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e at the time of IOP measurement. Histological analysis Eyes from at least 2 mice of the listed strains were fixed (4% paraformaldehyde or Fekete's acid-alcohol-formalin fixative) processed, paraffin embedded and sectioned as previously reported [15,79], except that the paraformaldehyde was buffered w
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al images of sections from all cases are available at . Mice were anesthetized deeply with Avertin (1.25% 2,2,2-tribromoethanol and 0.8% tert-pentyl alcohol in water, 0.5-1.0 ml ip) and perfused through the left ventricle with 0.9% sodium phosphate buffered (PB) saline (pH 7.4) followed by 1.25% glutarald
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the time of IOP measurement. Histological analysis Eyes from at least 2 mice of the listed strains were fixed (4% paraformaldehyde or Fekete's acid-alcohol-formalin fixative) processed, paraffin embedded and sectioned as previously reported [15,79], except that the paraformaldehyde was buffered with 0.1
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parate group of the same age. Anesthesia protocol avoids IOP alteration and allows detection of diurnal differences All IOPs were assessed using an anesthetic regime of 99 mg/kg ketamine and 9 mg/kg xylazine (defined as 1X). Initial experiments suggested that an almost identical dose (100 mg/kg ketamine and
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1 and BRCA2 are involved in recombinational repair of DNA damage. BRCA1 and BRCA2 form discrete nuclear foci during the S phase and after exposure to DNA damaging agents [9,10,11]. These foci are probably sites of repair of spontaneous and induced DNA damage [12,13,14]. Cell lines defective in either B
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97), was cloned in-frame into the EcoRI/XhoI sites of an expression vector pET32b (Novagen, Madison, WI). Sequences at the junctions were verified by DNA sequencing. Milligrams of fusion proteins between thioredoxin and DAZAP1 were prepared and purified on His-Bind metal chelation resins (Novagen, Madi
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and homologous recombination Abstract Two recent papers provide new evidence relevant to the role of the breast cancer susceptibility gene BRCA2 in DNA repair. Moynahan et al provide genetic data indicating a requirement for BRCA2 in homology-dependent (recombinational) repair of DNA double-strand br
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subnuclear RAD51 foci with normal efficiency, suggesting that these proteins are required for the formation of recombinase complexes at the sites of DNA damage [20,21]. Finally, genetic measurements of recombination frequency have shown that Brca1-/- embryonic stem (ES) cells are deficient in recombin
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ia the synthesis-dependent annealing mechanism. Figure 2 Potential roles of BRCA2 in promoting assembly of Rad51 at sites of DNA damage. Chromosomal DNA is shown as pairs of straight lines, Rad51 as open circles, and BRCA2 as grey bars. (a) Prevention of nonproductive DNA interactions. BRCA2–Rad51 int
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ole in assembly of RAD51 at damaged sites (Fig. 2b). Several proteins that interact with RAD51 are thought to 'mediate' assembly of RAD51 at sites of DNA damage (reviewed in [30]). RAD51 paralogs in particular, including XRCC2, XRCC3, RAD51B, RAD51C and RAD51D, may function as a complex that actively p
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ty gene BRCA2 in DNA repair. Moynahan et al provide genetic data indicating a requirement for BRCA2 in homology-dependent (recombinational) repair of DNA double-strand breaks. The second paper, by Davies et al, begins to address the mechanism through which BRCA2 makes its contribution to recombinationa
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BRCA2 as grey bars. (a) Prevention of nonproductive DNA interactions. BRCA2–Rad51 interaction is proposed to suppress RAD51–BRCA2 interactions until DNA damage is present. When damage occurs, Rad51 is recruited to damaged sites where is polymerizes into nucleoprotein filaments. In this model, BRCA2 is
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r by Davies et al, data provide evidence for a plausible mechanism of the dominant negative effect associated with expression of a single BRC repeat. DNA binding assays and electron microscopy methods were used to show that a BRC3 peptide interferes with the ability of RAD51 to assemble into oligomeric
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event a substantial fraction of Rad51 from being sequestered in a nonfunctional form. In a BRCA2-defective cell, mutant Rad51 becomes associated with DNA at random sites and is therefore not readily recruited to sites of damage. (b) Positive regulation.  Transient transfection of an I-SceI expression vector results in the production of a DSB in the first mutated copy of GFP. One or both DNA ends formed by the break invade(s) the homologous sequence in the second mutant GFP copy, resulting in repair of the DSB via a homology-mediated gene
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es of DNA damage. Chromosomal DNA is shown as pairs of straight lines, Rad51 as open circles, and BRCA2 as grey bars. (a) Prevention of nonproductive DNA interactions. BRCA2–Rad51 interaction is proposed to suppress RAD51–BRCA2 interactions until DNA damage is present. When damage occurs, Rad51 is recr
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clear sites with BRCA1 [9], a number of additional results have provided evidence that both BRCA1 and BRCA2 are involved in recombinational repair of DNA damage. BRCA1 and BRCA2 form discrete nuclear foci during the S phase and after exposure to DNA damaging agents [9,10,11]. These foci are probably si
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gene conversion occurs via the synthesis-dependent annealing mechanism. Figure 2 Potential roles of BRCA2 in promoting assembly of Rad51 at sites of DNA damage. Chromosomal DNA is shown as pairs of straight lines, Rad51 as open circles, and BRCA2 as grey bars. (a) Prevention of nonproductive DNA inter
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gaag) were designed from intronic sequences flanking Dazap1 exon 8. These primers amplified a 271 bp fragment from mouse but not hamster genomic DNA. DNA samples of a panel of 100 radiation hybrids containing mouse chromosome fragments in a hamster background were purchased from Research Genetics (Hunt
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clear foci during the S phase and after exposure to DNA damaging agents [9,10,11]. These foci are probably sites of repair of spontaneous and induced DNA damage [12,13,14]. Cell lines defective in either BRCA1 or BRCA2 are sensitive to damaging agents that form double-strand breaks (DSBs), as are other
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ve cells Pierce et al have designed a set of recombination substrates for measuring the level of homologous recombination in vivo (Fig. 1) [25]. The DNA substrate contains a pair of mutated GFP genes (GFP encodes the easily detected green fluorescent protein), one of which contains a restriction site
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tgtctgaag) were designed from intronic sequences flanking Dazap1 exon 8. These primers amplified a 271 bp fragment from mouse but not hamster genomic DNA. DNA samples of a panel of 100 radiation hybrids containing mouse chromosome fragments in a hamster background were purchased from Research Genetics
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ing of one of the eight BRC repeats from human BRCA2. Expression of a single BRC repeat (BRC4) had previously been reported to act as an inhibitor of DNA repair by Chen et al [27]. These investigators showed that expression of constructs containing the BRC4 repeat in MCF-7 cells enhances the radiosensi
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s and electron microscopy methods were used to show that a BRC3 peptide interferes with the ability of RAD51 to assemble into oligomeric filaments on DNA. A BRC4 peptide was also reported to inhibit the RAD51–DNA interaction. These experiments raise the possibility that the full-length BRCA2 protein co
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l population due to a mutated W (White spotted) gene. GC1 and MT4 are mouse germ cell and Sertoli cell lines, respectively, and gDNA is mouse genomic DNA. The PCR primers span over introns and produce much larger (if any) fragments from genomic DNA. To study the expression of the DAZAP1 protein, two a
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addition, suppression of RAD51–ssDNA interaction might be important for preventing RAD51 from binding to the regions of ssDNA that form during normal DNA replication. BRCA2 may thus promote RAD51 assembly into recombinational repair complexes via a negative regulatory mechanism (i.e. by blocking RAD51–
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onal DSB repair [22,23]. The similarity between phenotypes associated with BRCA1 and BRCA2 deficiency, together with data showing a similar effect of DNA damage on distribution of BRCA1 and BRCA2 in repair-proficient cells, led to the hypothesis that BRCA2, like BRCA1, is required for efficient recombi
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BRC3 peptide interferes with the ability of RAD51 to assemble into oligomeric filaments on DNA. A BRC4 peptide was also reported to inhibit the RAD51–DNA interaction. These experiments raise the possibility that the full-length BRCA2 protein could act as a negative regulator of inappropriate RAD51–DNA
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–DNA interaction. These experiments raise the possibility that the full-length BRCA2 protein could act as a negative regulator of inappropriate RAD51–DNA interaction. The final experiment reported by Davies et al [26] is particularly important with respect to understanding the mechanism(s) through whi
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gure 2 are not mutually exclusive. BRCA2 could prevent inappropriate RAD51 assembly in the absence of damage and promote functional RAD51 assembly at DNA lesions during the damage response. Further experiments are clearly required to clarify the functional interactions between BRCA2 and RAD51 during re
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li cell lines, respectively, and gDNA is mouse genomic DNA. The PCR primers span over introns and produce much larger (if any) fragments from genomic DNA. To study the expression of the DAZAP1 protein, two antibodies against mouse DAZAP1 were generated. The anti DAZAP1-C antibody was raised against a
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cluding the large size of BRCA2, promise to make biochemical characterization of BRCA2 function difficult. Abbreviations DSB = double-strand break; dsDNA = double-strand DNA; ES = embryonic stem; GFP = green fluorescent protein; ssDNA = single-strand DNA. References 1. Shinohara A, Ogawa H, Ogawa T.
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mage occurs. For example, previous studies indicated that RAD51 shows little binding preference for single-strand DNA (ssDNA) over double-strand DNA (dsDNA) [3,29]. This result could mean that regulatory factors, such as BRCA2, are required to suppress RAD51–dsDNA interactions to prevent sequestration of
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the large size of BRCA2, promise to make biochemical characterization of BRCA2 function difficult. Abbreviations DSB = double-strand break; dsDNA = double-strand DNA; ES = embryonic stem; GFP = green fluorescent protein; ssDNA = single-strand DNA. References 1. Shinohara A, Ogawa H, Ogawa T. Rad51 protein involv
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air complex when damage occurs. For example, previous studies indicated that RAD51 shows little binding preference for single-strand DNA (ssDNA) over double-strand DNA (dsDNA) [3,29]. This result could mean that regulatory factors, such as BRCA2, are required to suppress RAD51–dsDNA interactions to prevent sequestra
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rand DNA (ssDNA) over double-strand DNA (dsDNA) [3,29]. This result could mean that regulatory factors, such as BRCA2, are required to suppress RAD51–dsDNA interactions to prevent sequestration of RAD51 in a nonfunctional form. Alternatively, or in addition, suppression of RAD51–ssDNA interaction might b
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d by the neuron-specific nuclear RNA-binding protein, Nova-1. Nova-1 regulates the alternative splicing of the pre-mRNAs encoding neuronal inhibitory glycine receptor α2 (GlyR α2) [23]. The ability of Nova-1 to activate exon selection in neurons is antagonized by a second RNA-binding protein, brPTB (brain-
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equences at the junctions were verified by DNA sequencing. Milligrams of fusion proteins between thioredoxin and DAZAP1 were prepared and purified on His-Bind metal chelation resins (Novagen, Madison, WI). The proteins were mixed with Freund's adjuvant and injected into rabbits to generate the anti-DAZ
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derstanding of the molecular mechanisms controlling diurnal rhythms of IOP and that may be relevant to glaucoma. Car2 deficiency does not alter IOP Bicarbonate formation is important for aqueous humor secretion from the ciliary processes and carbonic anhydrase (CA) facilitates this secretion. There are multi
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ay, NJ). Fractionation of mouse testicular extracts Adult mouse testes were homogenized in a buffer containing 20 mM Tris, pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.3% NP-40, 40 U/ml of Rnasin ribonuclase inhibitor (Promega, Madison, WI), and a mixture of 10 protease inhibitors provided in the Protease Inhibit
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g for 10 minutes to pellet the mitochondria, aliquots of the supernatant were applied to 15–45% sucrose gradients in 20 mM Tris, 100 mM KCl and 5 mM MgCl2 and centrifuged in a Beckman SW41 rotor at 39,000 rpm for 2 hours at 4°C. Fractions of 0.5 ml were collected from the bottom of the tubes and analyze
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Recently, the Xenopus orthologue of DAZAP1, Prrp, was identified and characterized [16]. Prrp binds to a 340 nt sequence in the 3' UTR of Xenopus Vg1 mRNA. This Vg1 localization element (VLE) is sufficient for the migration and clustering of Vg1 mRNA to the vegetal cortex of mature oocyte. Prrp also int
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tting. Discussion RNA-binding proteins have been found to participate in many cellular functions, including RNA transcription, pre-mRNA processing, mRNA transport, localization, translation and stability [20]. A role for the DAZ family in the regulation of mRNA translation is supported by lines of cir
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ee-hybrid system to bind specifically to oligo(U) stretches interspersed by G or C residues, including a U-rich segment in the 5' UTR of mouse Cdc25C mRNA [13]. In an attempt to elucidate the function of the DAZ gene family and to understanding the mechanisms of its action, we used a yeast two-hybrid s
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and brain, and even less in other tissues. This pattern of expression is similar to that of the human DAZAP1 (14). RT-PCR analyses showed that Dazap1 mRNA was already present in fetal testes at embryonic day 15, similar to Dazl1 mRNA (Figure 3). The expression of both Dazl1 and Dazap1 persisted througho
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proteins may act together to facilitate the expression of a set of genes in germ cells. For example, DAZAP1 could be involved in the transport of the mRNAs of the target genes of DAZL. Alternatively, DAZL and DAZAP1 may act antagonistically to regulate the timing and the level of expression. Such an anta
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. Its predominant expression in testes suggests a role in spermatogenesis. Its subcellular localization indicates that it is not directly involved in mRNA translation. Background Spermatogenesis is a complex developmental process in which male germ cells progress through mitotic proliferation, meiotic
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mouse testes, DAZAP1 was found both in the nuclei and in the cytoplasm. Its absence from polyribosomes indicates that it is not directly involved in mRNA translation. Materials and methods Isolation of mouse Dazap1 cDNA clones Dazap1 cDNA clones were isolated from a mouse testis cDNA library (#93730
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RNA transcription, pre-mRNA processing, mRNA transport, localization, translation and stability [20]. A role for the DAZ family in the regulation of mRNA translation is supported by lines of circumstantial evidence, including the association of DAZL with polyribosomes [12]. The absence of DAZAP1 from p
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ed by Western blotting. Discussion RNA-binding proteins have been found to participate in many cellular functions, including RNA transcription, pre-mRNA processing, mRNA transport, localization, translation and stability [20]. A role for the DAZ family in the regulation of mRNA translation is supporte
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lso interacts through its proline-rich domain with two microfilament-associated proteins profilin and Mena, which may facilitate the anchoring of Vg1 mRNA to the vegetal cortex. The Vg1 RNA encodes a member of the transforming growth factor-β family that is required for generating dorsal mesoderm at the
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nds to a 340 nt sequence in the 3' UTR of Xenopus Vg1 mRNA. This Vg1 localization element (VLE) is sufficient for the migration and clustering of Vg1 mRNA to the vegetal cortex of mature oocyte. Prrp also interacts through its proline-rich domain with two microfilament-associated proteins profilin and M
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haracterization of the mouse Dazap1 gene and its protein product. The subcellular localization of DAZAP1 suggests that it is not involved directly in mRNA translation. Results Characterization of the mouse Dazap1 cDNA Mouse Dazap1 cDNA clones were isolated by library screening, and the 5' end of the
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ressed later, in the cytoplasm of pachytene spermatocytes [5]. Genetic and biochemical studies suggest a role for the DAZ family in the regulation of mRNA translation. Drosophila Boule mutants was defective in the translation of the meiosis-specific CDC25 homologue, Twine [11], and DAZL was found to be
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RNA-binding proteins is exemplified by the neuron-specific nuclear RNA-binding protein, Nova-1. Nova-1 regulates the alternative splicing of the pre-mRNAs encoding neuronal inhibitory glycine receptor α2 (GlyR α2) [23]. The ability of Nova-1 to activate exon selection in neurons is antagonized by a seco
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r to that of the human DAZAP1 (14). RT-PCR analyses showed that Dazap1 mRNA was already present in fetal testes at embryonic day 15, similar to Dazl1 mRNA (Figure 3). The expression of both Dazl1 and Dazap1 persisted throughout testes development, in both the prenatal and postnatal periods. Dazl1 and Da
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on resins (Novagen, Madison, WI). The proteins were mixed with Freund's adjuvant and injected into rabbits to generate the anti-DAZAPl-C antibody. An oligopeptide containing the last 19 ammo acid residues of the mouse DAZAP1 was synthesized in vitro using the services of Bethyl Laboratories (Montgomery, TX). Th
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ntron insertion sites. Generation of anti-DAZAP1 antibodies Antibodies were generated against both a recombinant protein produced in E. coli and an oligopeptide synthesized in vitro. The insert of a Dazap1 cDNA clone P21, which encoded the C-terminal portion of DAZAP1 (starting from aa #197), was cloned in-fr
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ntibody was raised against a recombinant protein containing the C-terminal proline-rich portion, and the anti DAZAP1-P antibody was raised against an oligopeptide containing the last 19 amino acid residue at the C-terminus. Both antibodies recognized in vitro synthesized DAZAP1 in an immunoprecipitation assay (
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was conjugated to KLH as carrier and injected into a goat. The anti-DAZAP1-P antibody thus produced was purified on an affinity column containing the oligopeptide antigen. Western blotting Mouse tissues were homogenized in the RIPA lysis buffer (150 mM NaCl, 1.0% NP-40, 0.5% DOC, 0.1% SDS, 50 mM Tris, pH 8.0)
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t negative effect associated with expression of a single BRC repeat. DNA binding assays and electron microscopy methods were used to show that a BRC3 peptide interferes with the ability of RAD51 to assemble into oligomeric filaments on DNA. A BRC4 peptide was also reported to inhibit the RAD51–DNA interact
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ontaining the last 19 ammo acid residues of the mouse DAZAP1 was synthesized in vitro using the services of Bethyl Laboratories (Montgomery, TX). The peptide was conjugated to KLH as carrier and injected into a goat. The anti-DAZAP1-P antibody thus produced was purified on an affinity column containing the
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The second paper providing evidence, by Davies et al [26], is a biochemical study of the interaction between the homologous recombinase RAD51 and a peptide consisting of one of the eight BRC repeats from human BRCA2. Expression of a single BRC repeat (BRC4) had previously been reported to act as an inhib
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ge has occurred and factors required for 'productive' assembly of RAD51 at damaged sites are in place). As mentioned previously, the ability of a BRC peptide to repress the RAD51–BRCA2 interaction could reflect a role of full-length protein in suppressing unwanted RAD51–BRCA2 interactions. Yet another pot
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on microscopy methods were used to show that a BRC3 peptide interferes with the ability of RAD51 to assemble into oligomeric filaments on DNA. A BRC4 peptide was also reported to inhibit the RAD51–DNA interaction. These experiments raise the possibility that the full-length BRCA2 protein could act as a neg
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, Piscataway, NJ). Fractionation of mouse testicular extracts Adult mouse testes were homogenized in a buffer containing 20 mM Tris, pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.3% NP-40, 40 U/ml of Rnasin ribonuclase inhibitor (Promega, Madison, WI), and a mixture of 10 protease inhibitors provided in the Prot
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n at 10,000 × g for 10 minutes to pellet the mitochondria, aliquots of the supernatant were applied to 15–45% sucrose gradients in 20 mM Tris, 100 mM KCl and 5 mM MgCl2 and centrifuged in a Beckman SW41 rotor at 39,000 rpm for 2 hours at 4°C. Fractions of 0.5 ml were collected from the bottom of the tu
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ontaining 20 mM Tris, pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.3% NP-40, 40 U/ml of Rnasin ribonuclase inhibitor (Promega, Madison, WI), and a mixture of 10 protease inhibitors provided in the Protease Inhibitors Set (Roche Molecular Biochemicals, Indianapolis, IN). Homogenates were centrifuged at 1,000 × g for 10 minutes to
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KCl, 5 mM MgCl2, 0.3% NP-40, 40 U/ml of Rnasin ribonuclase inhibitor (Promega, Madison, WI), and a mixture of 10 protease inhibitors provided in the Protease Inhibitors Set (Roche Molecular Biochemicals, Indianapolis, IN). Homogenates were centrifuged at 1,000 × g for 10 minutes to pellet cell debris and nuclei. Afte
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sites are in place). As mentioned previously, the ability of a BRC peptide to repress the RAD51–BRCA2 interaction could reflect a role of full-length protein in suppressing unwanted RAD51–BRCA2 interactions. Yet another potential role for BRCA2 in promoting RAD51-dependent recombinational repair is a posi
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absence of DAZAP1 from polyribosomes indicates that it is not directly involved in protein synthesis. This finding is different from two RNA-binding proteins, FXR1P and FXR2P, that were identified through their interaction with another polysomal-associated RNA-binding protein, the fragile X mental retardat
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tein (3418 amino acids) offers very little clue to its function, although there are eight repeated segments (termed BRC repeats) in the middle of the protein that are highly conserved among mammalian orthologs [7,8]. Breast cancer susceptibility genes and DNA repair Following the landmark discovery by Sc
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titutions and two deletions/insertions. The mammalian proteins shares 89% similarity and 81% identity with Xenopus Prrp (for proline-rich RNA binding protein) [16]. The two RBDs are especially highly conserved. They share 98% and 97% similarity and 97% and 92% identity, respectively, between DAZAP1 and Prr
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P and FXR2P are associated with the polyribosomes [22]. The significance of the interaction between DAZAP1 and DAZL/DAZ remains to be defined. These proteins may act together to facilitate the expression of a set of genes in germ cells. For example, DAZAP1 could be involved in the transport of the mRNAs of
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the expression of the DAZAP1 protein, two antibodies against mouse DAZAP1 were generated. The anti DAZAP1-C antibody was raised against a recombinant protein containing the C-terminal proline-rich portion, and the anti DAZAP1-P antibody was raised against an oligopeptide containing the last 19 amino acid r
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y virtue of their expression of GFP. Moynahan et al [24] have used such a GFP recombination substrate to demonstrate that cells with defective BRCA2 protein are deficient in their ability to repair the I-SceI-induced DSB through homologous recombination. Expression of I-SceI resulted in 1 out of 1400 cell
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mpletely independent of its defect in BRCA2. First, in the same study [24], recombination in a mouse ES cell line that expresses only truncated BRCA2 protein was measured. This line was found to have lower recombination efficiency than isogenic cells expressing full-length BRCA2 (the defect observed was ab
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striatum [45]. Another gene that falls into the Bsc10a interval is Macs, the gene encoding the myristoylated alanine-rich C kinase substrate (MARCKS protein). This molecule is important in cerebral development. MARCKS-deficient mice have a high incidence of exencephaly, agenesis of the corpus callosum, an
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mouse Dazap1 gene encoding an RNA-binding protein that interacts with infertility factors DAZ and DAZL Abstract Background DAZAP1 (DAZ Associated Protein 1) was originally identified by a yeast two-hybrid system through its interaction with a putative male infertility factor, DAZ (Deleted in Azoospermi
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via homologous recombination in the human pancreatic tumor cell line CAPAN-1. CAPAN-1 cells carry a deletion of BRCA2 on one homolog and codes for a protein truncated at amino acid 1981 on the other homolog. The authors indicate that the level of I-SceI-induced recombination in CAPAN-1 is over 100-fold le
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t. Sedimentation was from left to right. The presence of DAZAP1 and DAZL in each fractions was analyzed by Western blotting. Discussion RNA-binding proteins have been found to participate in many cellular functions, including RNA transcription, pre-mRNA processing, mRNA transport, localization, translatio
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by centrifugation at 10,000 × g for 10 minutes. Protein concentration of the tissue extracts was determined by the Bradford method using the Bio-Rad Protein Assay system (Bio-Rad, Hercules, CA). About 50 μg of tissue extracts were separated on 10% SDS-polyacrylamide gels and blotted with either the anti-D
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, agenesis of the corpus callosum, and abnormalities other forebrain structure including widespread neocortical ectopias [46, 47]. The MARCKS-related protein gene is expressed in the striatum during early brain development in the rat [48]. The location of the QTL modulating striatal neuron number to the d
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e for direct RAD51 interaction. Cells lacking BRCA1/2 fail to form damage-induced subnuclear RAD51 foci with normal efficiency, suggesting that these proteins are required for the formation of recombinase complexes at the sites of DNA damage [20,21]. Finally, genetic measurements of recombination frequency
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sophila[7], C. elegans[8], and Xenopus[9] also affect the fertility in either males, females, or both sexes. The DAZ gene family encodes RNA binding proteins that are expressed specifically in germ cells. DAZ and DAZL are expressed in the nucleus and cytoplasm of primordial germ cells and spermatogonia, an
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R1P and FXR2P, that were identified through their interaction with another polysomal-associated RNA-binding protein, the fragile X mental retardation protein [21]. Both FXR1P and FXR2P are associated with the polyribosomes [22]. The significance of the interaction between DAZAP1 and DAZL/DAZ remains to be
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ed in CAPAN-1 cells, the same human tumor line examined by Moynahan et al [24]. It had been shown previously that the small amount of truncated BRCA2 protein expressed by CAPAN-1 cells was mislocalized to the cytoplasm, consistent with the fact that the truncation protein lacks a nuclear localization signa
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em, DAZAP1, is expressed predominantly in testes. It encodes a protein with two RNA binding domains and a proline rich C-terminal portion. The DAZAP1 protein interacted with both DAZ and DAZL in vitro. It also bound to RNA homopolymers. We now report our characterization of the mouse Dazap1 gene and its pr
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riginal URL. -------------------------------------------------------------------- Characterization of the mouse Dazap1 gene encoding an RNA-binding protein that interacts with infertility factors DAZ and DAZL Abstract Background DAZAP1 (DAZ Associated Protein 1) was originally identified by a yeast tw
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he two RBDs are especially highly conserved. They share 98% and 97% similarity and 97% and 92% identity, respectively, between DAZAP1 and Prrp. These proteins may therefore have a similar RNA binding specificity. The C-terminal proline-rich portions of DAZAP1 and Prrp are less conserved (81% similarity and
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se genomic DNA. The PCR primers span over introns and produce much larger (if any) fragments from genomic DNA. To study the expression of the DAZAP1 protein, two antibodies against mouse DAZAP1 were generated. The anti DAZAP1-C antibody was raised against a recombinant protein containing the C-terminal pr
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infertility factor, DAZ (Deleted in Azoospermia). In vitro, DAZAP1 interacts with both the Y chromosome-encoded DAZ and an autosome-encoded DAZ-like protein, DAZL. DAZAP1 contains two RNA-binding domains (RBDs) and a proline-rich C-terminal portion, and is expressed most abundantly in the testis. To under
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. Second, in an independent study, Powell was able to compare derivatives of CAPAN-1 that differed only in their ability to express full-length BRCA2 protein (S. Powell, personal communication, 2001). In these experiments, the derivative expressing full-length BRCA2 yielded 10-fold more recombinants than c
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NA-binding domains (RBDS) and a C-terminal portion that is rich in proline (Figure 1). It is highly conserved evolutionarily. The mouse and the human proteins differ in 9 amino acids only, with 7 substitutions and two deletions/insertions. The mammalian proteins shares 89% similarity and 81% identity with X
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tein product. Results The human and mouse genes have similar genomic structures and map to syntenic chromosomal regions. The mouse and human DAZAP1 proteins share 98% identity and their sequences are highly similar to the Xenopus orthologue Prrp, especially in the RBDs. Dazap1 is expressed throughout test
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nal inhibitory glycine receptor α2 (GlyR α2) [23]. The ability of Nova-1 to activate exon selection in neurons is antagonized by a second RNA-binding protein, brPTB (brain-enriched polypyrimidine tract-binding protein), which interacts with Nova-1 and inhibits its function [24]. DAZAP1 could function in a
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ng dorsal mesoderm at the blastula stage of Xenopus embryogenesis [25]. Sequence conservation between the RBDs of DAZAP1 and Prrp suggests that these proteins may bind to similar RNA sequences. However, a BLAST search of the GenBank for the 340 nt VLE sequence failed to identify any mammalian sequences with
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to the Xenopus orthologue Prrp, especially in the RBDs. Dazap1 is expressed throughout testis development. Western blot detects a single 45 kD DAZAP1 protein that is most abundant in the testis. Although a majority of DAZAP1 is present in the cytoplasmic fraction, they are not associated with polyribosomes
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f expression. Such an antagonistic interaction between two interacting RNA-binding proteins is exemplified by the neuron-specific nuclear RNA-binding protein, Nova-1. Nova-1 regulates the alternative splicing of the pre-mRNAs encoding neuronal inhibitory glycine receptor α2 (GlyR α2) [23]. The ability of N
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s a change of reading frame and results in a shorter Prrp with a different C-terminal end sequence. Figure 1 Evolutionary conservation of the DAZAP1 proteins. The amino acid sequences of the human and mouse DAZAP1s and the Xenopus Prrp are compared. The two RNA binding domains are boxed. Differences betwee
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s recognized in vitro synthesized DAZAP1 in an immunoprecipitation assay (data not shown). Western blotting of mouse tissue extracts detected a 45 kD protein that was present most abundantly in the testis, and to a lesser degree in spleen, liver, lung and brain (Figure 4). The protein was also present in t
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ation of BRCA2 function difficult. Abbreviations DSB = double-strand break; dsDNA = double-strand DNA; ES = embryonic stem; GFP = green fluorescent protein; ssDNA = single-strand DNA. References 1. Shinohara A, Ogawa H, Ogawa T. Rad51 protein involved in repair and recombination in S. cerevisiae is a R
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of DAZAP1 increased afterward, as the testes contained increasing number of proliferating and meiotic germ cells. Figure 4 Expression of the DAZAP1 protein in adult mouse tissues. Equal amounts of total protein from various tissue extracts were applied to a 10% SDS-polyacrylamide gel and western blotted
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nds Prrp, and possibly DAZAP1, may help to identify the natural substrates of DAZAP1. Conclusions DAZAP1 is an evolutionarily conserved RNA-binding protein. It is present at variable levels in many tissues. Its predominant expression in testes suggests a role in spermatogenesis. In mouse testes, DAZAP1 w
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share 85%, 90%, and 97% similarity with segments in the human 3' UTR, respectively. These segments probably contain regulatory elements. The DAZAP1 protein contains two RNA-binding domains (RBDS) and a C-terminal portion that is rich in proline (Figure 1). It is highly conserved evolutionarily. The mouse
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RI/XhoI sites of an expression vector pET32b (Novagen, Madison, WI). Sequences at the junctions were verified by DNA sequencing. Milligrams of fusion proteins between thioredoxin and DAZAP1 were prepared and purified on His-Bind metal chelation resins (Novagen, Madison, WI). The proteins were mixed with Fre
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he small amount of truncated BRCA2 protein expressed by CAPAN-1 cells was mislocalized to the cytoplasm, consistent with the fact that the truncation protein lacks a nuclear localization signal [28]. Davies et al showed CAPAN-1 cells to be defective in nuclear localization of RAD51, raising the possibility
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acts detected a 45 kD protein that was present most abundantly in the testis, and to a lesser degree in spleen, liver, lung and brain (Figure 4). The protein was also present in the ovary. The expression of DAZAP1 during germ cell development paralleled that of DAZL (Figure 5). It is present at a low level
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ce, including the association of DAZL with polyribosomes [12]. The absence of DAZAP1 from polyribosomes indicates that it is not directly involved in protein synthesis. This finding is different from two RNA-binding proteins, FXR1P and FXR2P, that were identified through their interaction with another poly
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clustering of Vg1 mRNA to the vegetal cortex of mature oocyte. Prrp also interacts through its proline-rich domain with two microfilament-associated proteins profilin and Mena, which may facilitate the anchoring of Vg1 mRNA to the vegetal cortex. The Vg1 RNA encodes a member of the transforming growth fact
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(DSBs), as are other cell lines defective in recombinational repair (reviewed in [15]). BRCA2 interacts with the RAD51 recombinase via direct protein-protein contacts [16,17,18,19]. Biochemical analysis also showed interaction between BRCA1 and RAD51, although these detected interactions may have been indi
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illigrams of fusion proteins between thioredoxin and DAZAP1 were prepared and purified on His-Bind metal chelation resins (Novagen, Madison, WI). The proteins were mixed with Freund's adjuvant and injected into rabbits to generate the anti-DAZAPl-C antibody. An oligopeptide containing the last 19 ammo acid
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ifferent from two RNA-binding proteins, FXR1P and FXR2P, that were identified through their interaction with another polysomal-associated RNA-binding protein, the fragile X mental retardation protein [21]. Both FXR1P and FXR2P are associated with the polyribosomes [22]. The significance of the interaction
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ty of Nova-1 to activate exon selection in neurons is antagonized by a second RNA-binding protein, brPTB (brain-enriched polypyrimidine tract-binding protein), which interacts with Nova-1 and inhibits its function [24]. DAZAP1 could function in a similar manner by binding to DAZL and inhibiting its functio
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em to isolate two human genes encoding DAZ associated proteins (DAZAPs) [14]. One of them, DAZAP1, is expressed predominantly in testes. It encodes a protein with two RNA binding domains and a proline rich C-terminal portion. The DAZAP1 protein interacted with both DAZ and DAZL in vitro. It also bound to R
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Both primer pairs annealed across intron insertion sites. Generation of anti-DAZAP1 antibodies Antibodies were generated against both a recombinant protein produced in E. coli and an oligopeptide synthesized in vitro. The insert of a Dazap1 cDNA clone P21, which encoded the C-terminal portion of DAZAP1 (
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erved evolutionarily. The mouse and the human proteins differ in 9 amino acids only, with 7 substitutions and two deletions/insertions. The mammalian proteins shares 89% similarity and 81% identity with Xenopus Prrp (for proline-rich RNA binding protein) [16]. The two RBDs are especially highly conserved. T
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end of the cDNA was isolated by 5' RACE [15]. The near fall length cDNA consists of a 53 bp 5' untranslated region (UTR), an open reading frame for a protein of 405 amino acid residues, and a 362 bp 3' UTR (GenBank Accession No: AF225910). The coding region shares 89% similarity with that of the human orth
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increasing number of proliferating and meiotic germ cells. Figure 4 Expression of the DAZAP1 protein in adult mouse tissues. Equal amounts of total protein from various tissue extracts were applied to a 10% SDS-polyacrylamide gel and western blotted with the anti-DAZAP1-P antibody. Figure 5 Western blot
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Z gene family and to understanding the mechanisms of its action, we used a yeast two-hybrid system to isolate two human genes encoding DAZ associated proteins (DAZAPs) [14]. One of them, DAZAP1, is expressed predominantly in testes. It encodes a protein with two RNA binding domains and a proline rich C-term
Text source name = 11597317.txt
gene to be discovered, and was isolated through positional cloning using data from families with inherited breast cancer [4]. Cells with mutant BRCA2 protein are, like many cancer cells, genetically unstable and accumulate gross chromosomal rearrangements [5,6]. The sequence of this large protein (3418 ami
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AP1 may act antagonistically to regulate the timing and the level of expression. Such an antagonistic interaction between two interacting RNA-binding proteins is exemplified by the neuron-specific nuclear RNA-binding protein, Nova-1. Nova-1 regulates the alternative splicing of the pre-mRNAs encoding neuron
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logous recombination in vivo (Fig. 1) [25]. The DNA substrate contains a pair of mutated GFP genes (GFP encodes the easily detected green fluorescent protein), one of which contains a restriction site for I-SceI, a yeast intron encoded endonuclease with an 18 base pair recognition site. Transient transfect
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ential role for BRCA2 in promoting RAD51-dependent recombinational repair is a positive role in assembly of RAD51 at damaged sites (Fig. 2b). Several proteins that interact with RAD51 are thought to 'mediate' assembly of RAD51 at sites of DNA damage (reviewed in [30]). RAD51 paralogs in particular, includin
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) at a concentration of 0.2 g tissue per ml of buffer. The homogenized samples were cleared of debris by centrifugation at 10,000 × g for 10 minutes. Protein concentration of the tissue extracts was determined by the Bradford method using the Bio-Rad Protein Assay system (Bio-Rad, Hercules, CA). About 50 μ
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breaks (DSBs), as are other cell lines defective in recombinational repair (reviewed in [15]). BRCA2 interacts with the RAD51 recombinase via direct protein-protein contacts [16,17,18,19]. Biochemical analysis also showed interaction between BRCA1 and RAD51, although these detected interactions may have b
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ant BRCA2 protein are, like many cancer cells, genetically unstable and accumulate gross chromosomal rearrangements [5,6]. The sequence of this large protein (3418 amino acids) offers very little clue to its function, although there are eight repeated segments (termed BRC repeats) in the middle of the prot
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nts on DNA. A BRC4 peptide was also reported to inhibit the RAD51–DNA interaction. These experiments raise the possibility that the full-length BRCA2 protein could act as a negative regulator of inappropriate RAD51–DNA interaction. The final experiment reported by Davies et al [26] is particularly importa
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BLAST search of the GenBank for the 340 nt VLE sequence failed to identify any mammalian sequences with significant homology. Further mapping of the RNA sequence within VLE that binds Prrp, and possibly DAZAP1, may help to identify the natural substrates of DAZAP1. Conclusions DAZAP1 is an evolution
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article's original URL. -------------------------------------------------------------------- Characterization of the mouse Dazap1 gene encoding an RNA-binding protein that interacts with infertility factors DAZ and DAZL Abstract Background DAZAP1 (DAZ Associated Protein 1) was originally identifi
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ch domain with two microfilament-associated proteins profilin and Mena, which may facilitate the anchoring of Vg1 mRNA to the vegetal cortex. The Vg1 RNA encodes a member of the transforming growth factor-β family that is required for generating dorsal mesoderm at the blastula stage of Xenopus embryoge
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erved. They share 98% and 97% similarity and 97% and 92% identity, respectively, between DAZAP1 and Prrp. These proteins may therefore have a similar RNA binding specificity. The C-terminal proline-rich portions of DAZAP1 and Prrp are less conserved (81% similarity and 71% identity). There is an insert
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DAZL and DAZAP1 may act antagonistically to regulate the timing and the level of expression. Such an antagonistic interaction between two interacting RNA-binding proteins is exemplified by the neuron-specific nuclear RNA-binding protein, Nova-1. Nova-1 regulates the alternative splicing of the pre-mRNA
Text source name = 11604102.txt
ressed most abundantly in the testis. Figure 3 Developmental expression of Dazap1 and Dazl in mouse testes. RT-PCR was performed on total testicular RNAs isolated from day 15 (El 5) and day 17 (El 7) embryos, new born mice (Day 0), and mice at various days after birth. Wv/Wv testes contain diminished g
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communication). The genomic structure of Dazap1, delineated here, should facilitate the generating of Dazap1 null mutation. DAZAP1 was shown to bind RNA homopolymers in vitro, with a preference for poly U and poly G. Its natural substrates have not been identified. Recently, the Xenopus orthologue of
Text source name = 11604102.txt
coding neuronal inhibitory glycine receptor α2 (GlyR α2) [23]. The ability of Nova-1 to activate exon selection in neurons is antagonized by a second RNA-binding protein, brPTB (brain-enriched polypyrimidine tract-binding protein), which interacts with Nova-1 and inhibits its function [24]. DAZAP1 coul
Text source name = 11604102.txt
human genes encoding DAZ associated proteins (DAZAPs) [14]. One of them, DAZAP1, is expressed predominantly in testes. It encodes a protein with two RNA binding domains and a proline rich C-terminal portion. The DAZAP1 protein interacted with both DAZ and DAZL in vitro. It also bound to RNA homopolyme
Text source name = 11604102.txt
Evolutionary conservation of the DAZAP1 proteins. The amino acid sequences of the human and mouse DAZAP1s and the Xenopus Prrp are compared. The two RNA binding domains are boxed. Differences between the human and the mouse sequences, and between the mouse and Xenopus sequences are marked by #'s and *
Text source name = 11604102.txt
n with two RNA binding domains and a proline rich C-terminal portion. The DAZAP1 protein interacted with both DAZ and DAZL in vitro. It also bound to RNA homopolymers. We now report our characterization of the mouse Dazap1 gene and its protein product. The subcellular localization of DAZAP1 suggests th
Text source name = 11604102.txt
VLE that binds Prrp, and possibly DAZAP1, may help to identify the natural substrates of DAZAP1. Conclusions DAZAP1 is an evolutionarily conserved RNA-binding protein. It is present at variable levels in many tissues. Its predominant expression in testes suggests a role in spermatogenesis. In mouse
Text source name = 11604102.txt
es [12]. The absence of DAZAP1 from polyribosomes indicates that it is not directly involved in protein synthesis. This finding is different from two RNA-binding proteins, FXR1P and FXR2P, that were identified through their interaction with another polysomal-associated RNA-binding protein, the fragile
Text source name = 11604102.txt
rose gradient. Sedimentation was from left to right. The presence of DAZAP1 and DAZL in each fractions was analyzed by Western blotting. Discussion RNA-binding proteins have been found to participate in many cellular functions, including RNA transcription, pre-mRNA processing, mRNA transport, localiz
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mbers of Drosophila[7], C. elegans[8], and Xenopus[9] also affect the fertility in either males, females, or both sexes. The DAZ gene family encodes RNA binding proteins that are expressed specifically in germ cells. DAZ and DAZL are expressed in the nucleus and cytoplasm of primordial germ cells and
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the level of expression. Such an antagonistic interaction between two interacting RNA-binding proteins is exemplified by the neuron-specific nuclear RNA-binding protein, Nova-1. Nova-1 regulates the alternative splicing of the pre-mRNAs encoding neuronal inhibitory glycine receptor α2 (GlyR α2) [23].
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stula stage of Xenopus embryogenesis [25]. Sequence conservation between the RBDs of DAZAP1 and Prrp suggests that these proteins may bind to similar RNA sequences. However, a BLAST search of the GenBank for the 340 nt VLE sequence failed to identify any mammalian sequences with significant homology. F
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n Azoospermia). In vitro, DAZAP1 interacts with both the Y chromosome-encoded DAZ and an autosome-encoded DAZ-like protein, DAZL. DAZAP1 contains two RNA-binding domains (RBDs) and a proline-rich C-terminal portion, and is expressed most abundantly in the testis. To understand the biological function o
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The 5' end of the cDNA was isolated by 5' RACE [15], using prdap11 (ttgcgggccatatccttg, #749–732) as the primer for cDNA synthesis from mouse testis RNA, and prdap37 (ttgttgccacgtgggcg, #734–718) and an adaptor primer as the primers for PCR amplification. The PCR products were cloned into a TA cloning
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finding is different from two RNA-binding proteins, FXR1P and FXR2P, that were identified through their interaction with another polysomal-associated RNA-binding protein, the fragile X mental retardation protein [21]. Both FXR1P and FXR2P are associated with the polyribosomes [22]. The significance of
Text source name = 11604102.txt
97% similarity with segments in the human 3' UTR, respectively. These segments probably contain regulatory elements. The DAZAP1 protein contains two RNA-binding domains (RBDS) and a C-terminal portion that is rich in proline (Figure 1). It is highly conserved evolutionarily. The mouse and the human pr
Text source name = 11604102.txt
ch fractions was analyzed by Western blotting. Discussion RNA-binding proteins have been found to participate in many cellular functions, including RNA transcription, pre-mRNA processing, mRNA transport, localization, translation and stability [20]. A role for the DAZ family in the regulation of mRNA
Text source name = 11604102.txt
with 7 substitutions and two deletions/insertions. The mammalian proteins shares 89% similarity and 81% identity with Xenopus Prrp (for proline-rich RNA binding protein) [16]. The two RBDs are especially highly conserved. They share 98% and 97% similarity and 97% and 92% identity, respectively, betwee
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BRCA2 function difficult. Abbreviations DSB = double-strand break; dsDNA = double-strand DNA; ES = embryonic stem; GFP = green fluorescent protein; ssDNA = single-strand DNA. References 1. Shinohara A, Ogawa H, Ogawa T. Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-lik
Text source name = 11597317.txt
d to suppress RAD51–dsDNA interactions to prevent sequestration of RAD51 in a nonfunctional form. Alternatively, or in addition, suppression of RAD51–ssDNA interaction might be important for preventing RAD51 from binding to the regions of ssDNA that form during normal DNA replication. BRCA2 may thus prom
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nction difficult. Abbreviations DSB = double-strand break; dsDNA = double-strand DNA; ES = embryonic stem; GFP = green fluorescent protein; ssDNA = single-strand DNA. References 1. Shinohara A, Ogawa H, Ogawa T. Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein.. Cell. 19
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recruited into a functional repair complex when damage occurs. For example, previous studies indicated that RAD51 shows little binding preference for single-strand DNA (ssDNA) over double-strand DNA (dsDNA) [3,29]. This result could mean that regulatory factors, such as BRCA2, are required to suppress RAD51–dsDNA in
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nctional repair complex when damage occurs. For example, previous studies indicated that RAD51 shows little binding preference for single-strand DNA (ssDNA) over double-strand DNA (dsDNA) [3,29]. This result could mean that regulatory factors, such as BRCA2, are required to suppress RAD51–dsDNA interacti
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al form. Alternatively, or in addition, suppression of RAD51–ssDNA interaction might be important for preventing RAD51 from binding to the regions of ssDNA that form during normal DNA replication. BRCA2 may thus promote RAD51 assembly into recombinational repair complexes via a negative regulatory mechan
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urified on an affinity column containing the oligopeptide antigen. Western blotting Mouse tissues were homogenized in the RIPA lysis buffer (150 mM NaCl, 1.0% NP-40, 0.5% DOC, 0.1% SDS, 50 mM Tris, pH 8.0) at a concentration of 0.2 g tissue per ml of buffer. The homogenized samples were cleared of deb
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taining the oligopeptide antigen. Western blotting Mouse tissues were homogenized in the RIPA lysis buffer (150 mM NaCl, 1.0% NP-40, 0.5% DOC, 0.1% SDS, 50 mM Tris, pH 8.0) at a concentration of 0.2 g tissue per ml of buffer. The homogenized samples were cleared of debris by centrifugation at 10,000
Text source name = 11604102.txt
etermined by the Bradford method using the Bio-Rad Protein Assay system (Bio-Rad, Hercules, CA). About 50 μg of tissue extracts were separated on 10% SDS-polyacrylamide gels and blotted with either the anti-DAZAP1-C antibody (at a 1/2,000 dilution) or the anti-DAZAP1-P antibody (at a 1/5,000 dilution).
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ply with Avertin (1.25% 2,2,2-tribromoethanol and 0.8% tert-pentyl alcohol in water, 0.5-1.0 ml ip) and perfused through the left ventricle with 0.9% sodium phosphate buffered (PB) saline (pH 7.4) followed by 1.25% glutaraldehyde/1.0% paraformaldehyde in 0.1 M PB (pH 7.40) over a period of 2 to 4 min. An additional
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analyses shows that DAZAP1 is not associated with polyribosomes. The post-mitochondrial supernatant of mouse testis extracts was analyzed on a 15–45% sucrose gradient. Sedimentation was from left to right. The presence of DAZAP1 and DAZL in each fractions was analyzed by Western blotting. Discussion RNA-
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uclei. After an additional centrifugation at 10,000 × g for 10 minutes to pellet the mitochondria, aliquots of the supernatant were applied to 15–45% sucrose gradients in 20 mM Tris, 100 mM KCl and 5 mM MgCl2 and centrifuged in a Beckman SW41 rotor at 39,000 rpm for 2 hours at 4°C. Fractions of 0.5 ml were
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se gradient analyses of the post- mitochondria fraction showed that, unlike DAZL, DAZAP1 did not co-sediment with polyribosomes (Figure 6). Figure 6 Sucrose gradient analyses shows that DAZAP1 is not associated with polyribosomes. The post-mitochondrial supernatant of mouse testis extracts was analyzed on
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[12]. Similar analyses showed that a majority of DAZAP1 in adult mouse testes was also present in the cytoplasmic fraction (data not shown). However, sucrose gradient analyses of the post- mitochondria fraction showed that, unlike DAZL, DAZAP1 did not co-sediment with polyribosomes (Figure 6). Figure 6 Su
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esia in rats [38,39]. The type of bedding used also may be important. We use wood shavings for bedding and wood shavings expose the mice to terpenes. Terpene administration or environmental exposure to terpenes in wood shavings alters drug resistance and decreases the effect of anesthetic agents in both ra
Text source name = 11532192.txt
may be important. We use wood shavings for bedding and wood shavings expose the mice to terpenes. Terpene administration or environmental exposure to terpenes in wood shavings alters drug resistance and decreases the effect of anesthetic agents in both rats and mice [40-45]. Risk factors for increased IOP
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of anesthesia in rats [38,39]. The type of bedding used also may be important. We use wood shavings for bedding and wood shavings expose the mice to terpenes. Terpene administration or environmental exposure to terpenes in wood shavings alters drug resistance and decreases the effect of anesthetic agents i
Text source name = 11319941.txt
lution digital images of sections from all cases are available at . Mice were anesthetized deeply with Avertin (1.25% 2,2,2-tribromoethanol and 0.8% tert-pentyl alcohol in water, 0.5-1.0 ml ip) and perfused through the left ventricle with 0.9% sodium phosphate buffered (PB) saline (pH 7.4) followed by 1.25% g
Text source name = 11604102.txt
oligopeptide antigen. Western blotting Mouse tissues were homogenized in the RIPA lysis buffer (150 mM NaCl, 1.0% NP-40, 0.5% DOC, 0.1% SDS, 50 mM Tris, pH 8.0) at a concentration of 0.2 g tissue per ml of buffer. The homogenized samples were cleared of debris by centrifugation at 10,000 × g for 10 m
Text source name = 11604102.txt
centrifugation at 10,000 × g for 10 minutes to pellet the mitochondria, aliquots of the supernatant were applied to 15–45% sucrose gradients in 20 mM Tris, 100 mM KCl and 5 mM MgCl2 and centrifuged in a Beckman SW41 rotor at 39,000 rpm for 2 hours at 4°C. Fractions of 0.5 ml were collected from the bott
Text source name = 11604102.txt
ham Pharmacia Biotech, Piscataway, NJ). Fractionation of mouse testicular extracts Adult mouse testes were homogenized in a buffer containing 20 mM Tris, pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.3% NP-40, 40 U/ml of Rnasin ribonuclase inhibitor (Promega, Madison, WI), and a mixture of 10 protease inhibitors
Text source name = 11532192.txt
containing white pine bedding and covered with polyester filters. For most experiments, the mice were fed NIH31 (6 % fat) chow ad libitum, and their water was acidified to pH 2.8 to 3.2. B6 mice develop diet induced diabetes when maintained on a high fat diet [76]. To ensure that we were analyzing the e
Text source name = 11319941.txt
f sections from all cases are available at . Mice were anesthetized deeply with Avertin (1.25% 2,2,2-tribromoethanol and 0.8% tert-pentyl alcohol in water, 0.5-1.0 ml ip) and perfused through the left ventricle with 0.9% sodium phosphate buffered (PB) saline (pH 7.4) followed by 1.25% glutaraldehyde/1.0


Work experience
09/2017 - Till now
→
LeadSoft
Role: Full-Stack Software Engeneer
Project Description: Product provides user friendly interface for sales managers, and an ability to easily create, test and manage new rules.
Responsibilities:
- creating parsers for sites
- creating CRM JSON API and documentation for that
- developing new functionality for CRM
- developing utilities to simplify and automatization some kinds of operations
- optimization of UI/UX in CRM
- optimization of deployment
- conducting code refactoring


Explore the most common RFID applications with the Nordic ID RFID Demo App. Use the Nordic ID RFID Demo App in combination with your Nordic ID reader* for easy demonstrations of RFID inventory, write and locate functionalities. Compile inventory lists of your tagged items and discover how RFID can guide you to a specific item. If your device includes a barcode scanner, the app also allows you to experience barcode reading.
*supported readers: Nordic ID EXA31 and EXA51
When using a battery powered RFID reader - the application will keep you informed about the remaining battery power of the reader. A small battery indicator is displayed on the bottom of the main display showing you the percentage of the remaining power.
Experience fast and accurate RFID inventory. Scan UHF RFID tags and compile your own inventory list. Nordic ID RFID will show you tag related statistics and allows you to drill down into tag details. Trigger the locate functionality straight from the tag details if needed.
The locate functionality allows easy location of UHF RFID tags from your inventory list. Sound signal and visual indicator will guide you right to a selected tag.
Select UHF RFID tags from your inventory list and re-write their EPC codes.
Carry out barcode reading with this application.
Note: The barcode functionality is only available for RFID readers featuring barcode scanning.Adjust and select application, reader and antenna tuning settings to fit your needs and requirements. Ensure your reader is always up to date with the newest features: access firmware settings to effortlessly update newest firmware versions on your UHF RFID reader


Awards
- Dr. Dasgupta received ACM SIGEVO Impact Award 2014 for research paper "Real-Values Negative Selection Algorithm with Variable-Sized Detectors" published at GECCO in 2004 and which is both highly cited and a seminal work according to the SIGEVO Executive Committee. He has more than 250 publications with 13000+ citations and having h-index of 54 as per Google scholar and listed among top computer scientist whose work impacted the research community (UCLA site). He believes that some of his recent works (like Cloud security insurance model and Negative authentication) will also make similar impact in coming years.
- He was nominated and designated as an ACM Distinguished Speaker for next three years to speak about the most important topics in the computing and IT world today. He has so far given more than 150 Invited Keynote/Plenary talks in academia, industry and governmental events on various research and educational topics.
- He is elevated to an IEEE Fellow for contribution in Immunological Computation and Bio-Inspired Cyber Security. This is the highest grade of membership and is recognized by the technical community as a prestigious honor and an important career achievement.
- Dr. Dasgupta received Technology Transfer Award for Developing Adaptive Multi-Factor Authentication Framework on October 27, 2016.
- In 2014, University of Memphis Magazine published a news article called "The world is their stage". The article talks about Dr. Dasgupta's life and work in bio-inspired computing.
- Dr. Dipankar Dasgupta has been named a fellow for IEEE (Institute of Electrical and Electronics Engineers).
- Dr. Dipankar Dasgupta has been named the Dr. Pat E. Burlison professor at the University of Memphis.
- Dr. Dasgupta received the Alumni Association's "Eye of the Tiger" award at the homecoming football game of the U of M at 6:00 p.m. on Saturday, August 30, 2014.
- He is the recipient of 2012 Willard R. Sparks Eminent Faculty Award.
- He received an 'Excellence in engaged Scholarship Award' by CASDRA(College of Arts and Sciences Distinguished Research Award), 2007.
- He was awarded Dunavant Professorship in 2007
- He received Best Paper Award by International Conference On Genetic and Evolutionary Computation (GECCO) in Seattle, Washington on July 8-12, 2006.
- Dr. Dasgupta received 'New Faculty Research Initiative Award (NFRIA)' by The University of Memphis in 1998.
- He also received Early Career Research Award by The University of Memphis in 1999.
- He received Best Paper Award at the International Conference on Intelligent Systems in June 19-21, 1996 at Reno, USA.
- He has been a Postdoctoral Research Fellow (January, 1994 - August, 1995).


Welcome to SnakeGame (Nostalgia Edition)
Click on arrow keys to direct the snake, eat as many fruits as possible and do not collide with the walls or the body of the snake.
The NewGame and 2x-Speed button will be visible after the game is over.
Score:
High Score:
New Game
2x-Speed


Changelog
Here's what's new in this update for the best Holt Soundboard app -
- You can add your favourite quotes to a seperate page! Long press on any of the buttons to add them to favourites
- A countdown to Season 6! Let the hype begin!
- The long requested share feature is coming very, very soon!
- A bug with the PayPal support button has been fixed! You can now help keep the app running!
- No new sounds have been added as I've been busy


public class SlotEvent extends AbstractSlot
String
instead
as an instance of an actual slot class (such as InSlot
from InstantIO, or Informer
from RSB).| Modifier and Type | Field and Description |
|---|---|
protected long |
time |
protected java.lang.Object |
value |
label, namespace, type
| Constructor and Description |
|---|
SlotEvent()
Constructs an empty event.
|
SlotEvent(java.lang.Object value,
java.lang.String slot,
java.lang.Class<?> type,
long time)
Constructs an event with all needed fields.
|
| Modifier and Type | Method and Description |
|---|---|
boolean |
equals(java.lang.Object obj)
Checks whether two slotEvent objects have equal values.
|
long |
getTime()
Gets the timestamp of this event.
|
java.lang.Object |
getValue()
Returns the data of this event.
|
int |
hashCode()
Returns the hash code for this
SlotEvent |
void |
setTime(long time)
Sets the timestamp for this event.
|
void |
setValue(java.lang.Object value)
Sets the value for this event.
|
java.lang.String |
toString()
Returns a
String representation of this event. |
getLabel, getNamespace, getScope, getType, setLabel, setNamespace, setType
public SlotEvent()
public SlotEvent(java.lang.Object value, java.lang.String slot, java.lang.Class<?> type, long time)
value
- the data of this eventslot
- the label of the slot used by the eventtype
- the data type (=class) of the valuetime
- the timestamp of the eventpublic java.lang.Object getValue()
public void setValue(java.lang.Object value)
value
- the value of this eventpublic long getTime()
public void setTime(long time)
time
- the timestamp to setpublic java.lang.String toString()
String
representation of this event.
The format is'{label}' of {type}, timed for {time}, value: {value}
If there is no label, {label}
will be just an empty string.
The {type}
will be the java class name or
"unknown type"
, if type is null
.
{time}
is the String
representation of the
timestamp.
{value}
is the String
representation of the
value. If the value is an array, then each element of the array will
be turned into a String
representation, with the format
defined by java.util.Arrays
.
This method is used mainly as part of error messages or for debugging.
It is in no way meant to parse events. To parse events into
String
s use XIOParser
.
toString
in class AbstractSlot
public int hashCode()
SlotEvent
hashCode
in class AbstractSlot
SlotEvent
public boolean equals(java.lang.Object obj)
equals
in class AbstractSlot
obj
- the reference object with which to comparetrue
if this object is the same as the
obj argument; false
otherwise


Player
Score
Controls
{{player.name}}
{{player.score}}
+
-
Reset Scores
Clear Triggered
=> {{nextState}}


--- layout: post title: My Game Reflection ---
The main challange I encountered was how to make your background an image. I figured this out by going back to the directions, and there was a link that took me to a code that showed how to put an image as a background.
I am really proud of all of the many pages in my game. It was a lot of work, and I did a lot of it on my own time. I have at least 20 pages inside of the game. Thats a lot!
I want to fisrt of all, finnish year one, and then extend it all the way to year 7.
Click here to go to my game!


Football: Klopp hails local hero Trent Alexander-Arnold ahead of 100th appearance
LONDON: Trent Alexander-Arnold has delighted Liverpool fans with not only his performances but the fact he is a locally-born player said manager Jurgen Klopp on Friday (Nov 1).
The 21-year-old right-back is set to make his 100th appearance for the Premier League leaders against Aston Villa on Saturday.
Klopp said Alexander-Arnold's rise to stardom had been in part due to his determination to set himself ambitious goals.
"He's so young and already so experienced," said Klopp at his eve of match press conference.
"It's a wonderful story for different reasons.
"I know that in Liverpool everyone was desperate to have a 'scouse' hero making his way into the first team.
"When he made his first appearance he said 'great, but it's not about having one game, it's about having 100'.
"Now we can talk about that, and there were not a lot of bad games in that 100."
Klopp, whose side lead Manchester City by six points going into this weekend's matches, praised Alexander-Arnold's attitude.
"I wish for him to stay healthy and fit, and then the future's bright for him and for us," said Klopp.
"He's a great role model. He's relaxed about that but feels responsible as well.
"It's a pleasure to work with him."


Global Functions in Global Namespace C++
in Sourcefile data.h
- uno_assignData
- extern "C"
sal_Bool uno_assignData(
-
| virtual |
abstract |
const |
volatile |
template |
static |
inline |
C-linkage |
| NO |
NO |
NO |
NO |
NO |
NO |
NO |
YES |
- Description
Assigns a destination value with a source value.
Widening conversion WITHOUT data loss is allowed (e.g., assigning a long with a short).
Querying for demanded interface type is allowed.
Assignment from any value to a value of type Any and vice versa is allowed.
- Parameters
| pDest |
pointer to destination value
|
| pDestTypeDescr |
type description of destination value
|
| pSource |
pointer to source value; if 0, then destination value will be assigned
to default value
|
| pSourceTypeDescr |
type destination of source value
|
| queryInterface |
function called each time an interface needs to be queried;
defaults (0) to uno
|
| acquire |
function called each time an interface needs to be acquired;
defaults (0) to uno
|
| release |
function called each time an interface needs to be released;
defaults (0) to uno
|
- Return
true if destination has been successfully assigned
- uno_constructData
- extern "C"
void uno_constructData(
-
| virtual |
abstract |
const |
volatile |
template |
static |
inline |
C-linkage |
| NO |
NO |
NO |
NO |
NO |
NO |
NO |
YES |
- Summary
Default constructs a value. All simple types are set to 0, enums are set to their default value.
- Parameters
| pMem |
pointer to memory of value to be constructed
|
| pTypeDescr |
type description of value to be constructed
|
- uno_copyAndConvertData
- extern "C"
void uno_copyAndConvertData(
-
| virtual |
abstract |
const |
volatile |
template |
static |
inline |
C-linkage |
| NO |
NO |
NO |
NO |
NO |
NO |
NO |
YES |
- Summary
Copy construct memory with given value. The size of the destination value must be larger
or equal to the size of the source value. Interfaces are converted/ mapped by mapping parameter.
- Parameters
| pDest |
pointer to destination value memory
|
| pSource |
pointer to source value
|
| pTypeDescr |
type description of source
|
| mapping |
mapping to convert/ map interfaces
|
- uno_copyData
- extern "C"
void uno_copyData(
-
| virtual |
abstract |
const |
volatile |
template |
static |
inline |
C-linkage |
| NO |
NO |
NO |
NO |
NO |
NO |
NO |
YES |
- Summary
Copy construct memory with given value. The size of the destination value must be larger
or equal to the size of the source value.
- Parameters
| pDest |
pointer to destination value memory
|
| pSource |
pointer to source value
|
| pTypeDescr |
type description of source
|
| acquire |
function called each time an interface needs to be acquired;
defaults (0) to uno
|
- uno_destructData
- extern "C"
void uno_destructData(
-
| virtual |
abstract |
const |
volatile |
template |
static |
inline |
C-linkage |
| NO |
NO |
NO |
NO |
NO |
NO |
NO |
YES |
- Summary
Destructs a given value; does NOT free its memory!
- Parameters
| pValue |
value to be destructed
|
| pTypeDescr |
type description of value
|
| release |
function called each time an interface pointer needs to be released;
defaults (0) to uno
|
- uno_equalData
- extern "C"
sal_Bool uno_equalData(
-
| virtual |
abstract |
const |
volatile |
template |
static |
inline |
C-linkage |
| NO |
NO |
NO |
NO |
NO |
NO |
NO |
YES |
- Summary
Tests if two values are equal. May compare different types (e.g., short to long).
- Parameters
| pVal1 |
pointer to a value
|
| pVal1TypeDescr |
type description of pVal1
|
| pVal2 |
pointer to another value
|
| pVal2TypeDescr |
type description of pVal2
|
| queryInterface |
function called each time two interfaces are tested whether they belong
to the same object; defaults (0) to uno
|
| release |
function to release queried interfaces; defaults (0) to uno
|
- Return
true if values are equal
- uno_type_assignData
- extern "C"
sal_Bool uno_type_assignData(
-
| virtual |
abstract |
const |
volatile |
template |
static |
inline |
C-linkage |
| NO |
NO |
NO |
NO |
NO |
NO |
NO |
YES |
- Description
Assigns a destination value with a source value.
Widening conversion WITHOUT data loss is allowed (e.g., assigning a long with a short).
Querying for demanded interface type is allowed.
Assignment from any value to a value of type Any and vice versa is allowed.
- Parameters
| pDest |
pointer to destination value
|
| pDestType |
type of destination value
|
| pSource |
pointer to source value; if 0, then destination value will be assigned
to default value
|
| pSourceType |
type of source value
|
| queryInterface |
function called each time an interface needs to be queried;
defaults (0) to uno
|
| acquire |
function called each time an interface needs to be acquired;
defaults (0) to uno
|
| release |
function called each time an interface needs to be released;
defaults (0) to uno
|
- Return
true if destination has been successfully assigned
- uno_type_constructData
- extern "C"
void uno_type_constructData(
-
| virtual |
abstract |
const |
volatile |
template |
static |
inline |
C-linkage |
| NO |
NO |
NO |
NO |
NO |
NO |
NO |
YES |
- Summary
Default constructs a value. All simple types are set to 0, enums are set to their default value.
- Parameters
| pMem |
pointer to memory of value to be constructed
|
| pType |
type of value to be constructed
|
- uno_type_copyAndConvertData
- extern "C"
void uno_type_copyAndConvertData(
-
| virtual |
abstract |
const |
volatile |
template |
static |
inline |
C-linkage |
| NO |
NO |
NO |
NO |
NO |
NO |
NO |
YES |
- Summary
Copy construct memory with given value. The size of the destination value must be larger
or equal to the size of the source value. Interfaces are converted/ mapped by mapping parameter.
- Parameters
| pDest |
pointer to destination value memory
|
| pSource |
pointer to source value
|
| pType |
type of source
|
| mapping |
mapping to convert/ map interfaces
|
- uno_type_copyData
- extern "C"
void uno_type_copyData(
-
| virtual |
abstract |
const |
volatile |
template |
static |
inline |
C-linkage |
| NO |
NO |
NO |
NO |
NO |
NO |
NO |
YES |
- Summary
Copy construct memory with given value. The size of the destination value must be larger
or equal to the size of the source value.
- Parameters
| pDest |
pointer to destination value memory
|
| pSource |
pointer to source value
|
| pType |
type of source
|
| acquire |
function called each time an interface needs to be acquired;
defaults (0) to uno
|
- uno_type_destructData
- extern "C"
void uno_type_destructData(
-
| virtual |
abstract |
const |
volatile |
template |
static |
inline |
C-linkage |
| NO |
NO |
NO |
NO |
NO |
NO |
NO |
YES |
- Summary
Destructs a given value; does NOT free its memory!
- Parameters
| pValue |
value to be destructed
|
| pType |
type of value
|
| release |
function called each time an interface pointer needs to be released;
defaults (0) to uno
|
- uno_type_equalData
- extern "C"
sal_Bool uno_type_equalData(
-
| virtual |
abstract |
const |
volatile |
template |
static |
inline |
C-linkage |
| NO |
NO |
NO |
NO |
NO |
NO |
NO |
YES |
- Summary
Tests if two values are equal. May compare different types (e.g., short to long).
- Parameters
| pVal1 |
pointer to a value
|
| pVal1Type |
type of pVal1
|
| pVal2 |
pointer to another value
|
| pVal2Type |
type of pVal2
|
| queryInterface |
function called each time two interfaces are tested whether they belong
to the same object; defaults (0) to uno
|
| release |
function to release queried interfaces; defaults (0) to uno
|
- Return
true if values are equal
- uno_type_isAssignableFromData
- extern "C"
sal_Bool uno_type_isAssignableFromData(
-
| virtual |
abstract |
const |
volatile |
template |
static |
inline |
C-linkage |
| NO |
NO |
NO |
NO |
NO |
NO |
NO |
YES |
- Description
Tests whether a value of given type is assignable from given value.
Widening conversion WITHOUT data loss is allowed (e.g., assigning a long with a short).
Querying for demanded interface type is allowed.
Assignment from any value to a value of type Any and vice versa is allowed.
- Parameters
| pAssignable |
type
|
| pFrom |
pointer to value
|
| pFromType |
type of value
|
| queryInterface |
function called each time an interface needs to be queried;
defaults (0) to uno
|
| release |
function called each time an interface needs to be released;
defaults (0) to uno
|
- Return
true if value is destination has been successfully assigned
Top of Page
Copyright © 2012, The Apache Software Foundation, Licensed under the Apache License, Version 2.0. Apache, the Apache feather logo, Apache OpenOffice and OpenOffice.org are trademarks of The Apache Software Foundation. Other names may be trademarks of their respective owners.


What is an NFT?
NFT stands for non-fungible token.
Non-fungible tokens or NFTs are cryptographic assets on blockchain with
unique identification codes and metadata that distinguish them from each
other. Unlike cryptocurrencies, they cannot be traded or exchanged at
equivalency. This differs from fungible tokens like cryptocurrencies,
which are identical to each other and, therefore, can be used as a
medium for commercial transactions.
The distinct construction of each NFT has the potential for several use
cases. For example, they are an ideal vehicle to digitally represent
physical assets like real estate and artwork. Because they are based on
blockchains, NFTs can also be used to remove intermediaries and connect
artists with audiences or for identity management. NFTs can remove
intermediaries, simplify transactions, and create new markets.
Google Trends
Hover over the image below to further explore the popularity trends of
NFTs.
Wanna learn more?
Click on the images below to explore more about NFTs.
How did we make NFTs?
We used online digital art software.
Vectary
Vectary is a free 3D designing and modeling website. More specifically, it is a CAD application with many features from
similar programs such as Fusion360 and Solidworks. CAD is an acronym for computer-aided design and is used to describe programs which help
in creating models that take up 3D spaces. Possible applications for CAD are CGI animation, 3D modeling for 3D prints and CNC cut-outs,
and 3D art. Unlike most CAD software on the market today, Vectary is completely accessible without the need of a download or license
(for educational/recreational purposes at least). It uses rudementary shapes and angles to create objects that eventually resemble
everyday objects, like a cup of bubble milk tea. Our team used it
for the 3D NFTs which you can find in the
Marketplace. Almost all of our designs
were made from scratch using Vectary.
We also used a plugin known as Model-Viewer, which was developed under the Copyright of Google for the purpose of displaying 3D models in websites.
We feature Model-Viewer in many instances as it was used to display all of our website’s 3D models.
Pixilart
Pixilart is a 2D Design software, specifically catered towards artists with a zeal towards pixelated art. Pixilart has an easy to use
interface that is similar to other design and art software. It allows users to draw, make shapes and other wide variety of features. While you
may not see pixel art as much as 3D art, we used it extensively at the beginning of the program. It also helped us design and plan our 3D models.
Google Trends
Hover over the image below to further explore the popularity trends of
Vectary and Pixilart.
And a special thanks to...
We could not have done this without you :)


Focal points should use this view to compare themselves to other projects. Click the graph column if you want historical views for your project.
This would take you to a page of historical charting of each of the metrics.
This would open a dialog to allow to define community actions required for each project.


// Copyright (c) 2013-2014, the Dart project authors. Please see the AUTHORS file // for details. All rights reserved. Use of this source code is governed by a // BSD-style license that can be found in the LICENSE file. /// Use the `route` Pub package, and associate callbacks with URL patterns. import 'dart:io'; import 'package:route/server.dart'; import 'package:route/url_pattern.dart'; // Pattern for all posts (plural). final postsUrl = new UrlPattern(r'/posts\/?'); // Pattern for a single post('/post/24', for example). final postUrl = new UrlPattern(r'/post/(\d+)\/?'); // Callback for all posts (plural). servePosts(req) { req.response.write("All blog posts"); req.response.close(); } // Callback for a single post('/post/24', for example). servePost(req) { var postId = postUrl.parse(req.uri.path)[0]; req.response.write('Blog post $postId'); req.response.close(); } // Callback to handle illegal urls. serveNotFound(req) { req.response.statusCode = HttpStatus.NOT_FOUND; req.response.write('Not found'); req.response.close(); } void main() { HttpServer.bind(InternetAddress.LOOPBACK_IP_V4, 8080).then((server) { var router = new Router(server) // Associate callbacks with URLs. ..serve(postsUrl, method: 'GET').listen(servePosts) ..serve(postUrl, method: 'GET').listen(servePost) ..defaultStream.listen(serveNotFound); }); }


class DropboxApi::Metadata::WriteMode
Your intent when writing a file to some path. This is used to determine what constitutes a conflict and what the autorename strategy is.
In some situations, the conflict behavior is identical:
- If the target path doesn't contain anything, the file is always written; no conflict. - If the target path contains a folder, it's always a conflict. - If the target path contains a file with identical contents, nothing gets written; no conflict.
The conflict checking differs in the case where there's a file at the target path with contents different from the contents you're trying to write. The value will be one of the following datatypes:
-
`:add`: Do not overwrite an existing file if there is a conflict. The autorename strategy is to append a number to the file name. For example, “document.txt” might become “document (2).txt”.
-
`:overwrite`: Always overwrite the existing file. The autorename strategy is the same as it is for add.
-
`:update`: Overwrite if the given “rev” matches the existing file's “rev”. The autorename strategy is to append the string “conflicted copy” to the file name. For example, “document.txt” might become “document (conflicted copy).txt” or “document (Panda's conflicted copy).txt”.
Constants
- VALID_WRITE_MODES
Public Class Methods
@example
DropboxApi::Metadata::WriteMode.new :add
@example
DropboxApi::Metadata::WriteMode.new :overwrite
@example
DropboxApi::Metadata::WriteMode.new :update, "a1c10ce0dd78"
@example
DropboxApi::Metadata::WriteMode.new({ ".tag"=>"update", "update"=>"a1c10ce0dd78" })
# File lib/dropbox_api/metadata/write_mode.rb, line 45 def initialize(write_mode, options = nil) case write_mode when Hash @write_mode = write_mode when String, ::Symbol @write_mode = { '.tag' => write_mode } @write_mode[write_mode.to_s] = options unless options.nil? end @write_mode['.tag'] = @write_mode['.tag'].to_sym check_validity end
Public Instance Methods
# File lib/dropbox_api/metadata/write_mode.rb, line 60 def check_validity unless valid_mode? @write_mode['.tag'] raise ArgumentError, "Invalid write mode<EMAIL_ADDRESS>end if @write_mode['.tag'] == :update && @write_mode['update'].nil? raise ArgumentError, 'Mode `:update` expects a `rev` number' end end
# File lib/dropbox_api/metadata/write_mode.rb, line 70 def to_hash @write_mode end


This document lists the regular expression syntax used by this library. The syntax validation for the expressions uses Perl expression syntax.
| Syntax |
Credit goes to J. Korpela for the following documentation. His website containing great information on the Perl language can be found at "http://www.cs.tut.fi/~jkorpela/perl/".
The information presented here is from the following web page "http://www.cs.tut.fi/~jkorpela/perl/regexp.html". Parts of the web page have been modified or removed to have proper formatting in the IDE's web browser.
This document presents a tabular summary of the regular expression (regexp) syntax in Perl, then illustrates it with a collection of annotated examples.
Metacharacters
To present a metacharacter as a data character standing for
itself, precede it with In the table above, the characters themselves, in the first column, are links to descriptions of characters in my The ISO Latin 1 character repertoire - a description with usage notes. Note that the physical appearance (glyph) of a character may vary from one device or program or font to another. |
Repetition
Read the notation a’s as “occurrences of strings,
each of which matches the pattern a”. Read
repetition as any of the repetition expressions listed above
it. Shortest match means that the shortest string matching the pattern is
taken. The default is “greedy
matching”, which finds the longest match. The
repetition |
\
|
|
\w |
matches any single character classified as a “word” character
(alphanumeric or “_ ”) |
\W |
matches any non-“word” character |
\s |
matches any whitespace character (space, tab, newline) |
\S |
matches any non-whitespace character |
\d |
matches any digit character, equiv. to [0-9] |
\D |
matches any non-digit character |
[
...]
Different meanings apply inside a character
set (“character class”) denoted by [
...]
so that,
instead of the normal rules given here, the following
apply:
[ characters] |
matches any of the characters in the sequence |
[ x- y] |
matches any of the characters from x to y (inclusively) in the ASCII code |
[\-] |
matches the hyphen character “- ” |
[\n ]
| matches the newline; other single
character denotations with \ apply normally, too
|
[^ something] |
matches any character except those that
[ something] denotes; that is,
immediately after the leading “[ ”, the circumflex
“^ ” means “not” applied to all of the rest |
| expression | matches... |
|---|---|
abc |
abc (that exact character sequence, but anywhere in the
string) |
^abc |
abc at the beginning of the string |
abc$ |
abc at the end of the string |
a|b |
either of a and b |
^abc|abc$ |
the string abc at the beginning or at the end of the
string |
ab{2,4}c |
an a followed by two, three or four b ’s
followed by a c
|
ab{2,}c |
an a followed by at least two b ’s followed
by a c |
ab*c |
an a followed by any number (zero or more) of
b ’s followed by a c
|
ab+c |
an a followed by one or more b ’s followed by
a c |
ab?c |
an a followed by an optional b followed by a
c ; that is, either abc or ac |
a.c |
an a followed by any single character (not newline)
followed by a c
|
a\.c |
a.c exactly
|
[abc] |
any one of a , b and c |
[Aa]bc |
either of Abc and abc |
[abc]+ |
any (nonempty) string of a ’s, b ’s and
c’s (such as a , abba ,
acbabcacaa ) |
[^abc]+ |
any (nonempty) string which does not contain any of
a , b and c (such as
defg ) |
\d\d |
any two decimal digits, such as 42 ; same as \d{2} |
\w+ |
a “word”: a nonempty sequence of alphanumeric characters and low lines
(underscores), such as foo and 12bar8 and
foo_1 |
100\s*mk |
the strings 100 and mk optionally separated
by any amount of white space (spaces, tabs, newlines) |
abc\b |
abc when followed by a word boundary (e.g. in
abc! but not in abcd ) |
perl\B |
perl when not followed by a word boundary (e.g.
in perlert but not in perl
stuff ) |
| See also |
String Functions Index
Main Menu


This example shows how to use
allowReconection()
reconnect()
...to reestablish a connection into a Room.
Room
Open Developer Tools for log messages.
Commands


By @astronomyblog. Stats last updated 15 April 2019.
Below are box and whisker plots for UK comedy panel shows. The left-most end represents the episode with the smallest proportion of women. The right-hand side represents the episode with the highest proportion of women. The middle coloured bar shows the "inter-quartile range" which are the middle 50% of episodes.
Including episodes : :


- Followers
- Following
List Jobs
Available at {{job.job_open| date: 'dd MMMM yyyy'}} until {{job.job_close| date: 'dd MMMM yyyy'}}
Fasilitas : {{job.job_facilities}}
Read MoreSearch not found!
New Applicant
Below is a list that has applied jobs to your company. Check it below!
{{data.student.full_name}} -
applied your job as {{data.job.jobtype.type_name}}
with status {{data.status.status}}. Download and check her/his CV for more informations first in download CV


John Turner is a recent college graduate from the Mendoza College of Business at the University of Notre Dame with a major in marketing. I’ve always had a passion for computers and technology due to its evolving nature. Balancing school and football at the highest level has taught me a lot of prioritizing, time management, dedication, and persistance in order to achieve your goals. I’ve traded my cleats in for a computer to chase my ambitions of being an architect in the tech world.
My Services!HTML & CSS knowledge to build functioning websites with JavaScript and JQuery.
Learning Java, Proficient in Java Core and creating basic web apps. Understand basics of Object Oriented Programming.
Able to create,branch,merge,pull and push to repositories.
Created several applications with Spring, Deployed to Heroku, and also have created multiple projects with Gradle and Maven, SQL also used in projects. All made with love!!
Do you have any questions or concerns? That's great! Give me a call,send an email, or message and I will get back to you as soon as possible!
317-797-5013


The cascade is a fundamental feature of CSS.
It is an algorithm defining how to combine properties values originating from different sources.
See the Pen Css specificity by Albino Tonnina (@albino) on CodePen.
<link rel="stylesheet" type="text/css" href="styles.css" />
External styles are styles that are written in a separate document.
<style type="text/css"> p { color: #00f; } </style>
Embedded styles are styles that are embedded in the head of the document.
<a href="#" style="text-decoration: none; color: blue;">
Inline styles are styles that are written directly in the tag on the document.


From now you will be able to exchange with other musicians on their musical opinions and create new links with these passionated people thanks to a wide online network.
You could also develop your artistic sense by stimulating your hearing dring the visit. Associating arts, you may enjoy a new source of inspiration !
If you are a passionate musician, Art Collab is for you. Reinvent your museum exerience : dare mixing music with painting and build a new network in your field.
With ArtCollab you will have the opportunity to find the perfect match between a music and a masterpiece in museums all around the world. Then feel free to share, exchange and get inspired with other users’ reactions.
When you are facing a masterpiece in a museum you just have to take a picture of it in the app. Then you have the choice between five different musics that ArtCollab considers as suitable for the masterpiece you snaped : vote for the best one ! If none of the five is perfectly appropriated, feel free to add to your own.
Share contents freely on your personal page in order to promote your musical and visual association or to showcase your new proposal. You can also discover other users creations on the ArtCollab network.
You can have access to what users chose between the five musics suggested by the app. Discover if you followed the crowd or not at all ? Then let’s launch debates between users and meet new friends through the ArtCollab tchat.


Authentication
This step must be done on your system, to use the library we need to instantiate an AccessToken
object passing the code returned from the RDStation API authentication flow.
For the demo we will start a ngrok tunnel to pass the HTTPS address to the RDStation App Store App configuration, the allowed callback URLs.
{% if code %}RDStation Authentication Code: {{code}}
{% endif %}
{% endif %}


Contact Information:
Present Address:
Permanent Address:
Mobile:
Email:
Country:
Bangladesh
U.S.A
U.K
Nepal
Japan
Has Passport:
Yes
No


Importer Options
The SketchUp Ruby API provides access to several importers that are shipped with SketchUp. Lists of valid options supported by each importer are given below.
Note
The option merge_coplaner_faces
was a typo in SketchUp 2018. This option will still work in the current SketchUp, but has been changed to merge_coplanar_faces
.
All Importers
show_summary
- Boolean to indicate whether to show the summary dialog.
3ds Max (3DS)
units
- Specifies the import units.- values:
"model"
,"inch"
,"foot"
,"yard"
,"mile"
,"mm"
,"cm"
,"m"
, or"km"
- values:
merge_coplanar_faces
- Boolean to indicate whether to merge coplanar faces.
Collada (DAE)
validate_dae
- Boolean to indicate whether to validate the input file.merge_coplanar_faces
- Boolean to indicate whether to merge coplanar faces.
Digital Elevation Model (DEM/DDF)
max_points
- Integer to specify the number of TIN points.generate_texture
- Boolean to indicate whether to generate gradient textures.
3D Autocad (DWG/DXF)
import_materials
- Boolean to indicate whether to import materials. Added in SU2019.merge_coplanar_faces
- Boolean to indicate whether to merge coplanar faces.orient_faces
- Boolean to indicate whether to orient faces consistently.preserve_origin
- Boolean to indicate whether to preserve drawing origin.
Industry Foundation Classes (IFC)
- No options are supported for this type on import.
Google Earth (KMZ)
validate_kmz
- Boolean to indicate whether to validate the input file.merge_coplanar_faces
- Boolean to indicate whether to merge coplanar faces.
STereoLithography (STL)
units
- Specifies the import units.- values:
"inch"
,"feet"
,"mm"
,"cm"
,"m"
- values:
merge_coplanar_faces
- Boolean to indicate whether to merge coplanar faces.preserve_origin
- Boolean to indicate whether to preserve drawing origin.swap_yz
- Boolean to indicate whether to swap Y and Z coordinates (Y is up).


Qt WebEngine
Qt WebEngine provides functionality for rendering regions of dynamic web content.
The functionality in Qt WebEngine is divided into the following modules:
Provides public API shared by both QtWebEngine and QtWebEngineWidgets | |
Provides QML types for rendering web content within a QML application | |
Provides a web browser engine as well as C++ classes to render and interact with web content |
Articles and Guides
- Qt WebEngine Overview
- Qt WebEngine Features
- Qt WebEngine Platform Notes
- Qt WebEngine Licensing
- Qt WebEngine Debugging and Profiling
- Deploying Qt WebEngine Applications
- Porting from Qt WebKit to Qt WebEngine


A stack is...
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Soundcloud Playlists
5 Nov 2017
I like listening to music and my taste ranges from soulful blues to gang pimping hiphop to Jamaican roots to disintegration loops.
When I discover a a magical track I always repost it and add it to a playlist.I try to curate each playlist carefully and build them around a central theme


Functions to performs Bayesian regression model with mixture of two scaled inverse chi square as hyperprior distribution for variance of each regression coefficient.
| Version: | 1.0 |
| Depends: | R (≥ 2.10) |
| Published: | 2016-04-04 |
| Author: | Renato Rodrigues Silva |
| Maintainer: | Renato Rodrigues Silva <renato.rrsilva at ufg.br> |
| License: | GPL-3 |
| NeedsCompilation: | yes |
| CRAN checks: | BayesH results |
| Reference manual: | BayesH.pdf |
| Package source: | BayesH_1.0.tar.gz |
| Windows binaries: | r-devel: BayesH_1.0.zip, r-release: BayesH_1.0.zip, r-oldrel: BayesH_1.0.zip |
| OS X binaries: | r-release: BayesH_1.0.tgz, r-oldrel: BayesH_1.0.tgz |
Please use the canonical form https://CRAN.R-project.org/package=BayesH to link to this page.


The always_direct access rules define a class of requests that must always be forwarded directly to the origin server. For these, Squid doesn't query or otherwise consider any neighbor caches. See Section 10.4.4.
Syntax
always_direct allow|deny [!]ACLname ...
Default
No default
Example
acl LocalServers dst <IP_ADDRESS>/24 always_direct allow LocalServers
Related
acl, never_direct, prefer_direct, nonhierarchical_direct, minimum_direct_hops, minimum_direct_rtt, cache_peer_access


| | |
- convert(lines, comment_character, magic_character, allow_pep8=True)
- Convert Lines to Markdown
Remove whitespace and magic characters from lines and output valid
markdown.
Kwargs:
lines: The lines to be converted.
comment_character: The comment character of the language.
magic_character: The magic character marking lines as excerpts.
allow_pep8: Allow for a leading comment character and space to confrom
to PEP 8 block comments.
Returns:
A list of strings containing the lines converted.
- excerpt(lines, comment_character, magic_character, allow_pep8=True)
- Extract and Convert Matching Lines
Just a wrapper to extract() and convert().
Kwargs:
lines: A list containing the code lines.
comment_character: The comment character of the language.
magic_character: The magic character marking lines as excerpts.
allow_pep8: Allow for a leading comment character and space to confrom
to PEP 8 block comments.
Returns:
A list of strings containing the lines extracted and converted.
- extract(lines, comment_character, magic_character, allow_pep8=True)
- Extract Matching Lines
Extract all itmes starting with a combination of comment_character and
magic_character from a list.
Kwargs:
lines: A list containing the code lines.
comment_character: The comment character of the language.
magic_character: The magic character marking lines as excerpts.
allow_pep8: Allow for a leading comment character and space to confrom
to PEP 8 block comments.
Returns:
A list of strings containing the lines extracted.
- modify_path(file_name, postfix='', prefix='', output_path='', extension=None)
- Modify a Path
Add a postfix and a prefix to the basename of a path and change
it's extension.
Kwargs:
file_name: The file path to be modified.
postfix: Set the output file postfix.
prefix: Set the output file prefix.
extension: Set a new file extension.
output_path: Set a new file name or an output directory. If the path
given is not an existing directory, it is assumed to be a file path and
all other arguments are discarded.
Returns:
A string containing the modified path.
|


{% extends 'base.html' %} {% load bootstrap3 %} {% load static %}
Beautiful, free pictures.
Explore the world through collections of beautiful HD pictures free to use under the
Picky License