GRM2 and GRM3, respectively) are therapeutic targets for several psychiatric disorders. GRM3 may also be a schizophrenia susceptibility gene. mGluR2−/− and mGluR3−/− mice provide the only unequivocal means to differentiate between these receptors, yet interpretation of in vivo findings may be complicated by secondary effects on expression of other genes. To address this issue, we examined the expression of NMDA receptor subunits  and glutamate transporters (EAAT1-3), as well as the remaining group II mGluR, in the hippocampus of mGluR2−/− and mGluR3−/− mice, compared with wild-type controls. mGluR2 mRNA was increased in mGluR3−/− mice, and vice versa. NR2A mRNA was increased in both knockout mice. EAAT1 (GLAST) mRNA and protein, and EAAT2 (GLT-1) protein, were reduced in mGluR3−/− mice, whereas EAAT3 (EAAC1) mRNA was decreased in mGluR2−/− mice. Transcripts for NR1 and NR2B were unchanged. The findings show a compensatory upregulation of the remaining group II metabotropic glutamate receptor in the knockout mice. Upregulation of NR2A expression suggests modified NMDA receptor signaling in mGluR2−/− and mGluR3−/− mice, and downregulation of glutamate transporter expression suggests a response to altered synaptic glutamate levels. The results show a mutual interplay between mGluR2 and mGluR3, and also provide a context in which to interpret behavioral and electrophysiological results in these mice.Group II metabotropic glutamate receptors (mGluR2 and mGluR3, also called mGlu2 and mGlu3, encoded by  GRM2) and mGluR3 . Both are G–protein coupled receptors, whose activation inhibits adenylate cyclase and decreases cAMP formation. Their primary functions are thought to be as inhibitory autoreceptors and thence modulation of glutamatergic signaling  containing the dorsal hippocampus were collected on a Shandon cryostat at −20°C. Sections were collected on Superfrost Plus slides , three per slide, and stored at −70°C.mGluR2escribed . Brains 35S]dATP using the enzyme terminal deoxynucleotidyl transferase (TdT) . Labeled probe was purified from unincorporated nucleotides using Nick Columns  according to manufacturer's instructions. Sections were incubated overnight at 35°C in 225 μl hybridization buffer  containing 3× 106 counts per minute of labeled probe and 20 μM dithiothreitol. Posthybridization washes were conducted in 1× SSC for 3× 20 min at 55°C, followed by 2× 60 min at room temperature. Hybridized sections were apposed to Kodak Biomax MS film together with 14C microscales  for an optimal length of time determined by trial experiments (In situ hybridization histochemistry was carried out as described by eriments .35S]–labeled donkey antirabbit IgG (Amersham) at 1:500 in PBS–T and 1% donkey serum, for 1 h at room temperature. Following washes in PBS and MilliQ Ultrapure water to remove unbound secondary, slides were left to air–dry, before being placed against Kodak Biomax MS film with 14C microscales for 7 days.Sections were fixed in 4% paraformaldehyde/PBS for 5 min, and then washed twice in PBS. Nonspecific binding was blocked by incubating the slides in 200 μl of normal donkey serum , diluted 1:10 in PBS with 0.3% Triton X–100 (PBS–T), for 30 min at room temperature. Sections were incubated overnight at 4°C with rabbit polyclonal anti–EAAT1  diluted 1:200 in PBS–T, or with rabbit polyclonal anti-EAAT2 (Abcam) diluted 1:200 with PBS–T. Omission of primary antibody was used as a control. After three PBS washes to remove unbound primary, sections were incubated with 0.2 μCi/ml [35SnCi/g tissue equivalents using the 14C microscales. In the case of the neuronally expressed mRNAs, namely EAAT3 and the NMDAR subunits, measurements were taken over the stratum granulosum of the dentate gyrus, and over the stratum pyramidale of CA3 and CA1 subfields. In contrast, EAAT1 and EAAT2 mRNA and immunoreactivity were measured over a broadband distributed across the various strata of each subfield, reflecting the diffuse localization of these glial-expressed genes.Autoradiographic films were developed under safelight conditions and measured using the MCID Elite v7.0 image analysis system . Optical density values were calibrated to −/− and mGluR3−/− mice were conducted at different times (and compared with separate control groups), and were therefore analyzed separately.All data were explored for normality using the Kolmogorov–Smirnov test . Group differences (knockout vs. wild-type) in the hippocampal expression of each gene were analyzed using ANOVA across all subfields examined; if a significant effect of genotype or genotype–by–subfield interactions were observed, separate analyses were performed in each subfield. Experiments on the mGluR2The regional hippocampal distribution of each transcript was as reported previously: mGluR2 , mGluR3 Hybridization signals for mGluR2  and mGlu−/− mice compared with their wild–type littermates  = 14.81; P = 0.003; mGluR2 mRNA was increased in the mGluR3−/− mice compared with their wild–type littermates  = 23.14; P = 0.003) mGluR3 mRNA was increased in the mGluR2−/− mice there was no effect of genotype and no genotype–by–subfield interaction . By contrast, mGluR3−/− mice showed a main effect of genotype  = 15.22; P < 0.001) as well as a trend genotype–by–subfield interaction  = 3.68; P = 0.070). Subsequent analysis revealed that EAAT1 mRNA was reduced in both the dentate gyrus  = 9.77; P = 0.011) and CA1  = 7.38; P = 0.022) of mGluR3−/− mice compared with their wild-type littermates   ; Fig. 1D−/− mice, EAAT3 mRNA showed a main effect of genotype  = 14.80; P < 0.001) but no interaction with subfield . One–way ANOVA confirmed that EAAT3 mRNA was reduced throughout the mGluR2−/− hippocampus, significantly in CA1  = 13.27; P = 0.008) and at trend significance in dentate gyrus  = 4.69; P = 0.067) and CA3  = 3.98; P = 0.086)  (In mGluR2= 0.086) . EAAT3 m > 0.20) ;.−/− mice, there was no effect of genotype and no genotype–by–subfield interaction . In mGluR3−/− animals, there was an effect of genotype  = 15.62; P = 0.001), but no genotype-by-subfield interaction . EAAT1 immunoreactivity was reduced in a fairly uniform manner throughout the mGluR3−/− hippocampus  = 7.60; P = 0.02; CA1: F = 8.03; P = 0.018)  ; Fig. 1G−/− animals, with no effect of genotype, and no genotype-by-subfield interaction . In mGluR3−/− animals, by contrast, there was a main effect of genotype  = 24.78; P < 0.001) although no genotype–by–subfield interaction . Consistent with these findings, EAAT2 immunoreactivity was reduced in all subfields of the mGluR3−/− hippocampus  = 5.98; P = 0.035; CA3: F = 5.86; P = 0.036; CA1: F = 15.60; P = 0.003) (EAAT2 immunoreactivity was not changed in mGluR2= 0.003) ; Fig. 1H−/− mice, there was no effect of genotype on NR1 mRNA  but there was a genotype–by–subfield interaction  = 3.98; P = 0.033). The latter reflected a small decrease in the knockouts in CA3 and an increase in CA1; however, neither change was significant in the post hoc test  = 2.97; P = 0.119; CA1: F = 2.86; P = 0.125). NR1 mRNA was unaltered in mGluR3−/− mice, with no effect of genotype or no genotype–bysubfield interaction  (In mGluR2P > 0.2) .−/− mice, there was an effect of genotype on NR2A mRNA  = 4.44; P = 0.045), but no interaction with subfield  = 2.09; P = 0.145). One–way ANOVA confirmed that NR2A mRNA was increased in the dentate gyrus  = 10.17; P = 0.013), but not significantly in CA3 or CA1   = 11.14; P = 0.002), but no genotype–by–subfield interaction . Post hoc tests showed NR2A mRNA increased in the dentate gyrus  = 5.73; P = 0.04) and CA3  = 5.35; P = 0.046) but not CA1  = 1.89; P = 0.203) of mGluR3−/− mice (In mGluR2 > 0.20)  and 3A. −/− mice ; Fig. 3BF < 1.7; P > 0.20)  .−/− and mGluR3−/− mice has therefore been an important step forward. Use of these mice has, for example, showed a prominent role for mGluR2 in mossy fiber long term depression (−/− and mGluR3−/− mice. Our results show that knockout of either gene leads to upregulation of the remaining group II mGluR and upregulation of the NR2A subunit of the NMDA receptor, as well as to downregulation of glutamate transporters. However, the identity of the affected transporter differs between the mice: loss of mGluR2 leads to a decrease of the neuronal EAAT3, whereas deletion of mGluR3 results in reduction of the glial transporters EAAT1 and EAAT2.Determining the individual properties of the two group II mGluRs, mGluR2 and mGluR3, has been problematic because of a dearth of suitable selective ligands and antibodies. The generation of mGluR2pression  and for pression , and forpression . However−/− mice, and vice versa, which suggests a mutual—albeit partial—compensatory response to the loss of a group II mGluR, which may contribute to the relatively mild behavioral phenotype seen in both mice, particularly mGluR3 knockouts. Equally, the increase was only modest in percentage terms, which may explain why deletion of either gene is not without demonstrable electrophysiological and behavioral consequences (for review, see A notable finding was the increase of mGluR3 expression in mGluR2−/− and mGluR3−/− mice, the NR2A subunit was selectively upregulated, with no alteration in either NR1 or NR2B subunit mRNAs. This finding follows a previous report that the mGluR2/3 agonist APDC enhances NMDA-evoked responses in dissociated rat prefrontal cortex neurons, predominantly via NR2A-containing receptors (−/− and mGluR3−/− mice contain an NR2A rather than NR2B subunit. This difference will likely influence their properties in several ways, including calcium flux and channel opening kinetics, postsynaptic protein coupling, and perhaps long-term potentiation (Group II mGluRs have complex interactions with, and effects upon, NMDA receptors , and it eceptors . Our datntiation . The netA major role of group II mGluRs, especially mGluR2, is as inhibitory autoreceptors . Our res−/− mice but unchanged in mGluR2−/− mice. In contrast, the neuronal glutamate transporter EAAT3 was reduced in the mGluR2 knockouts but unchanged in their mGluR3−/− counterparts. It is tempting to relate this profile to the fact that mGluR2 is exclusively neuronally expressed (The second point concerns the identity of the specific transporters affected in the two mouse lines. The glial glutamate transporters EAAT1 and EAAT2 were downregulated in mGluR3xpressed , whereasxpressed . It is axpressed . Third, xpressed .−/− vs. mGluR3−/− mice supports the emerging evidence for specific differences in the expression and properties of the two receptors (−/− and mGluR3−/− mice will contribute to the understanding of the relative contribution of each receptor in the aetiopathogenesis and pharmacotherapy of schizophrenia and other psychiatric disorders in which they are increasingly implicated.In summary, this study has shown that deletion of either mGluR2 or mGluR3 genes produces changes in the hippocampal expression of several other genes involved in glutamatergic neurotransmission. Such changes should be considered when interpreting the phenotype of the mice in behavioral, electrophysiological, and pharmacological studies. The fact that the changes are broadly similar in both mice is consistent with the grouping of mGluR2 and mGluR3 together as group II mGluRs, and for a significant overlap in their functional roles. Equally, the distinct profile of glutamate transporter alterations in mGluR2eceptors . Further


C2-symmetric bisamidines 8 have been tested as chiral Brønsted bases in the Diels-Alder reaction of anthrones and N-substituted maleimides. High yields of cycloadducts and significant asymmetric inductions up to 76% ee are accessible. The proposed mechanism involves proton transfer between anthrone and bisamidine, association of the resulting ions and finally a cycloaddition step stereoselectively controlled by the chiral ion pair. Moderate to excellent stereoselectivities of products 3 have been reported using pyrrolidines 4 [5 [6 [7 in up to 70% ee, in spite of their limited Brønsted-basicity [The cycloadditions of anthrones 7 and bisamidines 8. Bisamidines 8, readily accessible from malonodinitrile in two steps, prefer the conjugated tautomeric form (enamine-imine) in the monoprotonated state, which is characterised by an almost planar structure [Our study was motivated by the structural similarity of bisoxazolines tructure   by bi8a [9 and bisimidate 10 in refluxing ethanol. Simple extraction in the presence of Na2CO3 afforded the neutral bases 8b–c and ent-8d in almost quantitative yield. The S,S configurated diamines 9b and 9c were prepared from L-(+)-tartaric acid  via the vicinal diazide using Saalfrank's procedure [9d was purchased as the dihydrochloride salt and then deprotonated by aqueous sodium hydroxide. As an “artefact” of the sequence rule, the S,S configurated diamine 9d leads to bisamidine ent-8d  and 1c  resulted from regioselective reductions of 1,8-dichloroanthraquinone [2 could be introduced by a Mitsunobu alkylation of maleimide [The anthrones aquinone –11. Alipaleimide . Alternaaleimide –14.1a and 2a was examined first by 1H NMR in CD2Cl2 at room temperature. In the absence of catalyst, no product could be observed after 4 days. 5 mol% of the bisamidinium salt 8a·H+ with tetrakisphenyl)borate (TFPB-) as weakly coordinating anion resulted in 7% yield of 3a after 4 h. In contrast, only 1 mol% of the free Brønsted base 8a led to a high rate increase in the first 30 min. After 90 min no further conversion was observed indicating product inhibition  and anthrone (1a). Using 0.25 equiv of catalyst at room temperature, isolated yields between 71% and 86% were obtained after 30 min. The best enantioselectivity, albeit low, was induced by amidine 8c (24% ee). As expected, in the presence of catalyst ent-8d product ent-3a was formed preferentially . Even higher yields were accessible in aromatic solvents, however, at the price of reduced stereoselectivity  retarded the cycloaddition but did not change enantioselectivities. After extended reaction times, excellent yields were still observed. Up to 39% ee was finally obtained at −70 °C. However, such conditions resulted in lower yields, even with increased catalyst loads and further extended reaction times. Best results, 96% yield and 36% ee with only 10 mol% of catalyst, were found at −40 °C . With other sterically hindered dienophiles such as N-tert-butylmaleimide (2c), the level of ee remained low (entry 4). The halogen-substituted anthrones 1b–c did not react with 2i at −70 °C. At room temperature, however, 1b and 2i were efficiently transformed into 3m by catalyst 8a with 76% yield and 54% ee. A single recrystallisation step afforded an almost enantiopure product (96% ee). The R,R configuration of compound 3m was determined by anomalous X-ray diffraction using a single crystal of 3m with 96% ee  shows significant proton transfer when bisamidine 8a is added to anthrones 1a, 1b, or 1c. A chiral contact ion pair A is formed and controls the stereochemical course of the Diels-Alder reaction with maleimides. In the last step, the catalyst-product-complex B dissociates and regenerates the unprotonated bisamidine.A mechanistic rationalisation is proposed in  2a 10, –8) and 8 and 8Ka H+ . FurtheC2-symmetric bisamidines were shown to be potent chiral Brønsted base catalysts for the Diels-Alder reaction of N-substituted maleimides and anthrones. Compared to bisoxazolines 7, much shorter reaction times under comparable conditions were sufficient with the more basic bisamidine catalysts 8 . Keeping in mind that even the less selective bisamidine 8a could induce up to 76% ee in favorable cases, replacing the phenyl moieties of 8a by t-Bu is an attractive option for future studies on bisamidine-mediated organocatalytic transformations.File 11H- and 13C-NMR spectra of anthrones 1, maleimides 2, Diels-Alder adducts 3, bisamidine hydrochlorides 8b–d·H+·Cl-, neutral bisamidines 8b–d and diamines 9b–c, plus copies of chromatograms obtained with chiral columns.Supporting information features characterisation data and copies of File 23kX-Ray data of compound File 33mX-Ray data of compound


Two consanguineous Pakistani families with autosomal recessive primary congenital glaucoma were recruited to identify the disease locus.Ophthalmic examinations including slit lamp biomicroscopy and applanation tonometry were employed to classify the phenotype. Blood samples were collected and genomic DNA was extracted. A genome wide scan was performed on both families with 382 polymorphic microsatellite markers. Two point LOD scores were calculated, and haplotypes were constructed to define the disease interval.Clinical records and ophthalmic examinations suggest that affected individuals in families PKGL005 and PKGL025 have primary congenital glaucoma. Maximum two-point LOD scores of 5.88 with D14S61 at θ=0 and 6.19 with D14S43 at θ=0 were obtained for families PKGL005 and PKGL025, respectively. Haplotype analysis defined the disease locus as spanning a 6.56 cM (~4.2 Mb) genetic interval flanked by D14S289 proximally and D14S85 distally.Linkage analysis localizes autosomal recessive primary congenital glaucoma to chromosome 14q24.2–24.3 in consanguineous Pakistani families. Glaucoma is the second leading cause of vision loss, and approximately 15% of blindness worldwide result from glaucoma . GlaucomGLC3A , GLC3B , and GLC3C (14q24.3), with pathogenic mutations only reported in the human cytochrome P450 gene  [CYP1B1 mutations have also been reported in patients with early onset of primary open-angle glaucoma. Additionally, autosomal dominant forms of PCG have been reported, and MYOC, a gene associated with primary open-angle glaucoma, is reported to play a possible role in the pathogenesis [Although rare, primary congenital glaucoma (PCG) is the most common form of glaucoma in infants with an overall occurrence of 1 in 10,000 births . It is pnd GLC3C q24.3, wi 601771) . It is sogenesis ,17.The current study is aimed to explore the genetic basis of PCG in the Pakistani population. A genome wide linkage analysis was performed, which showed segregation of PCG in two consanguineous Pakistani families. Microsatellite markers on chromosome 14q24.2–24.3 cosegregated with the disease phenotype and defined the disease locus as spanning a 6.56 cM (~4.2 Mb) genetic interval flanked by D14S289 proximally and D14S85 distally.Thirteen consanguineous Pakistani families with PCG were recruited to participate in this study to understand the genetic aspects of glaucoma at the Centre of Excellence in Molecular Biology . Institutional Review Board approval was obtained for this study from the Centre of Excellence in Molecular Biology (CEMB). The participating subjects gave informed consent consistent with the tenets of the Declaration of Helsinki. Both families described in this study are from the Punjab province of Pakistan.A detailed medical history was obtained by interviewing family members. All of the ophthalmic examinations including slit lamp biomicroscopy and applanation tonometry were completed at the Layton Rahmatullah Benevolent Trust (LRBT) hospital . Diagnosis of PCG was based on established criteria that include measurement of IOP, measurement of corneal diameters, and observation of optic nerve head where possible as well as symptoms of corneal edema including photophobia, buphthalmos, cloudy cornea, and excessive tearing. Patients with elevated IOP associated with other systemic or ocular abnormalities were excluded. Blood samples were collected from affected and unaffected family members. DNA was extracted by a non-organic method as described by Grimberg et al. .2, and 0.2 U of Taq DNA polymerase . Amplification was performed in a GeneAmp PCR System 9700 (Applied Biosystems). Initial denaturation was performed for 5 min at 95 °C followed by 10 cycles for 15 s at 94 °C, for 15 s at 55 °C, and for 30 s at 72 °C, and then 20 cycles for 15 s at 89 °C, for 15 s at 55 °C, and for 30 s at 72 °C. The final extension was performed for 10 min at 72 °C followed by a final hold at 4 °C. PCR products from each DNA sample were pooled and mixed with a loading cocktail containing HD-400 size standards (PE Applied Biosystems). The resulting PCR products were separated in an ABI 3100 DNA Analyzer and analyzed by using the GeneMapper software package (Applied Biosystems).A genome wide scan was performed with 382 highly polymorphic fluorescent markers from the ABI PRISM Linkage Mapping Set MD-10  having an average spacing of 10 cM. Multiplex polymerase chain reactions (PCRs) were performed in a 5 μl mixture containing 40 ng genomic DNA, various combinations of 10 μM dye labeled primer pairs, 0.5 μl 10X GeneAmp PCR Buffer II, 0.5 μl 10mM Gene Amp dNTP mix, 2.5 mM MgClLINKAGE Program Package  [Marshfield database. For the initial genome scan, equal allele frequencies were assumed while for fine mapping, allele frequencies were estimated from 100 unrelated and unaffected individuals from the Punjab province of Pakistan.Two point linkage analysis were performed using the FASTLINK version of MLINK from the dge, UK) ,20. Maxiprimer3 program . Amplifications were performed in 25 μl reactions containing 50 ng of genomic DNA, 2.5 μl 10X GeneAmp PCR Buffer II, 8 pmoles of each primer, 2.5 mM dNTP, 2.5 mM MgCl2, and 0.2 U Taq DNA polymerase. Amplification was performed in a GeneAmp PCR System 9700 (Applied Biosystems). PCR amplification consisted of a denaturation step at 96 °C for 5 min followed by 40 cycles, each cycle starting at 96 °C for 45 s followed by 57 °C for 45 s and 72 °C for 1 min. PCR products were analyzed on 2% agarose gel and purified by ethanol precipitation. The PCR primers for each exon were used for bidirectional sequencing using Big Dye Terminator Ready reaction mix (Applied Biosystems) according to manufacturer instructions. Sequencing products were precipitated and resuspended in 10 μl of formamide and denatured at 95 °C for 5 min. Sequencing was performed on an ABI PRISM 3100 Automated sequencer (Applied Biosystems). Sequencing results were assembled by the ABI PRISM sequencing analysis software version 3.7 and analyzed using Chromas software (version 1.45).Individual exons were amplified by PCR using primer pairs designed by using the The two families reported here, PKGL005 and PKGL025, are from the Punjab province of Pakistan. Ophthalmic examinations and medical history for both families concluded that a total of 11 affected individuals in both families have primary congenital glaucoma (PCG). The symptoms of PCG in affected individuals of PKGL005 appeared in the first three years of life. Visual acuity was confined to light perception and/or counting fingers. The cup to disc ratios of affected individuals 11 and 41 were 0.8 (OD) and 1.0/0.3 (OD/OS), and the recorded IOPs for individuals 11 and 41 were 32/20 mm Hg (OD/OS) and 38/30 mm Hg (OD/OS), respectively . On the Initially, all reported loci for PCG were excluded for linkage using closely spaced microsatellite markers (data not shown). A genome wide scan was completed with the ABI MD10 panel, which consisted of 382 polymorphic microsatellite markers and spaced at an average of 10 cM across the whole genome. During the genome-wide scan, LOD scores above 1.5 were obtained for markers D6S308, D10S59, D10S1652, D11S1314, D14S74, D14S68, D16S404, and D18S53 in PKGL005 and for markers D2S112, D3S1279, D9S1776, D14S74, and D21S263 in PKGL025. Of these markers, D6S308, D10S59, D10S1652, D11S1314, D16S404, and D18S53 have closely flanking markers yielding large negative LOD scores in PKGL005. Similarly, in PKGL025, D2S112, D3S1279, D9S1776, and D21S263 have closely flanking markers yielding large negative LOD scores. Linkage to markers other than chromosome 14q markers that showed LOD scores greater than 1.5 during the genome-wide scan was further excluded by haplotype analysis of closely flanking markers.NCBI and Marshfield databases were genotyped to define the linkage interval in these families. Two point LOD scores of 4.96, 5.60, 4.01, 4.84, 4.76, 5.88, 3.50, and 3.69 with D14S77, D14S43, D14S284, D14S1036, D14S85, D14S61, D14S59, and D14S1008 at θ=0 were obtained for PKGL005 , which encodes a flavin-dependent monooxygenase in Saccharomyces cerevisiae. This suggests a functional similarity with CYP1B1. We investigated the COQ6 gene to identify the mutation leading to the disease phenotype in these families by sequencing all coding exons, exon-intron boundaries, and the 5'-untranslated region, but we did not find any pathogenic mutations in this gene. Our sequencing results identified previously reported SNPs rs3213692 and rs2074930 in PKGL005 and rs17552038, rs3213692, and rs7141392 in PKGL025.The critical interval on chromosome 14q24.2–24.3 harbors coenzyme Q6 homolog  in two large consanguineous Pakistani families, mapped to chromosome 14q24.2–24.3. Maximum LOD scores of 5.88 and 6.19 with markers D14S61 and D14S43 at θ=0 for families PKGL005 and PKGL025, respectively, the lack of LOD scores above 2.0 for any markers other than chromosome 14q in the entire genome scan, and the disease haplotype segregating with the disease phenotype in both families strongly suggest that the PCG locus maps to chromosome 14q24.2–24.3 in these families. Haplotype analysis of these two families refines the disease interval to a 6.56 cM (~4.2 Mb) region flanked by markers D14S289 and D14S85. Localization of the disease interval to 14q24.2–24.3 in two consanguineous Pakistani families strongly suggests genetic heterogeneity of primary congenital glaucoma.GLC3A), 1p36 (GLC3B), and 14q24.3 (GLC3C) whereas mutations associated with PCG have only been reported in the CYP1B1 gene [GLC3C was localized to chromosome 14q24.3 flanked by markers D14S61 and D14S1000 as shown in GLC3C. As both families in this study come from similar geographical and racial backgrounds, haplotype analysis of both families strongly suggests that the region flanked by markers D14S289 and D14S85 harbors the disease causing gene. However, we cannot rule out the possibility that the disease phenotype in these two families is caused by two different mutations, and the pathogenic mutation for PKGL005 may be present in a gene localized in a region overlapping with the GLC3C locus.To date, three PCG loci have been mapped to chromosomes 2p21 , WD repeat domain 21A (WDR21A), and ceh-10 homeo domain containing homolog (CHX10). COQ6 is a lipid soluble antioxidant and an obligatory component of the respiratory chain and uncoupling proteins [COQ6 in Saccharomyces cerevisiae encodes a flavin-dependent monooxygenase, suggesting a functional similarity with CYP1B1, a mixed-function monooxygenase that belongs to the cytochrome P450 1B subfamily [The critical interval on chromosome 14q24.2–24.3 harbors 97 genes including coenzyme Q6 homolog  [CHX10 is homeobox transcription factor gene that is expressed in progenitor cells of the developing neuroretina and in the inner nuclear layer of the mature retina. In humans, CHX10 mutations are associated with microphthalmia with cataracts and iris abnormalities, isolated microphthalmia with coloboma 3, isolated microphthalmia 2, and isolated microphthalmia with cloudy corneas [CHX10 cause microphthalmia, progressive degeneration of the retina, and an absence of the optic nerve in mice [WDR21A belongs to the WD repeat protein family. Members of this family are involved in a variety of cellular processes including cell cycle progression, signal transduction, apoptosis, and gene regulation. Mutations in a (POAG) . In cont corneas -29. Simi in mice . We are In summary, we have localized autosomal recessive PCG to chromosome 14q24.2–24.3 in two consanguineous Pakistani families. Identification of the PCG causing gene at this locus will help to unveil the underlying molecular complexity of primary congenital glaucoma and will be a valuable addition to the existing repertoire of glaucoma genetics, particularly of PCG. Finally, it will be helpful in screening for carrier status and genetic counseling of PCG families especially in the Pakistani population to prevent severe visual impairment and blindness.


Bacillus anthracis, is composed of three proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). PA binds a receptor on the surface of the target cell and further assembles into a homo-heptameric pore through which EF and LF translocate into the cytosol. Two distinct cellular receptors for anthrax toxin, TEM8/ANTXR1 and CMG2/ANTXR2, have been identified, and it is known that their extracellular domains bind PA with low and high affinities, respectively. Here, we report the crystal structure of the TEM8 extracellular vWA domain at 1.7 Å resolution. The overall structure has a typical integrin fold and is similar to that of the previously published CMG2 structure. In addition, using structure-based mutagenesis, we demonstrate that the putative interface region of TEM8 with PA  is responsible for the PA-binding affinity differences between the two receptors. In particular, Leu56 was shown to be a key factor for the lower affinity of TEM8 towards PA compared with CMG2. Because of its high affinity for PA and low expression in normal tissues, an isolated extracellular vWA domain of the L56A TEM8 variant may serve as a potent antitoxin and a potential therapeutic treatment for anthrax infection. Moreover, as TEM8 is often over-expressed in tumor cells, our TEM8 crystal structure may provide new insights into how to design PA mutants that preferentially target tumor cells.Anthrax toxin, which is released from the Gram-positive bacterium  Bacillus anthracis, a Gram-positive, spore-forming, rod-shaped bacterium that secretes three toxin proteins: lethal factor (LF), edema factor (EF), and protective antigen (PA) Anthrax is a lethal infectious disease caused by 83, 83 kDa) binds cell surface receptors and is cleaved by cellular furin-like enzymes 63, 63 kDa) then oligomerizes into a heptameric structure known as a prepore and interacts with EF and/or LF, which are located between two adjacent PA monomers Anthrax toxin enters target cells through a multi-step mechanism. First, full-length PA , TEM8 is only weakly detected in these tissues but abundant in tumor endothelial cells and the vasculature of developing embryos By using a genetic approach, two PA cell surface receptors have been identified: TEM8/ANTXR1  and CMG2/ANTXR2 (capillary morphogenesis protein 2/anthrax toxin receptor 2) d = 170 and 780 pM for the Mg2+- and Ca2+-bound complexes, respectively) than TEM8  Moreover, the PA-binding affinities of TEM8 and CMG2 are strikingly different. CMG2 was reported to have approximately 1000-fold higher affinity for PA  motif in vWA domains can exist in either of two conformations, much like the integrin I domain: closed (low-affinity ligand binding state) and open (high-affinity ligand binding state) 4Cl) treatment, which raises the endosomal pH The pH threshold for conversion of the PA prepore to the pore and toxin translocation is also receptor-specific. The pH required for CMG2-associated toxin pore formation (pH 5.0) is lower than that of TEM8 (pH 6.0) 2+ ion and a bound pseudo-ligand  in its MIDAS site. Based on structural analysis, we discuss a probable structural explanation for the difference between TEM8- and CMG2-mediated toxin interaction and pore formation. We also carried out a systematic mutational analysis of TEM8 using cell protection assays, surface plasmon resonance (SPR), and pH-dependent SDS-PAGE to verify our hypothesis.Here we report the high resolution structure of the TEM8 vWA domain. We found that the vWA domain contains a chelated MgWe obtained only one qualified crystal after screening more than 200 crystals in two crystallization conditions. The crystal belongs to the P1 space group and diffracted up to 1.7 Å resolution. The crystal structure was solved using the molecular replacement method from synchrotron data . The model was built from residues Ala38 to Cys220; Eight residues (MSHHHHHH) of N-terminal affinity tag were not modeled due to poor electron density and the following two residues (SM) linked to the target protein can be determined in the electron density map and refined as residue 36 and 37 in the final model. The structure model was refined to a final R-factor of 0.194 and R-free of 0.232  in the asymmetric unit  . Three mi.e., β1, residues 42–50; β2, 77–85; β4, 73–79; β5, 173–179; and β6, 196–199) and one short antiparallel β strand  form a central β-sheet. The hydrophobic residues of the β-sheet form a hydrophobic core on each side, surrounded by six amphipathic α-helices   . It shou200–217) . The exiThe extracellular domain of TEM8 shares a common topology with a wide variety of intracellular enzymes and cell adhesion molecules. Not surprisingly, CMG2 is the most similar structure to TEM8, according to the 3D structure similarity search engine DALI 2+-coordinated MIDAS motif that assumed an integrin-like open conformation.The TEM8 vWA domain structure also showed high similarity with domain I of Integrin CR3 in the open conformation  than a typical α-integrin–ligand (∼1300 Å2), and it was believed that this larger surface is responsible for the higher affinity of CMG2-PA binding Although TEM8 and CMG2 share high sequence identity, they widely differ in binding affinities for PA and in pH thresholds for forming SDS-resistant pores. The previously reported CMG2-PA complex structure depicts extensive contact between CMG2 and PA domains 2 and 4 2+ ion from the MIDAS site of CMG2 is directly involved in PA-binding. Based on the sequence alignment of the two receptors, there is a two residues width gap (from 135 to 136 of CMG2) between the TEM8 and CMG2 vWA domains. Interestingly, most of those interface residues in CMG2 are not conserved in the corresponding TEM8 sites . We can divide these non-conserved residues into four regions in TEM8: Part 1 (residues 56 and 57) is located in helix α1 (the corresponding part in CMG2 interacts with domain 2 of PA); Part 2 (residues 87 and 88) is located in the β2–β3 loop; Part 3  is located in the α2–α3 loop; and Part 4 (residues 152–156 and Tyr158) is located in the β3–β4 loop  and multiple site mutants M1 (L56A and H57N) and M2 . As stated in the E. coli expression system and were excluded from further studies. We purified the soluble mutants  and indirectly tested their PA binding affinity using a J774 A.1 cell protection assay and the SPR method.We designed a number of TEM8 single point mutants (50) compared to WT TEM8. Their protective ability even exceeded the CMG2-R40 protein , in the absence of the only disulfide bond between Cys39 and Cys218, and was one-third as strong as the CMG2-S38 variant  containing the disulfide bond. In contrast, the H57N, R88Q, L157V, and F158P point mutants had decreased protective ability. Further, the Part 4 H154D mutation resulted in a mild improvement of the PA-binding ability, and Part 2 mutant T87S and Part 3 mutant D117E had almost the same protective effect as WT TEM8. These results indicated that TEM8 Parts 1 and 4 are likely to be the main contributors to the PA affinity differences between TEM8 and CMG2  among the mutants tested, which is close to the affinity of CMG2-R40 (2.4 nM). The M1 mutant also showed a PA binding affinity (5.3 nM) similar to that of CMG2. For comparison, the KD value of TEM8 WT was 29.8 nM. We should mention that this WT TEM8 KD we measured was 4-fold lower than previous reported (130 nM), but this difference is acceptable considering SPR system characteristics D values of 49.7 nM and 167 nM, respectively, indicating that these Part 2 mutants have 2- to 5.5-fold lower PA binding affinity than the WT.Additionally, we used SPR to directly test whether the key residues identified in the cell protection assay have different PA binding abilities . In parte.g., Lys51, Tyr119, and Glu122) in TEM8. All of these mutants, especially K51A, reduced the protective effect (KD decreased by 22-fold)  , Table 122-fold) . Accordi22-fold) . In addiIn order to explain why the key residues of the two receptors identified above play important roles in the difference of PA-binding ability, we superimposed the TEM8 structure onto the CMG2 molecule in the CMG2-PA complex structure, thus creating a putative TEM8-PA complex model . The TEME. coli. We suspect that mutations in this loop may have negative effects on overall stability. Furthermore, TEM8 Leu157 is located at a position similar to CMG2 Val155  in TEM8; the corresponding residues in CMG2 are 152–158 (DGLVPSY) . In the 2 Val155  but appe2 Val155 . In our 2 Val155 . However2 Val155 .The other two parts of the receptor interface with PA, Part 2 (87–88) and especially Part 3 (113–122), bury a large amount of surface area upon PA binding. The α2–α3 loop of TEM8 forms a hydrophobic ridge that inserts into a groove formed by a β-sandwich of the immunoglobulin-like fold of PA domain 4. Non-intuitively, some of the TEM8 single mutations in Part 2 and Part 3 showed no significant effect on protective ability and PA binding. Regardless, previous research also showed that the CMG2 S113L and V115G mutants  cause no change in protective ability in vivo and pH 6.8–7.1 in vitro; and CMG2, pH 5.0 in vivo and pH 5.7–5.8 in vitro) in vitro system, when PA was bound to TEM8, formation of the SDS-PAGE resistant oligomer occurred at pH 6.8, and when bound to CMG2, its formation occurred at pH 5.6. In a previous report, Gly153 and Leu154, which contact PA domain 2, and residue Gln88, which contacts PA domain 4 , are major determinants of the lower pH threshold requirement associated with CMG2 i.e., K51A, L56A, R88Q, H154D, M1, and M2) were analyzed. M2 (154–158) showed a striking change in the pH threshold of pore formation, with a value as approaching to that of CMG2. Meanwhile, all single point mutations did not cause a significant change in the pH threshold of pore formation  and Part 4 (residues 153 and 154) significantly affected binding affinity and partially influenced the pH threshold of pore formation. Moreover, we found that the TEM8 L56A mutant strikingly increased the PA binding affinity and hence can be used as a good decoy antitoxin. In addition, based on our PA-TEM8 complex model, we analyzed why the PA M662R/R659S mutant preferentially binds to TEM8 over CMG2, highlighting the possibility to create more effective PA mutants based on our TEM8 structure.Escherichia coli strain BL21 (DE3) with a higher yield (more than two-fold) and was less susceptible to precipitation than the WT protein. The crystal structure of TEM8 C177A that we solved later in this study revealed that residue 177 was buried in a hydrophobic core and isolated, similar to the corresponding CMG2 structure. Thus, it is reasonable to deduce that this single point mutation would not induce a large scale conformational change at the MIDAS site and related interface with PA. Hereafter, for simplicity, we refer to the TEM8 C177A single point mutant as WT.The cDNA sequence encoding the human TEM8 (GenBank ID NP_115584.1) vWA domain (residues 38–220) was cloned into the pHAT2 vector (EMBL) for expression as an N-terminal His-tag fusion protein . In order to reduce aggregation of the recombinant protein, we also introduced a C177A mutation in the wild type (WT) TEM8 clone and subsequent mutant variants. Recombinant C177A protein was expressed in the soluble fraction in The TEM8 extracellular vWA domain crystals were grown by the hanging-drop vapor diffusion method at 22°C by mixing 1 µl of 5 mg/ml protein solution with 1 µl of reservoir solution (0.1 M sodium citrate trihydrate (pH 5.6), 0.2 M ammonium acetate, and 20% (w/v) polyethylene glycol 4000). We also directly added 0.1 M hexammine cobalt (III) chloride (Hampton Research) as an additive to the hanging drop (10% volume ratio) during optimization. The shape of the final crystals was laminary, and the crystals attained their maximum size (0.1×0.1×0.02 mm) after 10 d.a = 65.9 Å, b = 66.1 Å, c = 74.4 Å, α = 63.7°, β = 88.2°, and γ = 59.9°. Our attempt to process the data in higher symmetry space groups failed because of significantly worse mergeR values. The data collection statistics in the P1 crystal form are shown in Supplemental Data freeR = 23.2% and workR = 19.4%. The stereochemistry was of excellent quality, as validated by the program PROCHECK Prior to data collection, the crystals were plunged into liquid nitrogen and transported to the cold nitrogen stream of beamline 17A at the Photon Factory synchrotron facility . 20% (w/v) PEG 4000 in the reservoir solution was very suitable cryoprotectant for this crystal. Diffraction data were processed with the HKL2000 program at 1.7 Å resolution, and the crystal belongs to space group P1, with unit cell dimensions 570 of oxidized MTT was measured using a Microplate Reader Model 550 , and the data were analyzed with Prism software  as the percentage viability of control wells containing LF without PA. IC50 values were determined by nonlinear regression sigmoidal dose-response analysis with variable slopes. Each assay was performed in triplicate, and the assay was repeated at least four times independently. Data from representative assays are shown in Murine monocyte–macrophage cells J774A.1  were plated at a density of 30,000 cells/well in 96-well plates and cultured for 24 h before toxin treatment. A dilution series of TEM8 and its mutants, combined with PA proteins (100 ng/ml) and LF (100 ng/ml), was applied to the cells to a final volume of 100 µl/well. Cell viability was assayed 4 h after treatment by replacing the medium with 100 µl solution containing 1 mg/ml MTT , which was removed after a 1 h incubation at 37°C. The blue pigment  produced by viable cells was dissolved in 50 µl/well of 0.5% (w/v) SDS and 25 mM HCl in 90% (v/v) isopropanol, and the plates were vortexed. The A63 heptamer prepore (25 µg). MgCl2 was added to the reaction to a final concentration of 1 mM, and the mixture was left at room temperature for 20 min to allow complete binding. Reactions were divided into aliquots and incubated for 1 h with equal volumes of the following buffers: 1 M MES (pH 6.0), 1 M MES (pH 6.2), 1 M MES (pH 6.4), 1 M MES (pH 6.6), 1 M MES (pH 6.8), or 1 M HEPES (pH 7.0). Samples were then mixed with 2% SDS loading buffer of for 20 min. Their molecular weights were analyzed on a 3–12% Tris-Glycine gel in SDS running buffer. The protein complex bands were visualized by Coomassie Blue staining and digitalized with the computer program Bandscan (Glyko Inc).Receptor protein (15 µg) was added to PASurface plasmon resonance, to measure the binding between PA and receptor variants, was performed using the Biacore 2000 system. Monomeric PA83 was covalently linked to the carboxylated dextran matrix. It was diluted to 4 µM in sodium acetate buffer (pH 5.0), injected onto the activated surface at a flow rate of 5 µl/min for 65 min, and then blocked with ethanolamine. The vWA domains of the receptor variants  were diluted to various concentrations (50–800 nM) in HEPES buffered saline , 150 mM NaCl, and 0.005% (v/v) TWEEN-20) with additional 1 mM Mg2+, and serial injections were made at 20 µl/min at 25°C. After sample analysis, CM5 baselines were regenerated with 10 mM/L glycine HCl (pH 2.0) for 15 s and borate buffer (10 mM sodium tetraborate (pH 8.5) and 1 M NaCl) for 15 s.Figure S1The MIDAS site of one TEM8 vWA domain molecule was blocked by the adjacent molecule in the crystal cell. Protein molecules are shown in ribbon presentation. Only two adjacent TEM8 vWA domain molecules are shown and labeled as A, B in the crystal. The MIDAS site of A molecule (purple) is highlighted with a pink background. The MIDAS site of molecule A is blocked by molecule B (red) from above by steric hindrance. PA cannot interact with A's MIDAS site.(2.90 MB TIF)Click here for additional data file.Figure S2Sedimentation Velocity Data for TEM8. Curve represents a sedimentation velocity run for TEM8. This curve has only one peak (19.5 kD) that is close to the TEM8 vWA domain monomer's theoretic molecular weight, which demonstrates the TEM8 vWA domain molecule to exist as monomer in the solution. Sedimentation velocity (SV) experiment was conducted with an Optima XL-L analytical ultracentrifuge (Beckman-Coulter Instruments). An An60Ti rotor and standard six-sector equilibrium centerpieces were used. Freshly prepared TEM8 was further purified and buffer-exchanged into sedimentation buffer  using a gel filtration column. c(M) distribution for TEM8  obtained from sedimentation velocity experiments, at 20°C and a speed of 40,000 rpm. Absorbance scans were carried out at a wavelength of 280 nm, and 98 scans were collected at 2 min intervals.(0.75 MB TIF)Click here for additional data file.Table S1Data collection and refinement statistics.(0.05 MB DOC)Click here for additional data file.


However, recent bioinformatic analyses of EST sequences suggested widespread trans-splicing in mammals. These apparently spliced transcripts generally lacked canonical splice sites, leading us to question their authenticity. Particularly, the native ability of reverse transcriptase enzymes to template switch during transcription could produce apparently trans-spliced sequences.Trans-splicing, the in vitro system for the analysis of template switching in reverse transcription. Using highly purified RNA substrates, we show the reproducible occurrence of apparent trans-splicing between two RNA molecules. Other reported non-canonical splicing events such as exon shuffling and sense-antisense fusions were also readily detected. The latter caused the production of apparent antisense non-coding RNAs, which are also reported to be abundant in humans.Here we report an We propose that most reported examples of non-canonical splicing in metazoans arise through template switching by reverse transcriptase during cDNA preparation. We further show that the products of template switching can vary between reverse transcriptases, providing a simple diagnostic for identifying many of these experimental artifacts. Reverse transcriptases (RTs) are enzymes that synthesize complementary DNA (cDNA) from an RNA template, and have evolved in retroviruses to convert single stranded viral RNA into double stranded DNA for integration into host genomes. They are an invaluable tool for molecular biology, being used to copy RNA into DNA for analysis by PCR (RT-PCR), microarrays and high throughput sequencing. It is possible to sequence RNA directly, however, most of the experimentally determined RNA sequences, and all high-throughput data, have been generated by RT-based protocols.RTs lack a proof reading activity , we arbitrarily selected GenBank sequence M14410, a fusion between KRE29 and HXK1, as a substrate for in vitro analysis  polymerase to add a poly(A) tail. These substrates were then used in vitro for reverse transcription as above but primed from oligo(dT). This produced the same pattern of products seen in previous experiments, which now depended on the presence of poly(A) polymerase (SPT7 RNA still occurs in the absence of poly(A) polymerase due to priming of the oligo(dT) on short, encoded oligo(A) stretches in the substrate. These data show that template switching events detected in random hexamer primed RT reactions also occur during oligo(dT) primed cDNA synthesis.Our proposed mechanism for the formation of sense-antisense fusions requires two RT enzymes to be active on the same RNA molecule. This will occur frequently if reverse transcription is primed from random hexamers, but is expected to be less common when oligo(dT) is used to prime synthesis from the poly(A) tail. To test the effect of this change, lymerase . The pp1SPT7 reactions reduced the aberrant products in some experiments, but clearly did not eliminate template switching  and cloned into pGEM-T (Promega). Oligonucleotides used were HXK1 F1/R1 for HXK1, KRE29 F1/R1 for KRE29 and SPT7 F1/R1 for SPT7; sequences of oligonucleotides are given in XhoI and 1 µg transcribed using T7 RNA polymerase (NEB) for 2 h at 37°C. Gels were run in 1x TBE, acrylamide gels contained 8 M urea. Gels were stained with SYBR Safe and imaged using a Fuji FLA5100 scanner. RNA was eluted from acrylamide gel slices by crushing and soaking for 4 h in 0.5 M NaOAc/1 mM EDTA/0.1% SDS, followed by phenol/chloroform extraction and ethanol precipitation with 1 µg glycogen. PCR reactions on reverse transcribed material were performed with Phire (NEB), details of cycle number are given in individual figure legends. Annealing temperature was 50°C for IGS1 and HXK1/KRE29 PCR and 53°C for SPT7 PCR. For poly(A) tailing, 50 ng RNA was incubated with 5U poly(A) polymerase (NEB) and 1 mM ATP, then cleaned on QIAQuick columns (QIAgen).Substrates for 18 was added in place of hexamers, and reactions were heated to 42°C prior to enzyme addition. Superscript III reactions were performed as per manufacturer's instructions at the indicated temperatures. DNA template controls were cDNA from 500 ng HeLa RNA produced as above, with 0.5 ng gel purified XhoI-PvuI fragments of the template plasmids. Where indicated, Actinomycin D  was added to Superscript II RT reactions after the 65°C step at 6 µg ml−1 from a 1 mg ml−1 stock solution.Superscript II RT: 0.5 ng substrate RNA, 500 ng HeLa RNA (Invitrogen), 125 ng random hexamers and 0.5 µl 10 mM dNTPs in 6.5 µl total volume were denatured at 65°C for 5 min before 2 min on ice. 2 µl 5x first strand buffer and 1 µl of 0.1 M DTT were added followed by 0.5 µl (100 U) Superscript II (Invitrogen). Reactions were incubated 10 min at room temperature, 42°C for 50 min and 70°C for 15 min. For oligo(dT) priming, 250 ng oligo(dT)AMV RT: 0.5 ng RNA, 500 ng HeLa RNA (Invitrogen) and 125 ng random hexamers in total volume 8.25 µl were heated 5 min at 70° and 5 min on ice. 1.25 µl 10 mM dNTPs and 2.5 µl 5x buffer were added followed by 0.5 µl (5 U) AMV (Promega). Reactions were incubated for 1 h at 37°C.Table S1Sequencing information. All sequences obtained in this project are shown. Regions of the sequence have been colour coded red, green and blue to indicate that they emanate from different molecules or different regions of the same molecule. Overlapping regions are shown in purple. Sequences were obtained either by direct sequencing of band-purified PCR products, or for complex PCR products by sequencing multiple clones ligated in pGEM-T.(0.04 MB DOC)Click here for additional data file.


Clostridium botulinum intron. These results demonstrate the suitability of the native affinity purification method for the preparation of group I introns. We hope these findings will stimulate a broader application of this strategy to the preparation of other large RNA molecules.The study of functional RNAs of various sizes and structures requires efficient methods for their synthesis and purification. Here, 23 group I intron variants ranging in length from 246 to 341 nucleotides—some containing exons—were subjected to a native purification technique previously applied only to shorter RNAs (<160 nucleotides). For the RNAs containing both exons, we adjusted the original purification protocol to allow for purification of radiolabeled molecules. The resulting RNAs were used in folding assays on native gel electrophoresis and in self-splicing assays. The intron-only RNAs were subjected to the regular native purification scheme, assayed for folding and employed in crystallization screens. All RNAs that contained a 3′ overhang of one nucleotide were efficiently cleaved off from the support and were at least 90% pure after the non-denaturing purification. A representative subset of these RNAs was shown to be folded and self-splicing after purification. Additionally, crystals were grown for a 286 nucleotide long variant of the  T. maritima Ffh protein (TmaM)) immobilized on a resin, and then recovered by elution in the presence of imidazole  using the first-generation method. We present a version of the original protocol that allowed purification of small-scale transcription reaction products incorporating radiolabeled nucleotides. These RNAs were subjected to folding assays and catalytic activity assays. We also applied the standard native affinity purification method to 12 sequence variants of a 287 nt long group I intron from the bacterium Anabaena sp. PCC7120, A.s.; Clostridium botulinum, C.b.; Derbesia marina, D.m.; Bryopsis plumosa, B.p.) Scytalidium dimidiatum, S.d.) td) A.s., D.m., B.p.) or shortened to 75 nucleotides . Variants of the td and C.b. introns were also designed for crystallization purposes. td[Im] contained a mutated P6a domain . The C.b.[Im1–12] variants corresponded to 12 variants of the C.b. group I intron that possessed 5′ and 3′ ends circularly permuted to the P6a region and further engineered in both the P6a helix and loop L8 . Attempts to synthesize the S.d.[P] template at different temperatures (50–65°C) and using the three different DNA polymerases were unsuccessful; the only achievement was a PCR product containing the first 200 nt of the expected sequence . The failure of the S.d.[P] template to be engineered by this technique could be accounted for by a combination of its length (503 base pairs)—thus increasing the number of overlapping primers to 10 .Whereas amplifications of the 20 nt andS1. Thetd[Pm1] to td[I]; td[Pm1] template (td[Pm1a] and td[Pm1b]) that resulted from random PCR errors were further transcribed and purified. The corresponding RNAs were shown to retain some activity (see below). In summary, the templates that were optimally synthesized were about 300 nucleotides long (corresponding to five overlapping primers) and were amplified in one round using a high-fidelity DNA polymerase.PCR-amplification from overlapping primers randomly generated mutations within or close to the overlapping regions. Typically, only 25% of the clones sequenced following ligation to the pRAV12 plasmid contained the expected sequence Table 1.TmaM protein and its coupling to a support to generate the affinity resin were performed as described The preparation of the A.s.[Pm1], B.p.[P], and D.m.[P]; B.p.[P], D.m.[P], A.s.[P], C.b.[Im5]), while variants possessing no overhang  or a 5′ overhang (C.b.[Im3]) give moderate to low (<10%) cleavage yields . C.b.[Im4] was an exception to this rule, as only 12% were cleaved by HDV, in spite of the two-nucleotide 3′ overhang , because of a poorer yield (20%) at that lower magnesium concentration . Hence, that condition was not employed for the purification of other sequences. The other attempts failed to purify the A.s.[Pm1] variant , in spite of >75% binding to the resin .Of the three RNAs that could not be satisfactorily purified, one had the longest sequence tested  indicated that the RNAs in absence and in presence of the 5′-CGAdT-3′ oligonucleotide were folded after native purification, without the need of any additional annealing protocol . The td[Pm1b] variant that contains the A918G mutation and the deletion of G78 was self-splicing, although the second step of the self-splicing reaction was only 50% at best after 1 hour  ; 25% MPD (well solution). In these conditions, single crystals grew to sizes suitable for diffraction analysis . The space group was trigonal (P3 family), with cell parameters a = 194.1 Å, b = 194.1 Å, c = 415.5 Å, α = β = 90°, γ = 120°. The resolution improved slightly at a synchrotron, to ∼8.0–8.5 Å . Because we were unable to reduce the data collected at the synchrotron, we sought out to obtain crystals using the C.b.[Im11] variant purified by a conventional denaturing polyacrylamide gel electrophoresis (PAGE) technique.The resolution of the C.b.[Im11] variant looked similar to that of their natively-purified counterparts, although they tended to have sharper edges . Since about 10 times more crystals of the PAGE-purified RNA were tested than of the natively purified RNA, it cannot be ruled out that similar resolutions could have been achieved with crystals of the natively purified RNA. It is also possible that original crystals of the natively-purified RNA were grown from a solution that contained partially degraded RNA. In fact, in order to preserve the native folding of this intron, we initially stored the RNA at 4°C in the presence of 5 mM MgCl2 (in 10 mM Na cacodylate pH 5.0). We later found out that storage of the RNA at 4°C even in 1 mM MgCl2 was inducing severe degradation after a few weeks . We subsequently stored the RNA at −20°C in absence or in presence of 1 mM MgCl2, at pH 5.0, which did not promote RNA degradation  nor altered the folding. Finally, spherulites and urchins were obtained in similar conditions for two other variants purified by PAGE, C.b.[Im3] and C.b.[Im8] . In any case, attempts at annealing crystals to increase resolution The crystals of the PAGE-purified C.b.[Im6–12] variants enabled optimal cleavage by HDV. The sequence of this overhang did not seem to be critical, as variants ending with a single A , a single C . Fortunately, not all introns self-cleaved during the purification procedure  likely disrupted the corresponding base triple involving P6 and the J3/4 junction in all group I introns td intron deprived of G78 could be rescued by the CYT-18 protein or by the StpA protein, which also binds to this location Finally, the coupling of the PCR strategy employed here and of the native purification method could help engineer random mutations in the specific regions of RNA molecules about 300 nt long (i.e. by including such areas within the overlap regions). Such mutants may lead to improvement in crystallization, as mutation of one nucleotide often strongly affects resolution Clostridium botulinum intron gave crystals of a similar quality than crystals obtained using a conventional denaturing purification technique, although the time span to obtain the RNA was significantly reduced. Together, the first and the second generation of the purification techniques may constitute unique tools for the preparation of co-transcriptionally folded RNAs of a large size.While developing a second generation of the affinity purification method Leu introns, one ribosomal intron, one tmRNA intron, and one intron in a protein coding sequence), and other properties relating to the purpose of this study. For example, three introns  had been previously shown to be self-splicing td and S.d. exons). Additional variants were synthesized to incorporate the intron only, for td (two variants missing the first seven nucleotides C.b. (12 circularly permuted variants possessing diverse P6a regions).Group I introns were selected based on their length (ranging from 167 nt to 393 nt), their exon context . Polymerase chain reactions (PCR) from overlapping DNA primers spanning an entire gene thus offered an alternative to cloning genomic DNA Constructs were designed to incorporate the following sequence: six random nucleotides, a restriction site for cloning at the 5′ end (EcoRI), the T7 polymerase promoter 2; either 0.5 U Taq DNA polymerase (Roche #11 146 165 001), or 0.5 U Pwo DNA polymerase (Inno-Train Diagnostik GbmH #GX02010), or 2.5 U Herculase DNA polymerase (Stratagene #600262); 1X reaction buffer supplied by the manufacturer. PCR were performed in a Robocycler Gradient 96 apparatus (Stratagene) using the following protocol: 4 min at 94°C (1 cycle); 55 s at 94°C, 55 s at 50–65°C, 1.5 min at 72°C (30–35 cycles); 10 min at 72°C (1 cycle). When only one round of PCR was necessary, these conditions were employed, in the presence of the generic primers described below for the second round.For the first round of PCR, constructs were amplified in 50 µL-reaction mixes containing: 0.4 pmoles of each overlapping primer (∼10 ng); 200–250 µΜ each dNTP; 1.0 mM MgCltd[P] construct: 20 µL of the amplified primers 1−4+20 µL of the amplified primers 3–7); 50 pmoles of each of the two generic primers (containing the first 25–30 nucleotides of the first and the last overlapping primer); 200–250 µΜ each dNTP; 1.0 mM MgCl2; either 0.5 U Taq DNA polymerase (Roche #11 146 165 001), or 0.5 U Pwo DNA polymerase (Inno-Train Diagnostik GbmH #GX02010), or 2.5 U Herculase DNA polymerase (Stratagene #600262); 1X reaction buffer supplied by the manufacturer. PCR were performed in a Robocycler Gradient 96 apparatus (Stratagene) using the same cycles as for round #1. The A.s.[Pm1], D.m.[P] and B.p.[P] variants were subsequently amplified for a third round in presence of a primer replacing the BbsI restriction site by a NcoI/NgoMIV site .When a second round of PCR was necessary, PCR products from the first round were combined as follows in 50 µL-reaction mixes: 20 µL of each of the two sets of amplified portions from the first round necessary to obtain the complete sequence of the construct . Gels were stained using ethidium bromide (Sigma #46067-50ML-F) and visualized using a UV-light transilluminator.The PCR amplified products were purified using the QIAquick PCR purification kit (Qiagen #28104), and digested using the appropriate restriction enzymes  #R0101L; and NcoI, NEB #R0193L—incomplete digestion products were typically obtained using BbsI, NEB #R0539L, which made us avoid using this enzyme). The digested products were then purified by agarose gel electrophoresis, and eluted in a final volume of 30 µL using the QIAquick gel extraction kit (Qiagen #28706). Ligation occurred for 35 min at room temperature (∼23–25°C) in a solution containing: 0.2 µg/µL pRAV12 vector C.b.[P] and the A.s.[P] constructs were independently amplified from plasmids containing the intron and the complete exons . Each PCR step was performed under the reaction conditions described above. The PCR products of the first step were diluted by 10-fold and 1 µL of this dilution was used as a template into a final 50 µL-reaction mix for the second step. The various 5′ and 3′ ends of the C.b.[Im2–12] variants ; 4.0 mM each rNTP; 12.0 mM MgCl2; 2.0 mM spermidine-HCl; 10 mM DTT; 0.1% Triton X-100; 4.0 ng/µL inorganic pyrophosphatase (Sigma #I1891-100UN); 40 U RNasin Plus RNase inhibitor (Promega #N261B); 2 µL T7 RNA polymerase (prepared in house); 50 mM Tris-HCl pH 7.5. The final radioactivity of the samples was measured by the number of counts per minute (cpm) for 1.0 µL of the RNA solution diluted into 3.0 mL ScintiSafe Econo 1 solution (Fisher Scientific #SX20-5), obtained using a scintillation counter (Beckman #LS3801).Plasmids were linearized using the BamHI restriction enzyme (NEB #R0136L) and were transcribed for 1 h at 37°C in 25 µL-reaction mixes containing: 1.0–4.0 µg/µL linearized plasmid; 50 µCi α-C.b.[Im1–12], td[I], or td[Im1] variants were transcribed in a similar reaction solution, but for 2 h at 37°C and in 1.5 mL–10.0 mL reaction mixes. Concentrations of the purified products were determined from the A260 and calculated extinction coefficients based on the nucleotide sequence . The RNAs were concentrated to ∼500 µM using a centrifugal filter unit with a 10,000 MWCO , and stored at 4°C or at −20°C in a buffer containing either 50 mM Na HEPES pH 7.5, 25 mM NaCl, 15 mM MgCl2, or 10 mM Na cacodylate pH 5.0–6.5, 10 mM NaCl and 0–5 mM MgCl2. The C.b. RNAs were used in crystallization assays without prior refolding, while the td variants were annealed for 10 min at 37°C prior to being utilized in crystallization assays.Linearized plasmids each containing one of the C.b.[Im1–12] variants used as a control for the crystallization and X-ray diffraction experiments were transcribed by run-off transcription from a plasmid linearized using the FokI enzyme (NEB #R0109S), as described above, but for 4 h at 37°C and in 10 mL-reaction mixes. The RNAs were ethanol precipitated for 16 h at −20°C in 70% EtOH, 100 mM Na acetate pH 5.3, centrifugated for 35 min at 13,500 g, dried for 10 min under vacuum (SpeedVac), and purified by denaturing gel electrophoresis . The gel slice corresponding to the RNA was crushed and soaked for 2 h at 4°C in 35 mL of an elution buffer containing: 1.0 mM EDTA; 10 mM Tris-HCl pH 7.5. The solution was filtered through a 0.2 mm filter, and concentrated to ∼200 µM using a centrifugal filter unit with a 30,000 MWCO (Millipore #UFC903096), and stored at 4°C or −20°C in a buffer containing 10 mM Na cacodylate pH 6.5, 10 mM NaCl and 5 mM MgCl2. The RNAs were used in crystallization assays without prior annealing.The gel-purified A.s.[Pm1] RNA that was deprived of the affinity tag. Here, the gel slice corresponding to the A.s.[Pm1] RNA was crushed and soaked for 20 h (with one buffer exchange after 2 h) at 4°C in 800 µL of an elution buffer containing: 1.0 mM EDTA; 250 mM NaCl; 10 mM Tris-HCl pH 7.5. Precipitation and filtration were performed as described in the previous paragraph, and the RNA was stored at −20°C in water. The final radioactivity of the RNA was measured as described in the section entitled: “In vitro transcription of body-labeled RNAs”.A similar procedure was applied to a 25 µL-run off transcription reaction of the radiolabeled TmaM protein was prepared and coupled to the activated support as described Figure S8). Over the course of this preparation, similar problems to that later reported were observed, including for example the precipitation of the protein after its concentration The WB), 10 mM MgCl2, 250 mM NaCl, 25 mM Tris-HCl pH 8.0; cleavage buffer (CB), 10 mM MgCl2, 250 mM NaCl, 200 mM imidazole, 25 mM Tris-HCl pH 8.0; regeneration buffer (RB), 1.0 M LiCl, 174 mM glacial acetic acid, 25 mM Na2EDTA; storage buffer (SB), WB containing 0.1% Na azide.The body-labeled transcription products were purified by nondenaturing purification as described C.b.[Im1–12] variants were purified without modification of the published protocol (except for adjustments to the corresponding transcription reaction volumes) The unlabeled transcription products of the C.b.[Im1–12] variants). The gels were either stained using SYBR Green II RNA gel stain , or dried under vacuum and placed for 18–30 h in phosphorimager screens (Molecular Dynamics). Both the stained gels and the screens were scanned using a phosphorimager (Amersham Bioscience/GE) and the gel images were analyzed using ImageQuant TL v. 2005 (Amersham Bioscience/GE).Products from the purification were analyzed by electrophoresis on denaturing gels . Care was taken to load gels with volumes of samples proportional to the total volume of each fraction (except for the 1  =  [(N1/26)/((N1/26) + (N2/nA2)] × 100 and y2  =  [(N3/nA3)/((N3/nA3) + (N2/nA2)] x 100, where:An approximate yield of cleavage from the resin was estimated by calculating the average between y1 = number of counts in the band corresponding to the tag removed from the support during the regeneration steps (found in lanes “r1–r5” on N2 = number of counts in the band corresponding to the uncleaved RNA removed from the support during the regeneration steps (found in lanes “r1–r5” on N3 = number of counts in the band corresponding to the RNA cleaved off from the support during the elution steps (found in lanes “e1–e5” and/or “pool A/B” on NA2 = number of adenines in the precursor/intron+tag RNA (the tag contains 26 adenines);nA3 = number of adenines in the precursor/intron RNA .n3/] × 100, where N3 is as described above, and:Purity was estimated by the ratio [ and further incubation for 5 min or 1.5 h at 32°C. A sample of the C.b.[Im1] variant was also similarly incubated in the presence of 100 µM 5′-CGAU-3′.Approximately 2,000 cpm of the body-labeled wild-type C.b.[Im1], C.b.[Im3] and C.b.[Im11] variants natively purified were incubated according to different annealing protocols, as described in the legend to Approximately 20,000 cpm of the td[I] and td[Im] RNAs were annealed for 10 min at 50°C followed by 10 min at 25°C, in a solution containing 5.0 mM MgCl2, and 25 mM Na HEPES pH 7.5. These samples were added to various concentrations  of an RNA hexamer (sequence: 5′-UUGGGU-3′) corresponding to the end of the 5′ exon and necessary for the formation of the P1 helix 2; run at 4°C). The gels were dried and analyzed as described above.Approximately 5,000 cpm of the body-labeled D.m.[P], B.p.[P] and td[Pm1b] RNAs were tested for self-splicing activity under a set of conditions previously reported to efficiently assay self-splicing of group I introns A, 15 mM MgCl2, 25 mM NaCl, 25 mM Na HEPES pH 7.5 (incubation at 32°C); B, 15 mM MgCl2, 25 mM NaCl, 25 mM Na HEPES pH 7.5 (incubation at 42°C); C, 15 mM MgCl2, 1.0 M NaCl, 25 mM Na HEPES pH 7.5 (incubation at 32°C); D, 200 mM MgCl2, 25 mM NaCl, 25 mM Na HEPES pH 7.5 (incubation at 32°C); E, 10 mM Mg(OAc)2, 0.4 M NaOAc, 25 mM Na HEPES pH 7.5 (incubation at 32°C). Reactions were stopped by the addition of a solution containing: 500 mM EDTA; 7.0 M urea; 0.02% bromophenol blue; 0.02% xylene cyanol; 1X TBE . Products were analyzed by denaturing polyacrylamide gel electrophoresis (PAGE) as described above.The C.b. and td variants were diluted to 5–60 µM in a solution containing the following components: substrate oligonucleotide , 0–80 mM NaCl or KCl, 10–20 mM MgCl2, 40 mM Na cacodylate pH 5.5 or pH 6.0. Hanging drops were set up in crystallization plates by mixing 0.5–1.5 µL of the RNA solution and 0.5–1.5 µL of a crystallization solution (0.5–1 mL in the well) from the following screens: Natrix (Hampton Research (HR) #HR2-116); PEG/Ion Screen (HR #HR2-126); the MPD Suite (Qiagen #130706); a modified version of the Nucleic Acid Mini-Screen (HR #HR2-118), containing 2.5–15% MPD in the drop (18–35% MPD in the well), 1 mM Co3+ hexamine or 1 mM spermine; a screen prepared in house, containing as precipitant either 5–30% 1,6-hexanediol, or 5–15% isopropanol, or 1.4–1.8 M (Li)2SO4, or 1.6–2.2 M (NH4)2SO4, and 2–10 mM Mg(OAc)2, 1 mM spermine, 2 mM Co3+ hexamine, 50 mM Na cacodylate pH 6.5. Free interface diffusion experiments were set up in Topaz chips at the 4.96 format  using 1 µL of RNA solution and 10 µL of crystallization solution per well according to manufacturer's instructions. The chips were incubated at 16°C.The native-affinity purified C.b.[Im11] variant were cryoprotected for 10 min in 60% MPD and flash-frozen in liquid nitrogen. Data were collected on an R-AXIS IV++instrument with CuKα X-ray radiation using 1° oscillation angle and 30–45 min exposures. A 20° wedge of data collected with 45 min exposure time per frame was reduced using d*TREK (51% completeness).The first crystals obtained for the C.b.[Im11] variant were flash-frozen in liquid nitrogen directly from the hanging drop, or were first cryoprotected for 5–20 min in various cryoprotecting solutions . Data were collected at the Howard Hughes Medical Institute 8.2.1 beamline at the Advance Light Source , using 0.5° oscillation range and 15 s exposures. A 20° wedge of data was reduced using MOSFLM version 7.0.4 Additional crystals of the natively-purified and Table S1Generic and overlapping primers used in this study.(0.03 MB XLS)Click here for additional data file.Figure S1PCR synthesis using overlapping primers. Products are visualized on agarose gels stained using ethidium bromide. Expected products are marked by a black triangle. Labels above each lane indicate the number of overlapping primers (C: control reactions containing only the generic primers). Labels below each lane refer to the DNA polymerase used during PCR . A star symbol indicates when 5.0 U instead of 2.5 U Herculase were used. The annealing temperature is shown in yellow on the top right corner of each gel.(2.13 MB TIF)Click here for additional data file.Figure S2C.b.[Im6], C.b.[Im7], C.b.[Im10], C.b.[Im11] and C.b.[Im12] variants. These variants were purified using the large-scale purification method (purification of 10-mL transcription reaction mixes). The assays were visualized on 6% denaturing PAGE, and stained using SYBR Green II. Labels above each lane refer to purification steps detailed in Purification assays of the (0.97 MB TIF)Click here for additional data file.Figure S3A.s.[Pm1] variant at lower MgCl2 concentration, using CaCl2 in place of MgCl2, and at pH 6.5–8.0. The results are visualized by denaturing PAGE as in Purification assays of the (4.10 MB TIF)Click here for additional data file.Figure S4D.m.[P] and B.p.[P] variants. The self-splicing activity was tested under five different buffer conditions Click here for additional data file.Figure S5C.b.[Im11] variants and crystalline materials obtained for the C.b.[Im3] and C.b.[Im8] variants. (A) Diffraction patterns of crystals grown using either the natively purified RNA (left), or the RNA purified by denaturing PAGE (right). The 7.0–8.5 angstrom resolution typically obtained for these crystals is indicated in red. (B) Spherulites (left) and urchins (right) obtained with the PAGE-purified C.b.[Im3] and C.b.[Im8] variants, respectively.Diffraction patterns of (3.66 MB TIF)Click here for additional data file.Figure S6C.b.[Im11] RNA stock after eight weeks of storage in various conditions. The RNA was in 10 mM Na cacodylate pH 5.0 in all cases. Approximately 10 pmoles of RNA were loaded in each lane. The assays were visualized on 6% denaturing PAGE, and stained using SYBR Green II.Analysis of the (0.29 MB TIF)Click here for additional data file.Figure S7C.b. intron into the P6a region. During the first PCR step, the wild-type C.b. intron (colored from the wild-type 5′ end to the P6a helix (red), and from the P6a helix to the wild-type 3′ end (brown)) is used as a template for two independent PCR rounds that contain different sets of primers. During the second PCR step, the product of each of these reactions is combined to suitable primers from step #1 in order to amplify the expected circularly permuted C.b. intron. The incorporated T7 promoter and restriction sites suitable for cloning are colored as in A two-step PCR strategy to circularly permute the 5′ and 3′ ends of the (0.53 MB TIF)Click here for additional data file.Figure S8TmaM-domain protein. (A) Cells prior and post-induction with 1.0 mM IPTG (ten 750-mL cultures were grown for 12 h at 31°C in LB medium). (B) Supernatent fraction and pellets of three cell lysates. (C) Fraction of protein eluted from Ni2+-affinity column and after TEV cleavage (three fractions were cleaved for 16 h at 25°C by a 1∶100 ratio (by mass) of TEV protease, in 10 mM DTT). (D) Chromatogram after purification of the TmaM-domain on the SP-Sepharose column . The protein eluted around 0.55 M NaCl in 10 mM MES pH 6.0. (E) Corresponding elution fractions. Fractions #8 and 9 were pooled, dialysed and coupled to the activated support . Gels shown in panels A, B, C, and E were from SDS-PAGE using 4–12% or 12% acrylamide (as specified under each gel), and were stained using SimplyBlue SafeStain (Invitrogen #LC6060); ladders: SeeBlue Plus 2 ((A)–(E); Invitrogen #LC5925), BenchMark Pre-stained ((E) only; Invitrogen #10748-010).Expression and purification of the (3.29 MB TIF)Click here for additional data file.


BaTiO3:Eu3+ films ~500 nm thick, crystallized after thermal treatment at 700 ºC. The powders revealed spherical and rod shape morphology. The optical quality of films showed a predominant band at 615 nm under 250 nm excitation. A preliminary luminescent test provided the properties of the Eu3+ doped BaTiO3.Transparent BaTiO Barium acetate and europium chloride were dissolved in water in the following molar ratio: Ba:Eu:H2O [1:0.05:40]. Titanium butoxide was mixed with acetylacetone in a molar ratio [1:1], and the reaction was continued under reflux at 60 °C for 6 h. PVP was dissolved in isopropyl alcohol in a molar ratio [1:18] for 2 hours at room temperature. The molar ratios were calculated with respect to Ba. The titanium and barium solutions were mixed and stirred for 2 h. Thereafter, the PVP solution was added to the previous solution drop by drop under vigorous magnetic stirring for 2 h at room temperature. In this step, a transparent and stable BaTiO3:Eu3+ sol was formed. Uniform coatings of BaTiO3:Eu3+ were dip coated on high polished and carefully cleaned silica substrates (Herasil from Heraeus®) with a constant withdrawal speed of 5 cm min−1 for five cycles. BaTiO3:Eu3+ films were dried at 100 °C between each coating under O2 flow for 10 min. In this step, one film was taken for HT-XRD studies. The BaTiO3:Eu3+ films were finally calcined at 700 °C for 10 min. The remaining solution was dried in order to obtain a xerogel at 100 °C for 24 h . After drying, the xerogel was thermally treated at 700 °C for 2 h in order to densify and crystallize the sol-gel powders.Europium doped BaTiO3.2.−1 in a flux of nitrogen. The HT-XRD studies were carried out on a Philips Xpert Pro diffractometer operating with the Cu Kα radiation and equipped with a high temperature chamber, over a temperature range from 25 to 1000 °C in air atmosphere. The structural phases of the crystallized powder and film were recorded on a Siemens D5000 powder diffractometer using the Bragg–Brentano configuration and the Cu Kα radiation. The IR transmittance spectra were recorded on the crystallized powders calcined at 700 °C using a FTIR 2000 Perkin-Elmer in the range 4,000-400 cm−1, with the aim of completing DRX and DTA-TGA experiments. In order to determine the powder microstructure and quality of the derived BaTiO3:Eu3+ crystallized sol-gel films, SEM images were obtained using a JEOL 3200 scanning electron microscope (SEM) with a field emission gun operating at 15 and 8 kV. The thickness of BaTiO3 films was determined via an Alpha-step IQ profilometer (Tencor Instruments). The fluorescence emission spectra for BaTiO3:Eu3+ film as obtained with a modular Spectra Pro (Acton Research) fluorometer with a PM tube R955 (Hamamatsu).The xerogel was investigated by Thermogravimetric (TGA)-Differential Thermal (DTA) and High-Temperature X-Ray Diffraction (HT-XRD) analyses. The thermograms were recordered from 23 °C to 1000 °C using a Mettler Toledo TGA/SDTA 851e apparatus at a scan rate of 2 °C min 4.3:Eu3+ (5 mol%) powder and film incorporating a viscosity modifier (PVP) in the sol. Both systems exhibited crystalline BaTiO3 phase at 700 °C within 2 h as revealed the HT-XRD studies. The powders were mainly spherical with some rod shapes; however, the films presented good surface morphology as detected by SEM. The obtained films exhibit the room temperature photoluminescence of the europium ions, with the predominant band at 615 nm (5D0→7F2 transition). The obtained thickness (~500 nm) of BaTiO3:Eu3+ films must be optimized to be promising for luminescent applications.The sol-gel method and dip-coating techniques have been successfully employed to prepare BaTiO


DTS), porosity (P), and phase composition (powder x-ray diffraction) were determined after the hardened specimens had been immersed in a physiological-like solution (PLS) for 1 d, 3 d, and 7 d. In CPC specimens prepared with water, calcium hydroxyapatite (HA) was formed and DTS and P were (9.03 ± 0.48) MPa and (37.05 ± 0.20) vol % after 1 d, respectively, and (9.15 ± 0.45) MPa and (37.24 ± 0.63) vol % after 3 d, respectively. In CPC specimens prepared with phosphate solution OCP and HA were formed and DTS and P were (4.38 ± 0.49) MPa and (41.44 ± 1.25) vol % after 1 d, respectively,(4.38 ± 0.29) MPa and (42.52 ± 2.15) vol % after 3 d, respectively, and (4.30 ± 0.60) MPa and (41.38 ± 1.65) vol % after 7 d, respectively. For each group DTS and P did not change with PLS immersion time. DTS was significantly higher and P was significantly lower for CPCs prepared with water. HA formation slightly increased with immersion time from 40 mass % after 1 d to 50 mass % after 3 d in CPCs prepared with water. OCP + HA formation increased with immersion time from 30 mass % after 1 d to 35 mass % after 3 d and to 45 mass % after 7 d in CPCs prepared with 0.5 mol/L phosphate solution.The osteoconductive and possibly osteoinductive characteristics of OCP increased the interest in preparation of bone graft materials that contain OCP in its composition. Calcium phosphate cements (CPCs) were prepared using a mixture of α-tricalcium phosphate (α-TCP) and dicalcium phosphate anhydrous (DCPA), with α-TCP/DCPA molar ratio of 1/1 and distilled water or 0.5 mol/L phosphate aqueous solution (pH = 6.1 ± 0.1) as the cement liquid. Hardening time was (30 ± 1) min for the CPC mixed with water and (5 ± 1) min for the CPC mixed with phosphate solution. Diametral tensile strength ( OCP is H)2; HA) ,4. Recenn muscle  and subcn muscle . It was n muscle –15. In an muscle , osteobln muscle  and in sn muscle ,18. The 3(PO4)2; α-TCP) and dicalcium phosphate dihydrate  in water. Bermudez et al., [4; DCPA) as the solid components and water as the cement liquid, but the hardening time of this CPC was relatively slow (30 min). Sena et al., [3 + Ca(H2PO4)2) and a phosphate aqueous solution (pH = 7.4), that was used as a pulp filler.The osteoconductive and possibly osteoinductive characteristics of OCP increased the interest in preparation of bone graft materials that contain OCP in its composition. Calcium phosphate cements (CPCs) that are mixtures of two (or more) powdered calcium and/or phosphate containing materials that harden with addition of an aqueous solution are good candidates for formation of OCP. Monma et al.,  reported et al.,  reported et al.,  also sugThe objective of this study was to prepare fast self-hardening calcium phosphate cement in which OCP is formed. Based on our experience in CPCs with different powder and cement liquid compositions –25 and hThe α-tricalcium phosphate (α-TCP) with Ca/P molar ratio of 1.50 was prepared by heating a mixture consisting of equimolar amounts of reagent grade calcium carbonate and dicalcium phosphate anhydrous (DCPA)  at 1100 °C for 8 h in a furnace  and quenched in air. The α-TCP was ground for 6 min in the planetary ball mill  obtaining 90 mass % of α-TCP particles between 13 μm and 20 μm in diameter with median particle size of 15.8 μm ± 1.2 μm in diameter  . Additional portion of DCPA was ground in ethanol in the planetary ball mill for 24 h. The ground DCPA particles had median diameter of 1.4 μm ± 0.2 μm (n = 3) and 90 mass % of particles had a size distribution between 1.1 μm and 1.6 μm in diameter.2PO4 and 0.5 mol/L Na2HPO4 solutions. CPC specimens were prepared by placing the powdered mixture on a glass mixing slab, dispensing the liquid on the powder using a micropipette, and mixing the paste using a stainless steel spatula for 30 s. The powder to liquid ratio (P/L) that produced paste with optimal consistency was 3 g/mL.The CPC powdered mixture with a Ca/P molar ratio of 1.33 was prepared by mixing 69.5 mass % of α-TCP and 30.5 mass % of DCPA (molar ratio of α-TCP/DCPA is 1:1). Distilled water or a 0.5 mol/L phosphate solution with pH = 6.1 were used as cement liquids. The phosphate solution was prepared by mixing equal volumes of 0.5 mol/L NaHHT) determinations, 400 mg of CPC powder was mixed with 133 μL of cement liquid. The paste was packed into a stainless steel mold with a cylindrical hole of 6 mm in diameter and 3 mm in height. A pressure of 0.7 MPa was applied for 10 s, after which both the top and bottom surfaces of the mold were covered with glass plates and kept in a 100 % humidity box at 37 °C. Because cement hardening occurred very fast when the phosphate solution was the cement liquid, it was desirable to complete the sample preparation procedure within the shortest possible time. In the present study, the sample preparation time starting from delivery of the liquid onto the cement powder to completion of packing the cement paste into the mold was less than 60 s. The HT was determined by a large Gilmore needle.For hardening time (DTS) determination 154.3 mg of the CPC powder was mixed with 51.4 μL of cement liquid. The paste was mixed for 30 s and placed into the cylindrical stainless steel mold as previously described [c(CaCl2)= 1.15 mmol/L, c(KH2PO4) = 1.20 mmol/L, c(NaCl) = 133 mmol/L, c(HEPES) = 50 mmol/L with pH of 7.4 adjusted by addition of KOH. The PLS was replaced with fresh PLS every day. The time periods between sample preparation and DTS measurements were 1 d, 3 d and 7 d. Before the DTS measurement, the diameter and height of each cylindrical specimen were measured with a micrometer. DTS was measured using a Universal Testing Machine  at a loading rate of 10 mm/min.For diametral tensile strength (escribed . Brieflyescribed  and keptescribed  at 37 °Cd) determinations, additional DTS specimens were dried for 24 h at 37 °C. Diameter (D), height (H) and mass (m) of dried pellet were measured and density (d) was determined using the equation d=4 m/(2Dπ H). Porosity (P), expressed as volume percentage of empty space in the pellet, was calculated from the determined density (d) and theoretical density td= 3.14 g/cm3 of HA [P= 100 × (1 −d/td).For density (m3 of HA  using eqThe phase composition and morphology of solid phases were determined using powder x-ray diffraction (XRD)  and scanning electron microscopy (SEM) , respectively. The amount of non-reacted  α-TCP and DCPA was estimated from reduced intensities of α-TCP and DCPA diffraction lines in CPC samples compared to the initial intensities in α-TCP and DCPA powdered mixture.A commercially obtained statistical analysis program, Kwikstat , was used to perform ANOVA . In this study, the standard deviation was considered as the standard uncertainty for each measured variable.DTS specimens had diameter in the range from 6.00 mm to 6.03 mm, height in the range from 2.67 mm to 2.83 mm, and mass in the range from 141.3 mg to 150.2 mg. The DTS, density and porosity for specimens kept in PLS and determined 1 d or 3 d after preparation are listed in The hardening time of the CPC paste with distilled water as a cement liquid was 30 min ± 2 min (n = 6). The There were no significant (p > 0.05) differences between the values determined at two different times for any of the three measured properties.In θ of 10.8°, 26.3°, 31.8°, 32.2°, and 32.9° indicated formation of apatitic precipitate (HA) after 1 d and 3 d  as the cement liquid was 5.0 min ± 0.5 min (n = 6). The θ= 4.67° in the XRD pattern. The intensity of this line slightly increased with time for specimens kept in PLS for 1 d (The XRD of powdered CPC specimens after 1 d (4 h in water vapor and 20 h in PLS) showed formation of OCP (or OCP-like precipitate with combined OCP and HA layers) and the presence of residual α-TCP and DCPA particles . The ind for 1 d , 3 d  shorter Previous studies have shown that phosphate solutions significantly shorten the hardening time of several HA-forming CPCs composed of widely different initial ingredients , 31, 32.HT of 32 min was almost identical as the HT of 30 min measured in the present study. Bermudez et al., [Some of the properties of the CPCs prepared with distilled water as the cement liquid determined in the present study can be compared with the findings of Bermudez et al.,  in which et al.,  reported et al.,  reported et al.,  and 0.5 DTS of the OCP-forming CPC reported by Bermudez et al., [DTS than HA-forming cements, regardless of particle sizes of the cement starting materials.The  et al.,  was abouAlthough OCP can readily form by the spontaneous hydrolysis of α-TCP at pH of about 6.1 ,34, our In the present studies the CPC reaction products were HA or OCP-like precipitate (OCP + HA), depending, respectively, on whether water or a phosphate solution was used as the cement liquid. In either case the large amounts of α-TCP and DCPA remained unreacted; about 55 mass % with phosphate solution after 7 d incubation in PLS, and about 50 mass % with water as the cement liquid after 3 d incubation in PLS. In CPCs composed of TTCP and DCPA, with particle sizes similar to those of α-TCP and DCPA, respectively, used in the present study, HA was the major phase in the product (about 85 mass %) after 1 d with only a small amount (15 mass %) of TTCP left unreacted , 25. TTCIn our preliminary studies powder mixtures consisting of the same α-TCP and DCPA but with Ca/P molar ratios of 1.25 and 1.40 were prepared. These powdered mixtures when mixed with 0.5 mol/L phosphate solution at P/L of 3.0 g/mL hardened in 5.0 min ± 0.5 min (n = 6) and produced OCP as a product. In other preliminary experiments the α-TCP and DCPA powder mixture with the Ca/P ratios of 1.33 were mixed with 0.5 mol/L phosphate solutions with pHs of 5.4 or 6.6 at P/L of 3 g/mL, and these pastes also hardened in 5.0 min ± 0.5 min (n = 6) and produced OCP precipitates. These preliminary findings indicated that fast hardening OCP forming CPCs with a range of Ca/P ratios and using cement liquids of different pHs can be formulated.in vitro and in vivo studies are warranted to investigate whether OCP forming CPCs may have greater osteoconductivity than CPCs that form HA, carbonated HA, Ca-deficient HA, or brushite as the major phase.A previous study  showed tThe α-TCP and DCPA reaction products were HA or OCP + HA, depending, respectively, on whether water or a phosphate solution was used as the cement liquid. In either case α-TCP and DCPA remained unreacted in the amount of about 50 mass % when water and about 55 mass % when 0.5 mol/L phosphate solution was the cement liquid. The presence of phosphate in cement liquid accelerated the cement hardening from 30 min to 5 min, and induced formation of OCP.