using different salt concentrations  and ROF2 P2∶427; 8 peptides used with 100% probability) .A PIP chromatography was employed in order to identify PI(3)P and PIPions see  resultedractions  A–C. Salfraction . Band excultures , followehttp://www.ncbi.nlm.nih.gov/structure) known for binding to the IP33 were identified  and aligned to β-spectrin (ROF1 is a multi-domain protein. ROF1 contains an FK506 Binding Domain (FKBD) with PPIase activity, (FKBD1), two degenerate FKBDs (FKBD2 and FKBD3) as well as tetratricopeptide repeat domains (TPR) and a potential calmodulin binding domain entified . In partspectrin .http://ekhidna.biocenter.helsinki.fi/dali_lite/start) and Cn3D (http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml) indicated possible binding sites for a phosphorylated inositol ring on the FKBD1 domain of ROF1 P P as a competing lipid reduced signal for both the PI(3)P and the PIP2 in the ROF1 and the FKBD1 assay P2 stereoisomer as demonstrated by the ability of the 2FKTPR construct to interact with both lipids P . A compeD1 assay . Although lipids . On the h lipids . AlthougBD1 does .A polyclonal antibody was raised against the bacterially expressed ROF1 as described in the A. thaliana wild type and mutant lines  stressed at 37°C for up to 4 hrs. Results showed  confirming antibody specificity as described above. In untreated plants and plants treated for a short period of time with high NaCl (0.4 M for 2 mins) ROF1 was mainly localized in the elongation zone of the root tip (seedlings were immersed in the solution washed briefly and then fixed)  . Howevern fixed) . The cytn fixed)  whereas n fixed)  probably−rof1 (Col- −rof1), Wassilevskija wild type (WS), WS−rof1 and WS−rof1/−rof2, WS complemented mutants (WSROF1CM) and WS ROF1 over-expressions (WSROF1OE) were confirmed by western blotting using the αντι-ROF1 for ROF1 detection , Columbia etection . Columbio the WT , D a sig2 M NaCl . The NaCl P and PIPFKBPs - immunophilins with PPIase activity 2 binding candidates in a differential screening approach which was followed in order to isolate novel binding proteins with a preference for the PIP2 [compared to the PI(3)P] aiming at the identification of a PIP2 binding domain P2 has been identified in plants] and no band enrichment was observed in this case P and PIPhis case . ROF1 co3), inositol 1,3,4,5-tetrakisphosphate (IP4), and phosphatidylinositol 4- and 4,5-phosphates. Both PIPs were suggested – by the same study- as potent ligands of this protein. It has also been reported that β-spectrin, an IP3 binding protein, shows structural homology with FKBP12 2 through its FKBD domains. It was also shown that the TPR/calmodulin binding domain is not necessary for binding to the PI(3)P and PIP2 P2 . The truIP2  indicateIP2 .The fact that ROF1 bound to the PIPs in the absence of the FKBD1 known to contain the PPIase activity of ROF1 indicates that the presence of an active PPIase domain is not necessary for this interaction as previously suggested for the IPBP12 2, phospholipids related to osmotic stress responses A. thaliana roots within 30 mins following 140 mM NaCl treatment Interaction with PI(3)P and PIPA. thaliana seedlings aiming at the detection of the endogenous protein was performed. Endogenous ROF1 was mainly localized in the elongation zone of the root tip  2 incubations of the 0.1S fractions were run on a denatured polyacrylamide gel and stained with colloidal Coomasie . Bands of interest were excised, reduced, carbamidomethylated, digested with trypsin and analysed by on-line nanoLC ESI-MS/MS.PIP binding candidates were identified using nanoLC ESI-MS/MS A. thaliana entries in Uniprot release 2011.01, using Mascot version 2.3.02 . The search parameters used specified trypsin as the digestion enzyme, with one missed cleavage allowed: a fragment ion mass tolerance of 0.15 Da and a parent ion tolerance of 150 ppm; carbamidomethylation of cysteine as a fixed modification; cyclisation of peptide N-terminal glutamine and oxidation of methionine, as variable modifications. Scaffold  was used to validate MS/MS based peptide and protein identifications. Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least 2 peptides each identified at greater than 50.0% probability.Tandem mass spectra were extracted, charge state deconvoluted and deisotoped by Mascot.dll version 1.6b25, and searched against the 50961 E. coli cells and sequenced prior to any further use. Ligation for further sub-cloning was performed using T4 DNA ligase .A ROF1 construct in the pVAV44 vector (gift from Prof. Gasser) was used as a template for downstream applications. For subcloning in suitable vectors, primers described in 600 reached 0.5, for 6 hrs using 0.75 mM IPTG. Bacterial pellets were collected and lysed using 200 µg/ml lysozyme in Tris or PBS buffer. Proteins were purified after two sequential incubations with GST and His columns  according to the manufacturer’s instructions, collected at the end of the second (nickel column) elution, aliquoted, snap frozen in liquid nitrogen and stored at −80°C in the presence of 10% glycerol. Full length expression of the GST/His tagged proteins was confirmed using the αντι-GST  and the αντι-His mouse monoclonal IgG2a .Identified candidates and their mutants were sub-cloned in the pALEX expression vector for protein overexpression 2, PIP2, PIP2 and PIP3 lipids were purchased from Avanti  and PI(3)P, PI(4)P and PI(5)P from Cayman . Lipids were diluted in chloroform/methanol and 1 µl  of each was spotted on a nitrocellulose membrane . After drying for 30 mins the membrane was incubated with 1% fat free-ultrapure BSA for 1 hr in TBS  for 1 hr at room temperature. Incubation was performed overnight at 4°C using 2 µg/ml purified GST tagged protein. GST-tagged proteins bound to the lipids were detected using αντι-GST. For the lipid competition assay, the same procedure was followed with the additional step of the competing lipid inclusion at a final concentration of 50 µM during the incubation procedure with 1 µg/ml purified GST tagged protein. Equal volume of chloroform/methanol was added in the control (no competing lipid addition) reaction.A lipid overlay assay was used for the detection of direct protein-lipid interaction. PI, PIPA rabbit polyclonal αντι-ROF1 was prepared and used in Western blotting, dot blotting and immunofluorescence microscopy. Αντι-ROF1 polyclonal antibody was prepared using bacteria overexpressed and purified ROF1. Digestion with factor Xa and Enterokinase  was performed according to the manufacturer’s instructions. Following digestion the entire amount of the collected protein was run on a polyacrylamide gel under denaturing conditions. Following Coomassie blue staining a clear band of the expected molecular size was cut from the gel and ROF1 was eluted from the polyacrylamide gel using the electroeluter 422 system . Rabbits were immunized with 300 µg denatured protein and each immunization was performed in three week intervals. Serum before and after immunization was collected. For cytochemical applications the third bleed was used. Western blotting was performed following SDS-PAGE and transferring to a nitrocellulose membrane. Secondary antibodies goat anti-rabbit and goat anti-mouse horseradish peroxidase-linked for the ECL system were from Chemicon (USA).Quanitative, Real Time PCR was performed as previously described ROF1 and ROF2 fragment sizes were 255 bps and for the Ubiquitin 192 bps. Thermocycler conditions for all primer pairs were as follows: 50°C for 2 mins, 95°C for 2 mins, 36 cycles of 95°C for 5 secs and 60°C for 40 secs. Melting curve was performed from 70–90°C with reading every 0.2°C and 10 secs hold between reads. Quantitation of gene expression was performed For protein detection following osmotic stress treatment, samples were collected as described for Real Time PCR. In any case, protein was extracted in Laemmli sample buffer and equal protein amount was loaded in each lane. Bromophenol blue was excluded from the sample buffer, in case the protein assay was performed following extraction using sample buffer, and was added before sample loading on the gel. Protein concentration was determined using the SDS/DTT compatible RC DC protein assay system . Western blotting was performed as described in the previous section.A. tumefaciens mediated transformation ROF1 was sub-cloned in the PVKH18En6 binary vector for GFP-tagged protein expression was confirmed by western blotting using a rabbit αντι-GFP polyclonal antibody .2O, seedlings (mainly roots) were placed on top of slides  and allowed to dry overnight before they were kept at −20°C. After removal from −20°C, slides were placed in a dark humid chamber and immediately hydrated with a PBS buffer before applying 2% driselase in PBS and incubate for 45 mins at 37°C. After washing the driselase solution thoroughly, samples were incubated with 10% DMSO, 0.5% NP40 in PBS buffer for 1 hr at room temperature. Following removal of the permebilisation solution, 5% BSA was applied for 1 hr at room temperature followed by 1 hr incubation with αντι-ROF1 (1∶300) in 5% BSA at 37°C. Samples were further incubated overnight at 4°C with the antibody. Following first antibody washing with PBS, incubation was performed in the Cy3 goat anti-rabbit IgG  for 4 hrs at 37°C. Following washing and DAPI  treatment, mounting solution (0.01% moviol in glycerol) was added and samples were observed under a fluorescence or confocal microscope.Entire seedlings (3–4 days old) were submerged in a Paraformaldehyde (PFA) solution prepared in 50 mM Pipes, 5 mM EGTA, 5 mM MgSO4 pH 7.0 KOH, for 1 hr with vacuum infiltration applied during the first 5 mins of incubation. Following washing in PBS and HLocalization studies of GFP-tagged ROF1 as well as immunofluorescence microscopy were performed using fluorescence and Confocal microscopy. Fluorescence microscopy was performed using an Axioskop 40 fluorescence microscope with a A-Plan Varel contrast 40x/0.65 . Images were captured using a ProgRes digital camera system from Jenoptic (Germany). For confocal microscopy two confocal microscopes were used; Live cell imaging was performed using a laser Scanning confocal Microscope TCS SP5 SN: 5100000134 with an HCX PL APO CS 63.0×1.20 water UV lens. A pinhole 1 and 1.6 zoom were used. Scan speed was 400 Hz. Resolution was 1024×1024 with 8 bits/pixel. Excitation was achieved with a 488 Argon laser for the GFP and a DPSS 561 for the RFP. Emission bandwidth for GFP was 504.0 nm–521.2 nm and the emission bandwidth for RFP 574.2 nm - 620.1 nm. Fixed tissue imaging was performed using an HC X PLAPO CS 40.0×0.85 dry lens. Pinhole was 2, zoom 1.7 and resolution 1024×1024. Excitation was achieved using a 405 nm Diode for DAPI and a 561 DPSS for Cy3 red fluorescence. Emission bandwidth for DAPI was 423 nm –475.6 nm and the emission bandwidth for Cy3 567 nm –619.4 nm. Image analysis was performed using the LAS AF software version 2.1.0 as well as the ImageJ (Java) software. Also, a Zeiss LSM 510 confocal microscope with a EC Plan-Neofluar 40x/1.30 oil DIC M27 objective was used. Scan zoom was 2. Resolution was 1024×1024 with 8 bits/pixel. Excitation was achieved with a 488 Argon laser. Emission filters were the BP 505–530 for GFP and LP 615 for chloroplast autofluorescence.−rof1 heterozygous plants on a Columbia background, salk_003069, Sail_276_E11 and WiscDsLox502D06 of the Wisconsin DsLox T-DNA lines, were obtained from NASC. Following screening, seeds obtained from the second generation were used in downstream applications. −rof1, −rof2, rof1−−/2, ROF1OE, and −rof1 complemented mutants on a Wassilevskija background as well as Wassilevskija wild type plants were also used and were a gift from Prof. Adina Breiman Columbia wild type as well as the A. thaliana seeds of the wild type and mutant lines were disinfected with 70% ethanol for 2 mins and 10% sodium hypochloride for 5 mins. Following a 4–5 day treatment at 4°C to break dormancy, they were seeded on 0.8% agar plates with 1x MS/vitamins containing –where required- NaCl or mannitol. For each characterized line 230–270 seeds were spread on a petri dish. Germination kinetics was determined by recording radicle emergence time after transfer from the stratification conditions to the growth chamber. Plates were scanned in an HP scanjet 4800 series and measurements were performed on the scanned images or under a stereoscope.−/2−rof1 seeds was recorded at different time points. Following stratification, seeds were germinated in 24 well plates on whatman paper, in water, under dark conditions, at 22°C in the presence or absence of 0.1 M NaCl and in the presence or absence of 33 µM wortmannin  or 60 µM LY294002  dissolved in DMSO. An equivalent amount of DMSO was added in the case of the inhibitor absence. The experiment was performed in replicates so that at the end of each time point seeds were collected, scanned and their germination percentage was assessed. The entire experiment was repeated 4 times.Germination percentage of WSROF1OE and WSFigure S1ROF1 domain organisation. Organisation of the FKBDs, the TPR and the calmodulin binding domain on the amino-acid sequence of ROF1. The polylysine (KKKLLK) and the DSSRDR motives are indicated.(PDF)Click here for additional data file.Figure S2Structural alignment of ROF1-β-Spectrin. Structural alignment of FKBP12 with β-spectrin, and with the FKBD1, FKBD2 and FKBD3 domains of ROF1. Blue highlight: residues where the IP3 binds to β-spectrin  or residues on the FKBP12 and the FKBD domains of ROF1 predicted with the potential for inositide binding. DSSP: DSSP algorithm for assigning aminoacid secondary structure . L: loop; H: α-helix; E: extended β- structure.(PDF)Click here for additional data file.Figure S3Anti-ROF1 characterization. (A) two different serum extractions (1st and 3rd) were used to detect the ROF1 overexpressed protein (1) and its truncated mutants 3FKTPR (2), 2FKTPR (3) and TPR (4) (see  (4) see . anti-GS(PDF)Click here for additional data file.Figure S4WT and mutant characterization using anti-ROF1. Characterization of individual lines of A. thaliana wild type (Columbia and WS), knock out , WSROF1CM and WSROF1OE.(PDF)Click here for additional data file.Figure S5Germination rate of WT and mutant lines. (A) Germination rate of the Columbia background (Col-WT) and ROF1 knock out (Col-−rof1) plants grown on MS medium, B) on MS medium containing 0.2 M NaCl, C) on MS media containing 0.4 M mannitol. D) Germination rate of the Wassilevskija background plants WS, WS−rof1,WS−rof1,WS−/2−rof1, WSROF1CM and WSROF1OE on MS medium. Y axis: % germination; X axis: time (hours). Three independent characterized lines (in each experiment) were used for each genotype. Results are average of three independent experiments performed for each treatment. Values are means and bars are SDs.(PDF)Click here for additional data file.Table S1Primers used for the introduction of restriction sites (highlighted) and subsequent cloning.(PDF)Click here for additional data file.Table S2Constructs of ROF1 and of its truncated mutants.(PDF)Click here for additional data file.Movie S1Plasmolysis effect on ROF1-GFP localization following 0.25 M NaCl.(AVI)Click here for additional data file.Movie S2Endogenous ROF1 localization using anti-ROF1– control.(AVI)Click here for additional data file.Movie S3Endogenous ROF1 localisation using anti-ROF1–0.2 M NaCl.(AVI)Click here for additional data file.Spectrum Report S1(XLSX)Click here for additional data file.


Opposite effects on P2 and N400 ERP components of list-wide semantic context are discussed in relation to recent proposals on the representational status of RSVP targets at processing stages prior to consolidation in visual short-term memory.Three target words  were embedded in a rapid serial visual presentation (RSVP) stream of non-word distractors, and participants were required to report the targets at the end of each RSVP stream. T2 and T3 were semantically related words in half of the RSVP streams, and semantically unrelated words in the other half of the RSVP streams. Using an identical design, a recent study reported distinct reflections of the T2–T3 semantic relationship on the P2 and N400 components of event-related potentials (ERPs) time-locked to T3, suggesting an early, automatic, source of P2 semantic effects and a late, controlled, source of N400 semantic effects. Here, P2 and N400 semantic effects were examined by manipulating list-wide context. Relative to participants performing in a semantically unbiased context, participants over-exposed to filler RSVP streams always including semantically related T2/T3 words reported a  In the vast majority of cases, cognitive studies of unconscious cognition have made use of techniques devised to prevent conscious access to one visual stimulus, traditionally referred to as Recent theorization in this field has highlighted a critical subdivision of these techniques according to the way in which conscious access to prime stimuli is limited Though often used to investigate distinct aspects of the functional and neural processing architecture, behavioral and electrophysiological results in the masked priming and RSVP/AB fields have separately converged on a set of assumptions concerning the representational status of unconscious visual stimuli. One such assumption is that unconscious stimuli access post-sensory stages of processing very rapidly Furthermore, both fields have contributed evidence imposing a revisitation of the theoretical link between unconscious perception and automatic processing. Contrary to former proposals of unconscious stimuli as naturally bound to automatic processing  characterized by its well-established susceptibility to semantic modulations, namely, N400  and target (T3) words were unmasked and clearly visible, via omission of leading non-word distractors. In one condition, filler RSVP trials always included semantically related T2/T3 words, and in the other condition filler RSVP trials always included semantically unrelated T2/T3 words. The two conditions were administered to two different groups of participants. List-wide context effects were compared between the two groups of participants by considering standard RSVP trials only, namely, those RSVP trials that were in common between the two groups.Curiously, the influence of list-wide context effects on the processing of unconscious stimuli has never been tested using the RSVP technique. Such test is the scope of the present investigation. The starting point was the study of Pesciarelli, Kutas, Dell'Acqua, Peressotti, Job, and Urbach The manipulation of list-wide semantic context was included in the present design in order to disentangle two opposite hypotheses about the functional interplay between P2 and N400 semantic modulations reported by Pesciarelli et al. vis-a-vis identical trials accompanied by fillers always including semantically unrelated T2/T3. P2 semantic effects should parallel the expected result of a magnification of N400 semantic priming effects for participants exposed to filler RSVP trials always including semantically related T2/T3. As for N400, P2 semantic effects in standard RSVP trials should also increase when the coherence of the semantic context is increased by intermixing standard RSVP trials and filler RSVP trials always including semantically related T2/T3. Behaviorally, the results should adhere to this hypothetical ERP picture, and be compatible with prior indications of the independence of semantic processing of T2/T3 from a T1-elicited AB, as well as with the cited findings of Bodner and Masson One hypothesis is that semantic effects on P2 were mere precursors of N400 semantic effects. On this hypothesis, participants exposed to filler RSVP trials always including semantically related T2/T3 should report a magnification of N400 semantic priming effects in standard RSVP trials. This would be so because N400 is held to index a semantic mismatch between T3 and semantic context established by a consciously perceived T2. If the coherence of the semantic context were increased by filler RSVP streams always including semantically related T2/T3, then the magnitude of an ERP response to a semantic mismatch between T2 and T3 embedded in standard RSVP trials should also increase, bringing about an amplification of N400 semantic effects in standard RSVP trials satiation, usually observed in the form of a progressive semantic responsiveness reduction – or habituation – as the proportion of semantically overlapping stimuli is increased , with an age ranging from 19 to 31 years (mean = 21 years) volunteered to participate in the present experiment. Participants were right-handed, native Italian speakers, without a history of neurological or mental disorders, with normal or corrected-to-normal visual acuity, and normal color vision. Written consent was obtained from each participant before the beginning of the experiment, as required by the Regulation of the Ethical Committee regarding cognitive/behavioral studies involving adult human participants.standard RSVP trials were one-hundred and twenty Italian 4-letters and 5-letters words selected from the VELI corpus The stimuli used for filler RSVP trials were 80 4-letters to 6-letters words selected from the same corpus as T3 words. Each T3 word was paired with two semantically related T2 words and two semantically unrelated T2 words, and 80 T1 words were selected to be semantically unrelated to both T2 and T3 words. Four separate lists of filler RSVP trials were generated by including identical T1 and T3 words. Two lists included 80 semantically related T2–T3 words, and two lists included 80 semantically unrelated T2–T3 words. Standard and filler RSVP trials included T1, T2, and T3 words and 19 non-words, each composed of a random sequence of 4 to 6 consonants. The structure of standard and filler RSVP trials was identical, except for the omission of the distractors in T1-1, T2-1, and T3-1 positions in filler RSVP trials. When displayed on screen, the maximum height and width of the stimuli were 0.5 and 3.2 degrees of visual angle. The background of the screen  was uniformly filled in gray. Distractors were displayed in black, T1 in green, and T2 and T3 in equiluminant red, each for a constant duration of 84 ms . T1, T2, and T3 always occupied the fifth, eighth, and fifteenth positions in the sequence of distractors. The stimulus onset asynchrony (SOA) between T1 and T2 was thus 252 ms, and the SOA between T2 and T3 SOA was 588 ms.The stimuli used for An example of the sequence of events on one RSVP trial is illustrated in Each trial began with three fixation crosses (‘+++’) at the center of the screen. Trials were self-administered using the spacebar on the keyboard connected to the same apparatus controlling stimuli presentation. A 700 ms blank interval following a spacebar press preceded the beginning of each RSVP stream. At the end of the RSVP stream, participants were instructed to type in, without time/speed pressure, T1, T2 and T3 in the same order as they appeared in the RSVP stream. Feedback was given at the end of each trial by replacing the ‘+’ in the position congruent with target order  with a ‘−’ sign. The experiment consisted of 240 standard RSVP trials and 160 filler RSVP trials, randomly distributed in 10 blocks of 40 trials preceded by 1 block of 15 practice trials. Each participant was exposed to standard RSVP trials. Half of the participants were exposed to filler RSVP trials including only semantically unrelated T2 and T3 words, and the other half to filler RSVP trials including only semantically related T2 and T3 words.Electroencephalographic (EEG) activity was recorded at a 250 Hz sampling rate from 19 tin electrodes held in place at standard 10/20 positions including Fp1, Fp2, Fz, F3, F4, F7, F8, C3, C4, Cz, P3, P4, Pz, O1, O2, T3, T4, P7, and P8 sites. Electrooculogram (EOG) activity was recorded from electrodes placed at the outer canthus and below each eye. EEG and EOG activities were referenced online to the left mastoid, and re-referenced off-line to the right and left mastoids. Data were amplified and bandpass filtered at 0.01–100 Hz, keeping impedance at each electrode site below 5 KΩ. Trials with artifacts due to eye movements, excessive muscle activity or amplifier saturation were eliminated off-line before event-related potential (ERP) estimation, using a 100 ms pre-stimulus averaged activity as baseline. ERPs affected by eye blinks were corrected using the algorithm proposed by Gratton, Coles, and Donchin Only data from 24 participants  were retained in the following analyses. Data from 12 participants had to be discarded because less than ten trials per cell of the design were left after removal of artifacts and standard RSVP trials associated with incorrect report of T1 or T3. Behavioral analyses were carried out on data from standard and filler RSVP trials, these latter differing in the two groups of tested participants, with one group exposed to semantically related filler RSVP trials , and the other group exposed to semantically unrelated filler RSVP trials . The ERP analyses considered only results from standard RSVP trials.F = 98.5, p<.0001, p2η = .81, of trial type, F = 280.9, p<.0001, p2η = .92, and a significant interaction between these factors, F = 123.9, p<.0001, p2η = .84. A separate analysis on filler RSVP trials showed that the proportion of correct responses did not differ across T1, T2, and T3 (F<1). Separate t-tests contrasting each combination of targets in standard RSVP trials showed a reduced proportion of correct responses to T2 and T3 relative to T1, t(23) = 13.5, p<.0001; t(23) = 2.8, p<.01, and a reduced proportion of correct responses to T2 relative to T3, t(23) = 13.0, p<.0001, consistent with a T1-triggered AB effect affecting T2.On average, the mean proportion of correct responses to T1, T2, and T3 was .90 in filler RSVP trials. The mean proportion of correct responses to T1, T2, and T3 was .74, .39, and .68, respectively, in standard RSVP trials. Individual mean values were submitted to ANOVA considering target  and RSVP trial type (standard vs. filler) as within-participant factors. The ANOVA indicated significant main effects of target, Standard RSVP trials were submitted to an additional ANOVA considering RP group (high vs. low) as a between-participant factor, and T2–T3 semantic relatedness  as a within-participant factor. The analyses were conducted separately for each target. A summary of the results is illustrated in Fs<1). The ANOVAs on T2 and T3 were conducted on data from standard RSVP trials associated with a correct report of T1.The ANOVA on the proportion of correct responses to T1 found no significant factor effects  = 28.6, p<.0001, p2η = .57, indicating modest priming effects . Priming effects on T2 by T3 are not unusual using this type of designs requiring memory maintenance and delayed report of targets, and are likely to reflect a bias on the part of participants to report and/or guess correctly a T2 semantically related to T3 on a small fraction of standard RSVP trials.The ANOVA on the proportion of correct responses to T2 revealed a significant main effect of T2–T3 relatedness, F = 64.4, p<.0001, p2η = .75, of T2–T3 relatedness, F = 26.6, p<.0001, p2η = .55, and a significant interaction between these factors, F = 20.5, p<.0001, p2η = .48. No other factor or interaction were significant in this analysis . As The ANOVA on the proportion of correct responses to T3 considered T2 status (missed vs. correctly reported) as an additional within-participant factor. The analysis revealed a significant main effects of T2 status, Individual ERP amplitude values in the P2 range  were submitted to an ANOVA considering RP group (high vs. low) as between-participant factor, and T2 status (missed vs. correctly reported), T2–T3 relatedness and electrode location as within-participant factors.F = 4.3, p<.05, p2η = .15, suggesting that semantic effects were generally larger in trials in which T2 was reported relative to trials in which T2 was missed, and the other between T2–T3 relatedness and group, F = 4.7, p<.04, p2η = .18, suggesting that semantic effects on P2 amplitude were reliable only in the low RP group. Separate analyses on P2 amplitudes were conducted for standard RSVP trials broken down by T2 status. A summary of the results when T2 was correctly reported is illustrated in The ANOVA revealed two significant interactions, one between T2–T3 relatedness and T2 status, F = 4.3, p<.05, p2η = .15. An additional analysis conducted on P2 amplitude values recorded on T2-correct standard RSVP trials indicated a significant main effect of T2–T3 relatedness for the low RP group, F = 4.9, p<.05, p2η = .2, but not for the high RP group, F<1. A summary of the results when T2 was missed is illustrated in When T2 was correctly reported, the ANOVA revealed a significant interaction between T2–T3 relatedness and group, , F = 3.2, p<.05, p2η = .15, of electrode location, F = 7.0, p<.007, p2η = .24, indicating a larger P2 amplitude at frontal electrode sites, and a significant interaction between T2–T3 relatedness and RP group, F = 4.8, p<.05, p2η = .18. An additional analysis conducted on P2 amplitude values recorded on T2-missed standard RSVP trials indicated a significant main effect of T2–T3 relatedness for the low RP group, F = 11.0, p<.005, p2η = .5, but not for the high RP group, F<1.When T2 was missed, the ANOVA revealed significant main effects of T2–T3 relatedness, To further explore the interactions obtained, net priming effects on T3-locked P2 amplitude values were calculated by subtracting ERPs recorded on standard RSVP trials including semantically unrelated T2–T3 targets from ERPs recorded on standard RSVP trials including semantically related T2–T3 ERPs. These difference ERPs are illustrated as scalp topographic maps in F = 4.4, p<.05, p2η = .16 and F = 4.8, p<.04, p2η = .17, respectively, reflecting non-nil P2 semantic effects for the low RP group only.Individual difference P2 amplitude values were submitted to ANOVA, considering RP group (high vs. low) as between-participant factor and electrode location as within-participant factor. As can be seen from t-tests indicated that semantic effects on P2 amplitude were evident for the low RP group in both T2-correct and T2-missed standard RSVP trials, t(12) = 2.3, p<.04, and t(12) = 2.1, p<.05, respectively, while in the high RP group no evidence of semantic priming in the form of non-nil P2 difference activity was observed  were submitted to ANOVA considering RP group (high vs. low) as between-participant factor, and T2 status (missed vs. correctly reported), T2–T3 relatedness, and electrode location as within-participant factors.F = 4.6, p<.04, p2η = .17, and the other between T2–T3 relatedness and group, F = 5.5, p<.03, p2η = .2. The former interaction revealed the fact that N400 effects were observed only for T2 reported trials, the latter that these effects were larger for the high RP than for the low RP group. Separate analyses on N400 amplitudes were conducted for standard RSVP trials broken down by T2 status. A summary of the results when T2 was correctly reported is illustrated in The ANOVA revealed two significant interactions, one between T2–T3 relatedness and T2 status, F = 13.0, p<.002, p2η = .37, of electrode location, F = 7.9, p<.0001, p2η = .26, and a significant interaction between T2–T3 relatedness and RP group, F = 3.8, p<.05, p2η = .15, reflecting magnified classic, centro-parietal, N400 semantic priming effects for the high RP group relative to the low RP group.When T2 was correctly reported, the ANOVA indicated significant main effects of T2–T3 relatedness, F = 4.9, p<.04, p2η = .18, but not when T2 was missed, reflecting larger N400 semantic effects for the high RP group when T2 was correctly reported, and nil N400 semantic effects when T2 was missed  = 2.6, p<.03 and t(12) = 4.2, p<.001, respectively. On T2-missed trials, no significant effect of T2–T3 relatedness was detected, ts< = 1. As is evident in Net priming effects on T3-locked N400 amplitude values were calculated by subtracting ERPs recorded on standard RSVP trials including semantically unrelated T2–T3 targets from ERPs recorded on standard RSVP trials including semantically related T2–T3 ERPs. These difference ERPs are illustrated as scalp topographic maps in s missed  and 6. WThe present investigation moved its steps from observing a general analogy between results produced using the masked priming and RSVP techniques when the influence of top-down factors on unconscious processing was considered. Previous work showed that this analogy was observed when manipulating temporal expectation, order predictability, and task-set using both techniques e.g., . List-wiBehaviorally, the proportion of correct target words report, though showing clear AB effects on T2, did not reflect context effects on T3. Furthermore, semantic priming effects on T3 were detected in standard RSVP trials only when T2 was correctly reported. Smaller priming effects were also detected on T2, reflecting a bias to report semantically related words at a stage of retrieval of consolidated T3 words from visual short-term memory, and not at encoding stages during RSVP processing. Compatibly with this view, T2-locked ERP responses showed no effects of the semantic relation between T2 and T3 in standard RSVP trials.encoding stages. Behavioral estimates, in contrast, are prone to the influence of factors affecting memory, such as fading of the memory traces, report order confusion, and guessing, especially when one or more targets were missed owing to an AB effect. An additional limitation of behavioral estimates of priming effects in the present context would also arise from the relative duration of priming effects, which have been shown to be sometimes short-lived Inconsistently with the predictions, null priming effects were reported for trials in which T2 was missed not only for the high RP group, but also for the low RP group. As already pointed out in the dilution of T3-locked P2 semantic effects and a magnification of T3-locked N400 semantic effects.The electrophysiological results suggest both the T3-locked ERP components of interest were modulated by list wide context. N400 priming effects were of greater magnitude in the high RP group than in the low RP group on T2-correct standard RSVP trials  of stimuli of a given alphanumeric class was increased, resulting in a reduced proportion of correct target responses when targets were clustered temporally relative to a condition in which they were separated by long intervals prime, words. Clearly visible T3, target, words were displayed consistently outside the AB time-window, and semantic priming effects were estimated both in the form of behavioral priming effects on T3 report accuracy, and in the form of T3-locked ERP waveforms' changes as a function of T2–T3 semantic relation. Vachon and Jolicœur prime stimulus generating a semantic context for an unlimited duration prior to each RSVP stream, and what was resource-limited via a T1-triggered AB perturbation was an unconscious T2, target, words.Congruently with work overviewed in the N400 semantic effects have a top-down origin, reflecting T3 integration within a pre-established, T2-influenced, semantic context. There is ground to believe that latency and peculiarities of this component may be tied to cross-talking among anterior brain areas, including fronto- and dorso-lateral circuitries and their symmetric connections with the anterior parts of both temporal lobes e.g., . The idemasked prime/target words. In the present study, filler RSVP trials displayed prime/target words that were clearly visible. Furthermore, Bodner's and Masson It is not straightforward to forge a link between the present findings and prior work in which list-wide semantic context was varied using masked priming Perhaps, this may be one of those cases where either technique (RSVP and masked priming) discloses its inherent limitations. Pros aside, one con of the masked priming technique is the impossibility to monitor the visibility of primes on a per-trial basis The results here reported show that the semantic priming in the AB is the result of two independent processes, one evident as an early modulation of the ERPs, 200 ms after target presentation, and reflecting bottom-up semantic activation triggered by the presentation of a target word. The other process manifested itself as later N400 amplitude modulations, likely reflecting context integration processes. Both processes were shown to be permeable to list-wide context. The early effect, which was independent of prime visibility, is present when the system is not biased in the processing of the RSVP trial and it disappears in conditions in which the task induces participants to generate expectations on the semantic relationship between the target words they have to report. The late effect, which was observed only when the prime was consciously reported, is magnified in such conditions. The dissociation between early and late semantic effects is inspiring for models of word recognition, as it suggests that a subset of processing stages up to and including an early stage of semantic processing are ballistically activated upon presentation of linguistic stimuli. The present results suggest also that these automatic stages are partially independent from those required to generate a word representation available for conscious report.