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A modified filamentous phage that contains a gene for a wild type phage coat protein and a gene for a synthetic phage coat protein is provided. Uses of the modified phage and kits containing the phage are also provided.
1. A modified filamentous phage expression vector comprising a gene encoding a wild type major coat protein of the phage; a leaky, inducible promoter; a gene encoding a synthetic major coat protein of the phage; and a directional cloning site for receiving an insert that comprises a ribosome binding site encoding region, a leader sequence that directs polypeptide expression to a bacterial periplasmic membrane and a polypeptide encoding region, wherein the directional cloning site is situated between the promoter and the gene encoding the synthetic major coat protein of the phage wherein the vector is designated JC-M13-88.
2. A modified filamentous phage expression vector comprising a gene encoding a wild type major coat protein of the phage; a leaky, inducible promoter; a gene encoding a synthetic major coat protein of the phage; and a directional cloning site for receiving an insert that comprises a ribosome binding site encoding region, a leader sequence that directs polypeptide expression to a bacterial periplasmic membrane and a polypeptide encoding region, wherein the directional cloning site is situated between the promoter and the gene encoding the synthetic major coat protein of the phage wherein the vector has the sequence of SEQ ID NO:1.
This application is a continuation-in-part of U.S. Provisional Patent Application No. 60/096,326, filed Aug. 12, 1998, the entire disclosure of which is incorporated herein by reference.
Funds used to support some of the studies reported herein were provided by the United States Government (Department of the Army ARL No DAAL03-92-G-0215). The United States Government may, therefore, have certain rights in the invention disclosed herein.
The field of this invention is phage display of polypeptides. More particularly, this invention relates to a modified filamentous phage that allows for displaying polypeptides on the surface in a range of densities.
Phage display of antibodies was initially based on systems developed for the display of peptides (Smith, Science 228, 1315-7, 1985). Antibody single chain variable domains were fused to the coat protein gene (gpIII), (McCafferty, et al., Nature 348, 552-554, 1990) resulting in all the gpIII molecules displaying fusion antibodies. However, the fusion of a polypeptide to the gpIII reduced the ability of the phage to infect bacteria and secondly the multivalent display at the tip of the phage resulted in avidity selection rather than affinity discrimination. Utilizing a phagemid vector (to present the gpIII-fusion) and helper phage rescue (to introduce the wild type gpIII), the valency of fusion display was reduced and infectivity restored (Bass, et al, Proteins: Structure, Function, and Genetics 8, 309-314, 1990). Likewise, the display of heterodimeric polypeptides such as antibody F(ab) fragments as either major (gpVIII) (Kang, et al., Proc. Natl Acad. Sci. USA 88, 4363-66, 1991), or minor (gpIII) (Barbas, et al., Proc. Natl. Acad. Sci. USA 88, 7978-82, 1991; Garrard, et al., Bio/Technol. 9, 1373-77, 1991) coat protein fusions has successfully utilized phagemid with helper phage rescue.
Phage display of antibody fragments and other polypeptides has gained acceptance as a useful tool in contemporary molecular immunology. The density of polypeptide display per filamentous phage particle is influenced by the choice of which phage coat protein is used as fusion partner and the type of vector system used. Molecules expressed from nucleotide sequences fused with the sole copy of gpIII on the phage genome such as fd or M13 resulted in a multivalent cluster display (tri-penta valent) and reduced infectivity of bacteria (McCafferty et al., Nature 348, 552-554, 1990; Smith, Science 228, 1315-7, 1985). Multivalent binding of phage with ligand would favor avidity selection and limit the ability to discriminate between modest gains in affinity (Cwirla, et al., Proc. Natl Acad. Sci. USA 87, 6378-82., 1990). This may be desirable when attempting to isolate ligand binding molecules of lower affinity. Phagemid vectors encoding phage coat protein fusion polypeptides used in conjunction with helper phage rescue, generated phage with restored infectivity and reduced valency permitting enrichment for high affinity interactions (Bass, S., et al. Proteins: Structure, Function, and Genetics 8, 309-314, 1990).
Both the high and low density display systems have uses in accessing ligands against target receptors or tissues. It would be desirable to create a phage display system in which the density of the displayed fusion moieties on the phage particle could be modulated from a few displayed copies to less than 1 per phage. To achieve this with existing vectors requires shuttling of inserts between gpIII phage and gpVIII/gpIII phagemid vectors. However this may also be attained by utilizing a single M13 phage based vector with a synthetic second copy of the gene encoding gpIII or gpVIII (i.e. pseudo wild type) as a fusion partner (Huse, et al., J. Immunol. 149, 3914-20, 1992), and manipulating the phage growth conditions to favor low or moderate rates of fusion incorporation into the phage filament. Incorporating the display expression cassette onto the phage genome may also have the added benefits of fusion expression being synchronous with phage morphogenesis. The present invention describe a phage vector in which a polypeptide is displayed on the phage surface. This display system was used to investigate the modulation of display fusion on phage resulting in optimal phage display as determined by relative panning enrichment efficacy.
The present invention discloses a phage vector for the display of polypeptides on the surface of a modified filamentous phage which permits facile manipulation of the valency of display. The gene encoding the polypeptide is fused to a synthetic copy of a major coat protein gene which permits incorporation into the phage during assembly of the filament.
Thus, in one aspect, the present invention provides a modified filamentous phage expression vector. That vector includes a gene encoding a wild type major coat protein of the phage; a leaky, inducible promoter; a gene encoding a synthetic major coat protein of the phage; and a directional cloning site for receiving a nucleotide insert. The insert is a nucleotide that includes a sequence that encodes a translation initiation site, contains a leader sequence that directs polypeptide expression to a bacterial periplasmic membrane and a polypeptide encoding sequence. The directional cloning site is situated between the promoter and the gene encoding the synthetic major coat protein of the phage, such that the polypeptide is expressed as a fusion protein with the synthetic major coat protein.
In preferred embodiments, the translation initiation site is a ribosome binding site, the promotor is the lac promoter, the leader sequence is ompA, the wild type major coat protein of the phage is gpVIII, the synthetic major coat protein of the phage is a synthetic gpVIII, and the polypeptide is a ligand-binding heterodimeric antibody. An especially preferred filamentous phage is M13. A preferred modified M13 vector of this invention is designated herein as JC-M13-88.
Preferably, the nucleotide insert of the modified filamentous phage is obtained from a pre-selection open reading frame expression and secretion plasmid (pORFES), preferably pORFES II or pORFES IV.
In a related aspect, the present invention provides a process for expressing a polypeptide. The process includes the steps of (a) inserting a nucleotide sequence containing a translation initiation site encoding region, a leader sequence that targets expression of a polypeptide to a bacterial periplasmic membrane and a polypeptide coding sequence that into a directional cloning site of a filamentous phage that contains a gene encoding a wild type phage major coat protein, an inducible promoter and a gene that encodes a synthetic phage major coat protein wherein the directional cloning site is located between the inducible promoter and the gene encoding the synthetic phage major coat protein; and (b) propagating the filamentous phage from step (a) in a bacterium. Preferred translation initiation sites, promoters, leader sequences, polypeptides and major coat proteins are the same as set forth above. A preferred directional cloning site comprises a pair of restriction enzyme sites. Exemplary such enzyme sites are XbaI and HindIII.
The density of the polypeptides displayed on phage may be modulated by phage altering growth conditions. Propagation is preferably carried out at a temperature of from about 25� C. to about 37� C. in the absence or presence of inducers that induce expression by way of the inducible promoter. Lowering the temperature of phage propagation reduced the overall phage yield, yet increased the quality of the antibody display. Likewise the addition of inducers during phage propagation reduced the phage yield but led to enhanced the recovery of phage during panning.
In the drawings that form a portion of the specification.
FIG. 1 shows a schematic illustration of JC-M13-88 phage display system. The VH/κ is inserted into the JC-M13-88 vector from pORFES as an XbaI-HindIII fragment. The VH/κ molecule is expressed as a fusion with the product of a synthetic gpVIII gene, which is not homologous with the wild-type gene present in the phage genome. Expression from dual ribosome binding sites (RBS) is controlled by the Lac promoter and the VH/κ-gPVIII fusion molecule is targeted for secretion through the bacterial membrane by the ompA leader sequence. The single stranded phage DNA is encapsulated by the wild type gpVIII and one or more VH/κ-gpVIII fusion molecules may also be incorporated into the phage protein coat.
FIG. 2 shows a schematic drawing of a pre-selection open reading frame expression and secretion plasmid (pORFESIV) and a partial nucleotide sequence of that plasmid.
FIG. 3 shows an analysis of VH/k-gpVIII incorporation into phage filament using electron microscopy. DB3 R100 antibody phage were grown at 25C in the presence of IPTG. Phage were applied to grids and probed with PHS-BSA gold conjugates. The distribution of gold particles per phage was determined by visual examination of phage particles.
FIGS. 4-1, 4-2 and 4-3 show the nucleotide sequence (SEQ ID NO:1) of a modified filamentous phage of this invention.
FIGS. 5-1 and 5-2 show the nucleotide sequence (SEQ ID NO:2) of pORFES II.
FIG. 6 shows the nucleotide sequence (SEQ ID NO:3) of pORFES IV.
This invention provides a modified filamentous phage and methods for using that phage to express and display polypeptides on its surface at varying density.
As is well known in the art, a filamentous bacteriophage, hereinafter a filamentous phage, is a member of a group of related viruses that infect bacteria. The term filamentous refers to the long, thin particles of an elongated capsule that envelope deoxyribonucleic acid (DNA) (the phage genome). Well known filamentous phage include fd, f1 and M13. Mature capsules of filamentous phage contain five encoded gene products known as coat proteins: gpVIII, gpIII, gpIV, gpVII and gpIX.
A modified filamentous phage of the present invention comprises a gene encoding a wild type phage coat protein and a gene encoding a synthetic form of a coat protein. In a preferred embodiment, the phage contains a gene that encodes a wild type coat protein and a gene that encodes a synthetic form of that same coat protein. In an especially preferred embodiment, the coat protein is major coat protein gpVIII.
The modified phage also contains an inducible promoter. Inducible promoters are well known in the art. An especially preferred such well known inducible promoter is the lac promoter. Situated between the promoter and the gene for the synthetic coat protein is a directional cloning site designed for receiving a nucleotide insert. As used herein the phrase "directional cloning site" indicates an insertion site that orients the insert such that expression of a polypeptide coding sequence in the insert will be under the control of the promoter and the polypeptide will be expressed as a fusion protein with the synthetic coat protein. A preferred directional cloning site is a pair of restriction enzyme sites that are the same as restriction enzyme sites at the ends of the nulceotide insert. An exemplary and preferred such restricition enzyme site pair is XbaI and HindIII.
The nucleotide insert to be received by the modified phage comprises a translation initiation site, a leader sequence and a polypeptide coding region. Translation initiation sites are well known in the art. An exemplary and preferred such initiation site is a ribosome binding site. Ribosome binding sites for use in filamentous phage are well known in the art (See, e.g., U.S. Pat. No. 5,658,727, the disclosure of which is incorporated herein by reference). A leader sequence is located upstream to the polypeptide coding region and acts as a signal that targets the polypeptide to the periplasmic membrane of a bacterium. Such leader, signal sequences and their use with filamentous phage are well known in the art (See. e.g., U.S. Pat. No. 5,658,727, the disclosure of which is incorporated herein by reference). An exemplary and preferred leader sequence is ompA.
In a preferred embodiment, the polypeptide is a ligand-binding heterodimeric receptor. Preferred heterodimeric receptors include immunoglobulins, major histocompatibility antigens of class I or II, lymphocyte receptors, integrins and the like heterodimeric receptors. Immunoglobulins (antibody molecules) can be in the form of Fab or Fv fragments, or other portions of an antibody molecule that contain regions of the variable domain of the heavy and light chains.
Preferably the receptor produced by the subject invention is heterodimeric and is therefore normally comprised of two different polypeptide chains, which together assume a conformation having a binding affinity, or association constant for the preselected ligand that is different, preferably higher, than the affinity or association constant of either of the polypeptides alone, i.e., as monomers. The heterodimeric receptor is referred to as a ligand-binding heterodimeric receptor to connote its ability to bind ligand.
One or both of the different polypeptide chains is preferably derived from the variable region of the light and heavy chains of immunoglobulin. Typically, polypeptides comprising the light (VL) and heavy (VH) variable regions are employed together for binding a preselected ligand.
A nucleotide insert for use with a modified phage of this invention can be prepared using any means known in the art. In a preferred embodiment, the insert is obtained from an open reading frame expression secretion vector designated herein as pORFES. A schematic illustration of one pORFES (pORFES IV) is shown in FIG. 2. Nucleotide sequences of preferred pORFES are shown in FIG. 5 (pORFES II, SEQ ID NO:2) and FIG. 6 (pORFES IV, SEQ ID NO:3).
As shown in FIG. 2, pORFES IV contains the β-lactamase gene preceded by the ompA leader sequence (underlined) with a 4�gly 1�ser linker (underlined). An additional base between the leader and the linker results in a frame shift of the β-lactamase. A NaeI site between the leader sequence and the linker allows subcloning of blunt ended DNA. The CAT gene in the vector backbone permits the vector propagation using chloramphenicol if the vector does not contain an insert restoring the frame of the β-lactamase. Only the ligation of inserts restoring the frame of the β-lactamase which do not contain a stop codon should result in carbenicillin resistance. The XbaI/HindIII cloning sites permit cloning into compatible sites of a modified phage display vector such as JC-M13-88.
Gel-purified polypeptide coding sequences are digested with 70 ng DNAse containing MnCl2 and the reaction terminated by adding EDTA. After precipitation, the fragments are treated with mung bean nuclease. The resulting mixture containing blunt ended fragments is ligated into restriction enzyme digested pORFES between the ompA leader sequence and the β-lactamase gene. Only the inserts which restore the correct open reading frame and are readily translocated into the periplasm along with the fused β-lactamase confer β-lactam antibiotic resistance. The ligation products are transfected into non-suppressing E. coli via electroporation and propagated overnight in Super Broth containing 100 μg/ml carbenicillin at 37� C. The transfection efficiency is monitored by plating aliquots on agar plates containing chloramphenicol with or without 100 carbenicillin. The pORFES library DNA is recovered from the overnight culture and inserts released by digestion with XbaI and HindIII.
A preferred modified filamentous phage of this invention is designated herein as JC-M13-88. A schematic illustration of this phage is shown in FIG. 1 and the nucleotide sequence (SEQ ID NO:1) of this phage is shown in FIG. 4.
The present invention further provides a kit for the expression of a polypeptide. The kit comprises a modified filamentous phage of this invention together with instructions for use of that phage. The kit can further include solvents and reagents for the use of that phage. A preferred phage for inclusion in the kit is the same as set forth above.
In a related aspect, the present invention provides a process for expressing a polypeptide. The polypeptide is expressed on the surface of a modified filamentous phage as set forth above. The polypeptide is expressed as a fusion protein with a synthetic form of a major coat protein of the phage. Suitable such coat proteins are disclosed above.
The process includes the step of inserting a nucleotide sequence containing a ribosome binding site encoding region, a leader sequence that targets expression of a polypeptide to a bacterial periplasmic membrane and a polypeptide coding sequence that into a directional cloning site of a filamentous phage that contains a gene encoding a wild type phage major coat protein, an inducible promoter and a gene that encodes a synthetic phage major coat protein. The directional cloning site is located between the inducible promoter and the gene encoding the synthetic phage major coat protein. The process further includes the step of propagating the filamentous phage in a bacterium. Preferred promoters, leader sequences, polypeptides and major coat proteins are the same as set forth above. A preferred directional cloning site comprises a pair of restriction enzyme sites. Exemplary such enzyme sites are XbaI and HindIII. A detailed description of expressing an antibody polypeptide in accordance with the present process can be found hereinafter in the Examples.
The density of the polypeptides displayed on phage using a process of this invention can be modulated by altering growth conditions. Propagation is preferably carried out at a temperature of from about 25� C. to about 37� C. in the absence or presence of inducers that induce expression by way of the inducible promoter. Lowering the temperature of phage propagation reduced the overall phage yield, yet increased the quality of the antibody display. Likewise the addition of inducers during phage propagation reduced the phage yield but led to enhanced the recovery of phage during panning. The present process does not require the use of a helper/plasmid and has advantages over existing methods of phage display.
Using a present process, the integration of VH/κ-gpVIII fusion(s) has no apparent influence on phage infectivity, and labeling studies suggested that the recombinant molecules are incorporated at a low density. Other groups have estimated the level of incorporation into the phage filament of polypeptides expressed as fusion with either gpIII or gpVIII using indirect methods (Bass, S., et al. Proteins: Structure, Function, and Genetics 8, 309-314, 1990; Garrard, et al., Bio/Technol. 9, 1373-77, 1991; Zhong,et al., J. Biol. Chem. 269, 24183-8, 1994). The values obtained represented the average number of coat protein fusion's within a phage population. Using electron microscopy with immunogold labeling the distribution of coat protein fusion's on phage prepared under conditions of higher density display was estimated. The maximum number of labels per JC-M13-88 phage particle was four, which is in contrast to earlier studies on phagemid pComb8/helper in which as many as 24 F(ab)-gpVIII molecules were displayed on the phage particles (Kang, A. S., et al., Proc. Natl Acad. Sci. USA 88, 4363-66, 1991). Using a phagemid/helper system the interval between fusion expression and helper rescue may be extended in early stages of phage preparation, thus permitting accumulation of fusion prior to phage production which permits some phage particles to take up as many as 20 copies of the fusion moiety (both helper and phagemid). In the phage only system the fusion moiety and phage morphogenesis are in concert which would preclude the incorporation of fusion approaching the extent that can be achieved with phagemids prior to helper rescue, additionally only phage displaying the fusion would encode it. One feature observed with JC-M13-88 display vector was the retention of intact inserts throughout the duration of the experiment in contrast to fusion's with gpVIII or III in phagemid vectors. In cells infected with JC-M13-88 phage the metabolic demand on infected cells is high, the addition of fusion-gpVIII molecules driven from a much weaker Lac promoter may not significantly perturb this demand, thus loss of insert expression may not confer any discernible growth advantage. However, in general it has been acknowledged that large foreign DNA inserts are often unstable in M13 based vectors (see discussion in Sam brook, et al., Molecular Cloning: A laboratory manual, 1989). Reducing the size of the foreign insert and of homologous nucleotide sequences within the phage genome may confer insert/phage stability. This was achieved in JC-M13-88 by replacing the Lac Z polypeptide of M13 mp18 with a non-phage leader sequence (McGuiness, et al., Nature Biotech. 14, 1149-54, 1996) and a synthetic second copy of the gpVIII.
Parameters influencing the yield of phage have been linked closely to conditions favoring bacterial growth. Optimal growth conditions for the bacteria should provide optimal production conditions for phage. Growth conditions during phage preparation such as temperature of phage production and induction of fusion antibody expression by IPTG were investigated. Lowering the temperature of phage propagation reduced the overall phage yield, yet increased the quality of the antibody display. Likewise the addition of IPTG during phage propagation reduced the phage yield but led to enhanced the recovery of phage during panning. The combination of lower temperature and the presence IPTG during phage propagation improved the recovery of phage in panning experiments by more than 100 fold relative to phage prepared at 37� C. in the absence of IPTG. The reduction in growth temperature and addition of IPTG may have a concerted effect on phage production and on antibody fusion assembly resulting in a higher level of display per phage particle. Enhancing the density of display in early rounds of panning may be beneficial in accessing lower affinity interactions. By adjusting the growth conditions in later panning rounds the qualitative features of the phage population may be altered to favor "lower" density display. A priori requirement for successful phage display of a polypeptide is the successful production, secretion and assembly of recombinant proteins in E. coli which can be influenced by a number of factors in addition to amino acid sequence (for reviews see Marston, et al, Escherichia coli. Biochem. J. 240, 1-12, 1986; Schein, Bio/Technol. 7, 1141-9, 1989). Reducing the temperature may facilitate the correct folding and assembly, and contribute to stability, of the displayed antibody. The present data show that producing a well characterized antibody phage at lower temperatures in the presence of IPTG increases the number of phage recovered in panning experiments, and increases the number of VH/k-gpVIII polypeptides incorporated into the phage filament. At all growth temperatures investigated, phage produced in the presence of IPTG were more readily recovered in panning experiments than phage produced without IPTG induction. In contrast, experiments with antibody-gpVIII phage produced using a phagemid/helper phage system the opposite situation was found (Kretzschmar & Geiser, Gene 155, 61-65, 1995). Hence by manipulating the propagation temperature and the induction of the fusion moiety it should be possible to prepare phage for accessing based on avidity (higher density display, 25� C., +IPTG) or 2D affinity (lower density display, 37� C., −IPTG).
The present process was also evaluated for the ability to discriminate between two variants of the antibody DB3 (W100 and R100), with widely disparate affinities for PCMO-BSA yet indistinguishable affinities for PHS-BSA. The recovery of DB3 R100 over W100 on either PHS-BSA or CMO-BSA implies that R100 has a higher affinity for both ligands.
The Examples that follow illustrate preferred embodiments of the present invention and are not limiting of the specification or claims in any way.
Alphabetical list of oligonucleotides. (restriction sites underlined),
Construction of modified M13. Nucleotide sequence of bacteriophage M13mpl8 (Messing, Methods Enzymol. 101, 20-79, 1983; Messing, Gene 100, 3-12, 1991; Yanisch-Peron, et al., Gene 33, 103-119, 1985), was amplified (94� C. for 2 min, 10 cycles; 94� C. for 30 s, 60� C. for 1 min, 68� C. for 6 min, followed by 20 cycles during which the extension time was increased by 20 s per cycle) from replicative form DNA using primers M13-JC117, M13-JC118 and the Expand™ Long Template PCR System (Boehringer Mannheim). The PCR amplified M13mp18 was treated sequentially with T4 DNA polymerase, T4 polynucleotide kinase, and finally T4 DNA ligase, using standard methods (Sam brook, et al., Molecular Cloning: A laboratory manual, 1989). E. coli XL1-Blue strain (Stratagene) were transformed with the ligated DNA and a standard M13 plaque assay was performed in the presence of IPTG and X-gal as described (Sam brook, et al., Molecular Cloning: A laboratory manual, 1989). Replicative form DNA was prepared from colorless plaques and the desired DNA construct identified by analyzing restriction enzyme digests. DNA coding for a pseudo gpVIII, which is not homologous to the wild-type gene but nearly identical in primary sequence, was PCR amplified (30 cycles; 94� C. for 30 s, 55� C. for 1 min, 72� C. for 1 min) from vector f88-4 ((Zhong, et al., J. Biol. Chem. 269, 24183-8, 1994) provided by G. P. Smith, Univ. Missouri, Mo.) using primers SynVIII-JC119 and SynVIII-JC120. The PCR product was cloned using the TA Cloning kit plasmid pCR™II (Invitrogen), and the sequence of the DNA insert was confirmed by DNA sequencing. DNA coding for the synthetic gpVIII was ligated into the modified M13. The modified phage containing the wild type and the pseudo wild type gpVIII was designated JC-M13-88.
Construction of DB3 VH/k Phage. The wild type anti-progesterone antibody DB3 (He, et al., Immunology 84, 662-668, 1995; Wright, et al., Nature 295, 415-417, 1982) has a tryptophan residue at position 100 of the VH domain (designated W100). In a mutant single chain VH/κ molecule position 100 is arginine (designated R100). The DNA sequences coding for the DB3 VH/κ W100 (He, et al., Immunology 84, 662-668, 1995) and R100 variants were PCR amplified using the oligonucleotide primers JC102 and DB3BlaB and ligated into the plasmid pORFES (open reading frame/expression/secretion as Nhe—HindIII fragments, and the ligation products were transformed into bacteria. The plasmid pORFES is a low copy number plasmid in which sequences are expressed as fusions with beta lactamase. Antibiotic selection has been used to screen libraries of peptides and antibody genes fused with the bla gene for open reading frames and successful periplasmic targeting in bacteria. Here, the sub-cloning of the DB3 via pORFES was necessary both to introduce restriction enzyme sites and to provide a second ribosome binding site and the ompA leader sequence for the correct expression of the DNA insert. The 1.1 kilo base pair DNA fragments coding for the DB3 VH/κ variants were excised from pORFES using XbaI and HindIII, then ligated into XbaI/HindIII treated JC-M13-88. Phage encoding and displaying the W100 and the R100 VH/κ molecules were generated.
Western blotting. M13mp18 phage or DB3 R100 phage were precipitated with PEG and re suspended in SDS PAGE sample buffer (Laemmli, Nature 277, 680-685, 1970). Polypeptides from 1�1012 pfu were fractionated on 12.5% polyacrylamide gel, then transferred electrophoretically to nitrocellulose paper (NCP)(Towbin, et al., Proc. Natl Acad. Sci. USA 76, 4350-4354, 1979). The NCP was incubated firstly in blocking buffer (4% bovine serum albumin [BSA] in phosphate buffered saline [PBS], 0.02% NaN3), then with goat anti-mouse kappa light chain alkaline phosphatase conjugate (Southern Biotech). After washing NCP in PBS 0.05% Tween-20 (PBS/T) labeled polypeptides were revealed using a chromogenic substrate as described (Harlow & Lane, Antibodies: A laboratory manual. Cold Spring Harbor Laboratory Press, New York, 1988).
Phage panning experiments. Unless stated otherwise phage were propagated in Escherichia coli (strain XL1-blue, Stratagene) in Superbroth ([3-N-morpholino] propane-sulfonic acid, 10 g l−1, Bacto tryptone, 30 g l−1, yeast extract, 20 g l−1, pH 7.0) at 37� C. in for 16 h, and recovered from the spent media using PEG-8000 as described (Sam brook, et al., Molecular Cloning: A laboratory manual, 1989). Multiwell removable strips (Maxisorp, Nunc) were coated overnight at room temperature (RT) with 100 μl/well PCMO-BSA, PHS-BSA, or goat anti-mouse kappa light chain IgG (Southern Biotech), each diluted to 10 μg ml−1 in 0.1 M carbonate buffer (pH 9.6). The coating solution was replaced with blocking buffer and after 2 h at RT 100 μl of phage diluted in 0.1% BSA in PBS were added to duplicate wells and incubated for 3 h at RT. Wells were washed �5 (5 min per wash) with PBS/T plus 0.1% BSA, and bound phage were eluted into 100 μl/well 0.1 M glycine (pH 2.2) for 15 min and neutralized by adding an equal volume of 1 M Tris-HCl (pH 8.0). In experiments requiring multiple rounds of phage panning against antigen the neutralized eluate was added to freshly prepared bacteria in order to generate phage required for the next round of he panning. Phage titer was assessed using standard methodology as described (Sam brook, et al., Molecular Cloning: A laboratory manual, 1989).
Oligonucleotide hybridization and plaque immunostaining procedures. Replicas of phage plaques were made by applying dry nitrocellulose filters (0.45 μm, Schleicher & Schuell) to agar plates. After 1 min filters were removed, air dried, then baked in a vacuum oven at 80� C. for 1 h. Filters were incubated in pre-hybridization buffer (0.25% non fat milk powder in 6�SSC) at 68� C. for 1 h. Two 15 bp oligonucleotides GAAGTAACGGTTGAC (SEQ ID NO:10) and GAAGTACCAGTTGAC (SEQ ID NO:11), designed to hybridize in a specific fashion to the DB3 R100 and W100 variants respectively, were end-labeled with digoxigenin using the Genius 5 3′ end labeling kit (Boehringer Mannheim) according to the manufacturers instructions. Labeled probes were added to the filters in pre hybridization buffer and incubated at 25� C. for 4 h. After washing with fresh pre hybridization buffer the filters were first incubated in blocking buffer, then transferred to PBS/T containing alkalinephosphatase conjugated anti-digoxigenin antibodies (Boehringer Mannheim). In plaque immunostaining experiments, nitrocellulose plaque replicas on nitrocellulose were made as above except the filters were left on the plates for 2 h at 37� C. before removing and blocking as above. Blocked filters were incubated with alkaline phosphatase conjugated anti-mouse or anti-human k light chain antibodies (Southern Biotech). All filters were washed and developed as described for western blotting except in hybridization experiments the development was carried out overnight at 4� C.
Electron Microscopy. Stable complexes of 5 nm gold particles with PHS-BSA or goat anti mouse kappa chain were prepared essentially as described (Horisberger & Rosset, J. Histochem. Cytochem. 25, 295-305, 1977; Slot, & Geuze, Eur. J. Cell Biol. 38, 87-93, 1985). To ensure the removal of any unbound protein the complexes were centrifuged through a 7% glycerol cushion as described (Slot, & Geuze, Eur. J. Cell Biol. 38, 87-93, 1985). The pelleted complexes were resuspended in PBS containing 0.1% BSA and 0.02% NaN3 and stored at 4� C. Phage were prepared at 25� C. with IPTG or at 37� C. without IPTG diluted into PBS containing 1% BSA and 2�109 pfu were applied to Formvar coated nickel grids (200 mesh). Unbound phage were removed by washing with PBS and then 1% BSA in PBS was applied for 10 min. Protein-gold complexes were centrifuged briefly (700�g, 2 min.), and applied to the grids undiluted (ca. 20 μg protein ml−1). After 30 min. the grids were washed with PBS, then stained with 1% uranyl acetate for visualization by electron microscopy. All incubations were done at RT in a humid atmosphere. Randomly selected areas on the grids were photographed in order to quantify the number of gold particles associated with the phage.
The JC-M13-88 display vector was constructed by modifying M13mp18. The b-galactosidase a-complementation peptide encoding region was replaced with the ompA leader, antibody VH/k and the synthetic gpVIII cassette as shown in FIG. 1.
Western blots of poly ethylene glycol (PEG)-precipitated antibody phage polypeptides probed with anti-mouse k light chain reagent revealed a single immunoreactive protein which migrated at ˜47 kilodaltons (kDa), which is similar to the predicted molecular weight of the DB3 R100 VH/k-gpVIII polypeptide (44,656 Daltons) Polypeptides from M13mp18 control phage were not immunoreactive. This finding confirmed that the DB3 VH/k-gpVIII moiety was successfully integrated into the phage particle (or at least coprecipitated with phage). Phage prepared at 25� C. with IPTG induction were used to determine the density of the antibody on the filament surface by electron microscopy. The number of progesterone-11α-ol-hemisuccinate-bovine serum albumin (PHS-BSA) labels per phage varied from one (˜34% phage) to four (˜1% phage) adopting a classical distribution pattern. About 50% of the phage did not display functional antibody. Whilst phage prepared at 37� C. without IPTG, were essentially devoid of fusion and the few phage that could be observed binding antigen or binding to goat anti-mouse kappa chain were monovalent (3b&c). Preparations of wild-type M13mp18 phage were not labeled by either the PHS-BSA or the BSA gold probes. The effect of growth temperature and fusion induction by IPTG on antibody phage yield and subsequent enrichment by binding to immobillized steroid was investigated. Lower growth temperature resulted in lower overall phage production. Cultures grown for 6 h at 37� C. or 16 h at 30� C. had similar titers of phage (˜1012 plaque forming units (pfu) ml−1), while cultures grown for 16 h at 25� C. resulted in a further ten fold reduction in titer. The addition of 1 mM IPTG to bacterial cultures reduced the final phage titer after equivalent growth periods by 3 to 4 fold. In a panning experiment with equivalent pfu of phage prepared at 25� C. with IPTG and at 37� C. without IPTG, a 100 fold difference was noted in the phage eluted (Table I).
xDB3R100 or M13mp18 phage were propagated using different growth conditions and 2�108 plaque forming units were applied to ELISA plates coated with either PCMO-BSA or BSA alone. After a fixed time interval the plates were washed and bound phage were eluted with acid and the output titer was determined.
*M13mp18 were propagated at 37� C. without IPTG.
#Figures represent the mean average of plaque forming units eluted from two separate experiments corrected for non-specific binding to BSA (2860+/−1690). Values for the experimental error are shown in ( ).
@The quantity of phage eluted is shown relative to the DB3 R100 phage produced at 37� C. without IPTG, here assigned a value of 1.
Phage prepared at the lower temperature with IPTG appeared to enrich more efficiently in the panning process. The amount of antibody incorporated into the phage was analyzed by western blotting, The addition of IPTG slightly increased the amount of fusion polypeptide. In phage propagated at 37� C. we observed a small amount of an immunoreactive species of approximately 20 kDa. This is likely due to VH/k-gpVIII polypeptide proteolysis during phage storage in which the VH/VL has been cleaved. In phage prepared at 30� C. and 25� C. this band was was not detected. Phage prepared at the lower temperature also appeared to have increased antibody fusion incorporation.
The recombinant wild type steroid binding antibody fragment derived from DB3 encodes a tryptophan at position 100 of the VH domain and is designated W100. The two phage is W100 and R100 differ in two nucleotides which change the codon at position 100 of the VH domain of the displayed DB3 from tgg to cgt (arginine) respectively. The affinities of the recombinant DB3 VH/k W100 and R100 variants for progesterone and related steroids were determined previously (W100 to PHS-BSA, Ka˜109 M−1, and to progesterone-3-carboxymethyloxime (PCMO)-BSA Ka˜107 M−1; the R100 mutant to PHS-BSA, Ka˜109 M−1, and to PCMO-BSA Ka˜1011 M−1). However, the authors were not able to distinguish either DB3 variant by binding to PHS-BSA by competition enzyme-linked immunoadsorbent assay (ELISA). A mixture of the W100 and the R100 VH/k phage was diluted into a 50,000-fold excess of a control phage (BR4) displaying a human VH/k, and repeatedly panned on either progesterone PCMO-BSA, PHS-BSA or an anti-mouse kappa light chain antibody. A mock panning of the phage mixture was carried out with plates coated with unmodified BSA. The recovery of phage displaying murine (DB3) or human (BR4) VH/k polypeptides was monitored by immunostaining of plaque replica filter lifts on nitrocellulose filters (Table II).
*An equal mixture of the murine DB3W100 and R100 phage was diluted into a 50,000 fold excess of the control phage BR4, which displays a human VH/κ antibody. ˜1�1010 mixed phage were used in panning experiments.
#This figure represents the increase in the ratio of DB3 phage over BR4 phage in the eluted phage relative to the initial phage mixture (DB3:BR4=1:50,000), here assigned a value of 1. Phage were propagated at 37� C. without IPTG.
The proportion of DB3 phage within the phage mixture was enhanced by over 700 fold following a single round of panning on steroid coated plates. Further rounds of panning increased this proportion although the first round produced the most dramatic increase. This may be due to the saturation of available binding sites during latter rounds of panning by DB3 phage. We utilized two independent panning ligands to differentiate between selection based on steroid binding (i.e. a functional combining site) and on anti-kappa light chain binding (i.e. epitope recognition). However, the similar enrichment rate of DB3 phage over the control phage by panning on PHS and PCMO was paralleled by panning against anti-mouse k light chain antibodies. This provides a correlate that the majority if not all of the VH/k molecules on the phage which the kappa epitope also retain steroid binding.
The proportion of phage encoding the DB3 R100 or W100 variants was determined by oligonucleotide hybridization to replica plaque filter lifts (Table III).
*A mixture containing equal numbers of the murine DB3W100 and R100 phage was diluted into a 50,000 fold excess of the control phage BR4, which displays a human VH/κ antibody. ˜1�1010 mixed phage were used in panning experiments.
nd—Not determined—After one round of panning the ratio of the murine DB3:human BR4 was too small to permit accurate analysis of this value.
+The initial phage mixture used in this experiment contained an equivalent number of R100 and W100DB3 phage.
#Figures represent the percentage of the murine DB3 phage eluted with the R100 mutation. Phage were propagated at 37� C. without IPTG.
In order to determine whether the enrichment was a function of steroid-ligand binding and/or a growth advantage of one DB3 variant over the other during propagation, a 1:1 mixture of the two phage in an excess of BR4 (human antibody phage) was panned against anti-mouse κ light chain antibodies. This enrichment being independent of steroid binding should permit other factors influencing phage display to be assessed. After two rounds of panning 82% of the DB3 phage were the R100 variant, this differential was maintained in subsequent round of panning. Although somewhat removed from the 50% figure expected, and probably due to a slight bias introduced in the early rounds of panning since the proportion difference did not increase in subsequent rounds. Whilst the proportion of R100 in the eluted phage was significantly greater following panning on both PHS and PCMO steroids and increased with each round of selection. Based upon our knowledge of the binding affinities of the DB3 VH/κ variants for the different steroids, our observation that the R100 variant was preferentially enriched over W100 by panning on PCMO-BSA was not unexpected. However, a similar enrichment after panning on PHS-BSA implies that the DB3 R100 appears to have a slightly higher affinity for this ligand than the DB3 W100. The "mock" panning on BSA did not enrich for DB3 antibody phage over the control phage BR4, confirming that any potential growth advantage over the BR4 phage by either of the DB3 phage variants was not significant. Also, and despite exhaustive analysis of the recombinant phage by immunostaining of plaque replicas filter lifts made throughout the panning experiments, we did not observe loss of the VH/κ gene. This finding suggests that single chain antibody likely inserts in JC-M13-88 appear to be stable.
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Latisse was effective and I stopped using it for a month and lost the results, as I anticipated. But, since resuming use about 6 months ago, the growth has been much less significant than the first time. Is this common?
Latisse Less Effective on Second Round
Doctor Answers 1
There is no evidence that Latisse is less effective on the second round
There is no evidence that Latisse is less effective on the second round. Make sure you are using the correct dossage and following the package insert exactly.
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Thermodynamics deals with the amount of heat transfer as a system undergoes a process from one equilibrium state to another. Heat transfer, on the other hand, deals with the rate of heat transfer as well as the temperature distribution within the system at a specified time.
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With a current lockout looming over the National Basketball Association, players have taken steps to play elsewhere, this winter.
Deron Williams, who joined the Nets by virtue of a blockbuster deal in 2011, was the first NBA player to agree to a deal outside of the U.S., joining a Turkish team on July 7 for $200,000 a month.
Last week, Williams' fellow guard in New Jersey, Jordan Farmar, followed suit, signing a deal with Maccabi Tel Aviv, a professional team based out of Israel.
Earlier this week, Dwyane Wade of the Miami Heat and Kevin Durant of the Oklahoma City Thunder, both announced that they will be playing somwhere this winter. If the NBA Players' Association and owners do not reach an agreement by late October -- the start of the 2011 season -- both players plan to find a team overseas to play with.
LeBron James, another superstar for the Heat, however, said Wedneday, that he will not be travelling overseas. James, whose heat fell in the NBA finals to the Dallas Mavericks, said that he is confident that there will be a NBA season in 2011 and that he will remain in the United States.
In other off-season moves for the Nets, Gernal Manager Billy King has announced that the Barclays Center, the Nets state-of-the-art arena in Brooklyn, is on pace to be ready for the 2012-2013 season.
Reports say that the arena is about 60 percent completed, and King said that he is hopeful that the new arena will bring an economic boom to Brooklyn.
Williams' contract with the Nets is set to expire after the 2011-2012 season -- with or without any games being played. He is expected to be the Nets top priority this off-season.


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Monique is based in the heart of Knightbridge having worked with the top therapists in London and working on high profile clients, she offers a wide range of high quality beauty treatments from waxing to hand and feet treatments, Deep tissue massage and holistic treatments.
However, her main passion lies with skin care, working with the top high tech products and researching in the best homemade organic ingredients so she knows exactly what goes onto your skin. All facials include a double cleanse, mild chemical peel, deep pore extractions lymphatic drainage facial massage ending with a hydrating mask.
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Allen's 3 helps Celtics hold off Blazers charge
NOTES: Injured Celtics C/F Jermaine O'Neal told coach Doc Rivers he hoped to begin working out again next week after being out with a sore left knee. O'Neal has played in just seven games this season. ... Rivers was named Eastern Conference coach of the month for October/November on Monday. Boston's 13-4 start tied Orlando for best in the East. ... The teams wore red warmups with ribbons to acknowledge National AIDS Day. The coaches wore red ties, and several players also wore ruby red shoes. ... Portland center Joel Przybilla missed his third straight game with the flu. ... After going just 5-for-18 from 3-point range the night before in a loss at Philadelphia, the Blazers opened the game by making 3 of 4 from beyond the arc.
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text: I Love Married With Children Sitcom Episode Name: Proud To Be Your Bud I love married with Children, my favourite episode when Bud fails with women, and he wanted to go the basement an stay there. Then his alter ego tells him if he builds it; it will come. It's so funny when he fights with his alter ego. In the same episode; his dad Al is trying to call about his dodge, and they keep asking to press a number. I love that episode. LittleLena 36-40, F 0 Nov 7, 2012 SEND TO A FRIEND: Me Too Your Response Please respond with authenticity, support, and respect Cancel You can join Experience Project now! Username: Password Email Birthday Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Gender Select Male Female Trans* Already a member? Sign In By clicking "Sign Up", you confirm that you agree to the Terms of Service of Experience Project, Inc. Login Login to respond Username: Password: Keep me logged in Not a member? Sign Up Sign Up Cancel


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This Web site was designed using Web standards.
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Karley Williams and Harley Cook are headed to the 2015 State Tennis Tournament as a doubles team. During the 2015 season, they were the #1 Doubles team. They have a record of 9-5. Williams and Cook made it to the State Tournament by being the runner-up team from the Region 4 Singles/Doubles Tournament hosted by MSAE in Fairfield. In this tournament, Williams/Cook were seeded #2. They beat a Holy Trinity team the 1st round. They . . . read more
On May 5th students from Fairfield's PLTW classes went on a field trip to the John Deere factory in Ottumwa and to the Indian Hills College campus. They toured the factory and saw the process of how large square balers, small square balers, round balers, and other machines were made. Students learned about certain jobs of certain . . . read more
The Intro to Computers Class and Entrepreneurship Class toured Cambridge Investment Research on Thursday, May 14. The students learned about their IT (Information Technology) Department as well as facts about the company. Mrs. Goudy, their instructor, appreciates that Cambridge allows the students to visit Cambridge each . . . read more
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July 3, 2012
My book The Hypo is available for pre-order from Fantagraphics! Please order it! Come on.
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Parosteal osteosarcoma dedifferentiating into telangiectatic osteosarcoma: importance of lytic changes and fluid cavities at imaging
- M. Azura,
- D. Vanel,
- M. Alberghini,
- P. Picci,
- E. Staals,
- M. Mercuri
- … show all 6 hide
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This study was performed to assess the imaging findings in cases of parosteal osteosarcoma dedifferentiated into telangiectatic osteosarcoma. Parosteal osteosarcoma is a low-grade well-differentiated malignant tumor. Dedifferentiation into a more aggressive lesion is frequent and usually visible on imaging as a central lytic area in a sclerotic mass. Only one case of differentiation into a telangiectatic osteosarcoma has been reported. As it has practical consequences, with a need for aggressive chemotherapy, we looked for this rather typical imaging pattern.
Materials and methods
Review of 199 cases of surface osteosarcomas (including 86 parosteal, of which 23 were dedifferentiated) revealed lesions suggesting a possible telangiectatic osteosarcoma on imaging examinations in five cases (cavities with fluid). Histology confirmed three cases (the two other only had hematoma inside a dedifferentiated tumor). There were three males, aged 24, 28, and 32. They had radiographs and CT, and two an MR examination.
Lesions involved the distal femur, proximal tibia, and proximal humerus. The parosteal osteosarcoma was a sclerotic, regular mass, attached to the cortex. A purely lytic mass, partially composed of fluid cavities was easily detected on CT and MR. It involved the medullary cavity twice, and remained outside the bone once. Histology confirmed the two components in each case. Two patients died of pulmonary metastases and one is alive.
Knowledge of this highly suggestive pattern should help guide the initial biopsy to diagnose the two components of the tumor, and guide aggressive treatment.
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- Wines, A, Bonar, F, Lam, P, McCarthy, S, Stalley, P (2000) Telangiectatic dedifferentiation of a parosteal osteosarcoma. Skeletal Radiol 29: pp. 597-600 CrossRef
- Geschickter, CF, Copeland, MM (1951) Parosteal osteoma of bone. A new entity. Ann Surg 133: pp. 790-807 CrossRef
- Campanacci, M (1999) Bone and soft tissue tumours. Springer, New York
- Mirra, JM (1989) Bone tumours: clinical, radiologic and pathologic correlations. Lea and Febiger, Philadelphia
- Fechner, RE, Mills, SE. (1993) Tumours of the bones and joints. Armed Forces Institute of Pathology, Washington DC
- Christopher DM, Fletcher K, Unni K, Martens F. Pathology and genetics of tumours of soft tissue and bone. World Health Organization Classification of Tumours: WHO 2002. 279-281.
- Okada, K, Frassica, FJ, Sim, FH, Beabout, JW, Bond, JR, Unni, KK (1994) Parosteal osteosarcoma: a clinicopathological study. J Bone Joint Surg Am 76: pp. 366-378
- Paget, J. (1854) Lectures on surgical pathology. Lindsay and Blackiston, Philadelphia
- Mervak, TR, Unni, KK, Pitchard, DJ, Mc Leod, RA (1991) Telangiectatic osteosarcoma. Clin Orthop 270: pp. 135-139
- Bertoni, F, Bacchini, P, Staals, EL, Davidovitz, P (2005) Dedifferentiated parosteal osteosarcoma: the experience of the Rizzoli Institute. Cancer 103: pp. 2373-2382 CrossRef
- Unni, KK, Dahlin, DC, Beabout, JW, Ivins, JC (1976) Parosteal osteogenic sarcoma. Cancer 37: pp. 2466-2475 CrossRef
- Bertoni, F, Present, D, Hudson, T, Enneking, WF (1985) The meaning of radiolucencies in parosteal osteosarcoma. J Bone Joint Surg 67: pp. 901-910
- Campanacci, M, Picci, P, Gherlinzoni, F, Guerra, A, Bertoni, F, Neff, JR (1984) Parosteal osteosarcoma. J bone Joint Surg Br 66: pp. 313-321
- Sheth, DS, Yasko, AW, Raymond, AK (1996) Conventional and dedifferentiated parosteal osteosarcoma: diagnosis, treatment and outcome. Cancer 78: pp. 2136-2145 CrossRef
- Title
- Parosteal osteosarcoma dedifferentiating into telangiectatic osteosarcoma: importance of lytic changes and fluid cavities at imaging
- Journal
Skeletal Radiology
Volume 38, Issue 7 , pp 685-690
- Cover Date
- 2009-07-01
- 10.1007/s00256-009-0672-3
- Print ISSN
- 0364-2348
- Online ISSN
- 1432-2161
- Publisher
- Springer-Verlag
- Additional Links
- Topics
- Keywords
- Telangiectatic osteosarcoma
- Parosteal osteosarcoma
- Fluid–fluid levels
- Industry Sectors
- Authors
- M. Azura (1) (2)
- D. Vanel (3) (5)
- M. Alberghini (4)
- P. Picci (1)
- E. Staals (1)
- M. Mercuri (1)
- Author Affiliations
- 1. Musculoskeletal Oncological Surgery Department, Istituto Ortopedico Rizzoli, Via Pupilli 1, 40136, Bologna, Italy
- 2. Department of Orthopaedic Surgery, University of Malaya, Kuala Lumpur, 59100, Malaysia
- 3. Radiology, Istituto Ortopedico Rizzoli, Via del Barbiano 1/10, 40136, Bologna, Italy
- 5. Anatomia Patologica, Istituti Rizzoli, Via del Barbiano 1/10, 40106, Bologna, Italy
- 4. Pathology, Istituto Ortopedico Rizzoli, Via di Barbiano 1/10, 40136, Bologna, Italy


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Garden Lake Resort
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Travel Blogs from Ely
Exploring Boundary Waters
... comfort.
We watched the bears during their feeding time and they seemed like intelligent creatures who given half the chance would take a peanut over a human, though I am still wary of them.
Next stop was the International Wolf Centre to learn more about these other misunderstood creatures. Whilst we had not spotted any in the wild during our national park odyssey they had probably spotted ...
Minneapolis to Ely
... in car feed.
Our destination being the town of Ely, which sits on the edge of an area called Boundary Waters, a series of lakes that are great for canoeing and spotting wildlife as well as serving as the border between the US and Canada.
It was in the high 50's when we arrived and raining, so no outdoor activities for us today. Headed off to the local steakhouse for some cow.
Canoe Camping with Carol and Karl
... leading the way through the tough spots.
All and all, it was a great eye-opening experience for Glenn, Tara, and Dave. The weather held out nicely considering the record setting rain in the area just before. Carol planned nice meals and Karl provided the overall experience and fishing opportunities. It was a wonderful chance for us to see and do something unlike anything we've done before!
Extreme Camping
We caught TONS of giant smallmouth bass! The weather was decent, except for a couple of super windy days. We managed to avoid any bears or scary wildlife...just lots of chipmunks! As usual, the scenery was great. There's just something so cool about nature that's virtually untouched by man.
The great outdoors!
... away, as were showers and a lovely, low stink, fish cleaning house. The best part of the park, in my opinion, were the boat docks, which are first come first serve. However, we were there on the busiest of weekends and there were plenty of docks to be had. But forget about logistics for a moment and imagine mile after mile of pine and birch filling the air with the smell of summers long since past. Listen and you'll hear ...


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Latest posts by Eleanor Staniforth
27 March 2015
The Arduous Journey of Colombian Migrants Headed for Chile
Global Voices, in collaboration with Connectas, Agenda Propia, Útero.pe, Vice and El Mercurio de Antofagasta, explored the challenges and history of migration in Latin America during a Google Hangout.
5 March 2015
Alleged Video of Soldiers Beating Civilian Shocks São Tomé and Príncipe
The alleged images show a young man sitting on the ground, wearing only underwear, as soldiers—apparently high-ranking members of the Armed Forces of São Tomé and Príncipe—beat him mercilessly.
25 November 2014
Meet Global Voices Contributor and Free Spirit María Angélica Marín
Volunteer María Angélica Marín has translated more than 1,300 stories. Juan Arellano asks her about her experiences with Global Voices and beyond.
24 November 2014
22 November 2014
Dream Trip Around the World Becomes a Nightmare for an American Tourist in East Timor
American Stacey Addison is detained in a prison in East Timor, located between Indonesia and Australia. The tourist was arrested after sharing a taxi with a passenger carrying drugs.
9 September 2014
How a Group of Squatters Convinced Brazilian Authorities to Seize a Vacant Building for Public Housing
Over the years, the building changed owners but remained empty. The National Movement to Fight for Housing has organized various occupations of the building over the years.
8 May 2014
50th Anniversary of Dictatorship in Brazil: A French Torturer in South America
Documents obtained by a researcher in France shed light on the activities of Paul Aussaresses, the executioner of Algiers, military attaché in Brazil in the 1970s.
28 March 2014
How Police Have Turned Rio de Janeiro's Maré Favela Into a Battlefield
Thirteen residents of Maré, the largest favela complex in Rio de Janeiro, died during a police operation there in June 2013.
4 March 2014
Singer Rubén Blades and President Maduro Exchange Words Over Venezuela
A recent publication by the popular Panamanian singer-songwriter Rubén Blades on the subject of the protests in Venezuela has given rise to controversy.


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Lumo 1 Light 8W
Lumo 1 Light 8W Product Description
The Lumo 1 Light 8W is a white 8 watt fluorescent strip light that has a high efficiency inverter incorporated to ensure there is minimal battery drain for the high levels of light created.
Features and Benefits of the Lumo 1 Light 8W
- White 8W fluorescent strip light
- High efficiency inverter for low battery drain
Additional InformationThis product is delivered directly from manufacturer.
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Through our Lord Jesus Christ, by faith we are judged righteous and at peace with God, since it is by faith and through Jesus that we have entered this state of grace in which we can boast about looking forward to God's glory. But that is not all we can boast about; we can boast about our sufferings. These sufferings bring patience, as we know, and patience brings perseverance, and perseverance brings hope, and this hope is not deceptive, because the love of God has been poured into our hearts by the Holy Spirit which has been given us.
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because my choice withdrew you from the world,
therefore the world hates you.
Remember the words I said to you: A servant is not greater than his master.
If they persecuted me, they will persecute you too;
if they kept my word, they will keep yours as well.
But it will be on my account that they will do all this,
because they do not know the one who sent me.'
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A cloud is a collection of water droplets high in the air. A cloud forms when air containing water vapor is cooled and the water vapor condenses onto dust and other small particles in the air.
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Evolutionarily conserved bias of amino-acid usage refines the definition of PDZ-binding motif
1 Laboratory for Memory and Learning, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
2 Launey Research Unit for Molecular Neurocybernetics, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
BMC Genomics 2011, 12:300 doi:10.1186/1471-2164-12-300Published: 8 June 2011
The interactions between PDZ (PSD-95, Dlg, ZO-1) domains and PDZ-binding motifs play central roles in signal transductions within cells. Proteins with PDZ domains bind to PDZ-binding motifs almost exclusively when the motifs are located at the carboxyl (C-) terminal ends of their binding partners. However, it remains little explored whether PDZ-binding motifs show any preferential location at the C-terminal ends of proteins, at genome-level.
Here, we examined the distribution of the type-I (x-x-S/T-x-I/L/V) or type-II (x-x-V-x-I/V) PDZ-binding motifs in proteins encoded in the genomes of five different species (human, mouse, zebrafish, fruit fly and nematode). We first established that these PDZ-binding motifs are indeed preferentially present at their C-terminal ends. Moreover, we found specific amino acid (AA) bias for the 'x' positions in the motifs at the C-terminal ends. In general, hydrophilic AAs were favored. Our genomics-based findings confirm and largely extend the results of previous interaction-based studies, allowing us to propose refined consensus sequences for all of the examined PDZ-binding motifs. An ontological analysis revealed that the refined motifs are functionally relevant since a large fraction of the proteins bearing the motif appear to be involved in signal transduction. Furthermore, co-precipitation experiments confirmed two new protein interactions predicted by our genomics-based approach. Finally, we show that influenza virus pathogenicity can be correlated with PDZ-binding motif, with high-virulence viral proteins bearing a refined PDZ-binding motif.
Our refined definition of PDZ-binding motifs should provide important clues for identifying functional PDZ-binding motifs and proteins involved in signal transduction.


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From the Publisher"...the tasks that are asked of the reader are challenging and require clear thinking. This text could be an exiting tool for self study or a non-traditional course that is not just based on lectures."—Zentralblatt MATH, Sep-14
"Each chapter ends with a section marked "Notes", typically about two pages long, which gives a somewhat broader perspective of the material covered in that chapter, typically placing each topic in historical context, and sometimes giving precise definitions and statements of theorems."—MAA.org, May 4, 2014


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Parental Involvement: A New Metric for NYC Schools
New York City schools will soon be rated based on how well they work with parents. This new metric was announced by Chancellor Dennis Walcott on October 26 in a meeting introducing an overhaul of how the city's education system engages with families. Walcott said the plan would start with 10 to 15 pilot schools that have yet to be chosen.
He didn't reveal how parent interactions would figure in formal school ratings but said they would be judged on whether they successfully communicate with families and "provide a diverse range of roles for parents in the school community."
Mayor Michael Bloomberg created the position of parent coordinators in an attempt to maintain a local link between parents and their schools when he first started his term. However, Mr. Walcott indicated that greater parental involvement is necessary to make sure students are ready for college.
Mr. Walcott said he would address the problems by opening "parent academies" in every borough. He did not lay out a specific vision for what these academies would look like, but he said they could provide services as simple as giving parents tips on working with their children on homework or help parents become school leaders. (See full article.)
Parental involvement is much more than attending parent-teacher conferences and helping children with homework. With increased pressure to improve schools, many educators are looking for effective ways to enlist the support of parents to create a learning community during the academic year as well as during the summer months. It is essential that educators give parents the tools they need to participate and demonstrate that learning is not confined to the classroom.
Teaching Safari, a division of Chicago School Supply, now offers professional development seminars that specifically address improving parental involvement.
Decrease Summer Learning Loss by Increasing Parental Involvement
Preparing Middle School Students for College
Schoodoodle.com offers a wide variety of instructional materials that can facilitate parental involvement for students in preschool through high school and enable parents to be true partners with educational organizations.
Filed under: Education News, For Parents, Teaching Safari, Uncategorized on October 30th, 2011


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OrderUp delivers $7 mln Series A
OrderUp said Wednesday that it has secured $7 million in Series A funding. Revolution Ventures led the round with participation from Tim O'Shaughnessy, co-founder of LivingSocial. In addition to the funding, Tige Savage of Revolution Ventures and Chris Brandenburg of Millennial Media have been added to OrderUp's board of directors. Also, the firm will open two offices in New York City and Boulder, Colorado. Based in Baltimore, OrderUp is an online and mobile food ordering and delivery service.


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Challenging the Ban on Compensating Bone Marrow Donors - Press Conference (October 28, 2009)
Challenging the Ban on Compensating Bone Marrow Donors - Press Conference (October 28, 2009)
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Every year, 1,000 Americans die because they cannot find a matching bone marrow donor. Minorities are hit especially hard. Common sense suggests that offering modest incentives to attract more bone marrow donors would be worth pursuing, but federal law makes that a felony punishable by up to five years in prison.
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A small group of well-funded industry insiders led by the American Society of Interior Designers has been relentless in its pursuit of ever more restrictive laws. Studies have shown that interior design regulations result in higher prices, less variety, and fewer employment opportunities, especially for minorities and older mid-career switchers.
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How arbitrary has government power grown? Take a listen to the Saga of San Tan Flat—the Arizona steakhouse where the government tried to ban outdoor dancing.
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ClassicsOnline Home » BEETHOVEN / MOZART: Violin Concertos (Szigeti) (1932, 1934)
In an age of great violinists, Joseph Szigeti was a giant; and unlike some of his peers, he did not content himself with repeated performances of the same pieces all the time. He sought out composers who were writing interesting new music, Stravinsky, Prokofiev, Bloch, Kodály and Bartók, and propagated their works through his devoted performances. Szigeti was the most distinguished of a line of superb violinists taught by Jenö Hubay. Born József Szigeti in Budapest on 5th September 1892, he was brought up in the Carpathian area of Hungary at Máramaros-Sziget, from which his family, originally called Singer, took their name. His father led a café band, one uncle, Deszo Szigeti (1880-1963), had studied with Jenö Hubay and was an established orchestral violinist in Paris and New York, even making a few solo records, another uncle was a bass player, and Uncle Bernat gave Jóska, as he was known to family, friends and early audiences, his first lessons. From the age of eleven to thirteen Szigeti was under Hubays tutelage and he left the Ferenc Liszt Academy in 1905 to make his Berlin and Budapest débuts. As Szulagi he played in a Frankfurt circus, then auditioned in Berlin for Joseph Joachim, instinctively deciding not to study with the old man, although he always had the low bow arm of that school Joachim had been Hubays first major teacher. Szigeti did perform a number of Joachims cadenzas, however, as he does in both concertos on this disc.
Szigeti made his London début at the Bechstein (now Wigmore) Hall as a thirteen-year-old (the public was told he was twelve). From 1907 he was based in England and his concerto début was made with the Bach Concerto in E major and the Tchaikovsky Violin Concerto with the New Symphony Orchestra under Thomas Beecham. While in Britain, until 1913, he toured with Nellie Melba and John McCormack, met Myra Hess and Ferruccio Busoni, gave the first performance of Hamilton Hartys Concerto in D minor and made his first records. They reveal a typical Hubay disciple, with a brilliant but brittle technique. He used very little vibrato in those days and this was one technique he worked on, during the decade from 1913 when he was off the international scene he spent much of World War I in Swiss sanatoria with tuberculosis. Hubay equipped his students with a wide vibrato and Szigeti was influenced by this tradition in developing his own sound; he always vibrated rather slowly and this trait could make his legato sound slightly concave. Although a gentle person off stage, he had a reputation for breaking strings when he got carried away by the music.
From 1917 to 1924 Szigeti taught in Geneva. In 1922 he played with the Berlin Philharmonic under Reiner and from 1924 was a regular visitor to the Soviet Union and England; but it was his Philadelphia début in 1925, with the Beethoven Concerto conducted by Stokowski, that sealed his fame. Now known as Joseph to English-speaking audiences, he was a far more cultured artist than the Jóska who had left Hubays class with a tiny repertoire of virtuoso works. Based in Paris with his Russian wife Wanda and their daughter Irène, he was one of the busiest violinists of the interwar years, playing concertos, especially those of Beethoven and Brahms, or appearing in recital with his piano partner Nikita Magaloff, who married Irène in 1939.
In the mid-1920s Szigeti became friendly with Béla Bartók and appeared with him in concert, introducing the Second Sonata to New York in December 1927. Szigeti also transcribed seven pieces from Bartóks For Children, which they played in recital, and recorded, as Hungarian Folk Tunes. At Königsberg in 1929, with Hermann Scherchen conducting, Szigeti gave the first performance of Bartóks First Rhapsody. In 1931 he toured the Far East, causing a sensation in Japan, and in 1933 he appeared on the same bill as Benny Goodman and Fletcher Henderson. In 1938 he introduced Blochs Concerto with the Cleveland Orchestra under Dimitri Mitropoulos. In 1940 Szigeti emigrated with Wanda to the United States, where he and Bartók gave a Library of Congress recital and with Benny Goodman performed and recorded Bartóks trio Contrasts, which Szigeti and Goodman had already performed in 1939 with Endre Petri. In 1944, with Claudio Arrau, Szigeti presented the cycle of Beethoven Sonatas at the Library of Congress. He was the only great violinist of his generation to revive his career after World War II and he appeared a number of times with Artur Schnabel; but after 1950 his playing declined. He last appeared in London as a violinist in 1954, breaking a G string at his final concert. He settled in Switzerland in 1960 and died in Lucerne on 20th February 1973, having devoted his last years to competition jury work, writing and teaching.
Tall, courtly and courteous, Szigeti was the thinking mans virtuoso, among the first to perform the Debussy and Ravel Sonatas, as well as pieces by Honegger, Roussel and Milhaud. He promoted sonatas by Bloch, Ives and Cowell and the concertos by Prokofiev (D major), Busoni, Casella, Berg and Martin. His style of playing was old-fashioned, in that he used downward slides which often sat oddly on the contemporary music he played. Yet this portamento helped to give his playing a singing, breathing, easeful quality. He owned two Guarnerius violins, his main concert instrument being the Pietro Guarneri of Mantua formerly played by Henri Petri.
Szigeti recorded a number of concertos for Columbia when he was at his peak. Mozarts Violin Concerto in D major, K.218, was one of three major recordings he made with Beecham, who had been one of the first musicians to impress him in his London period. Truth to tell, Beecham, the supposed Mozart lover, serves up an accompaniment that is brusque in some places and slapdash in others; but while Szigeti is playing, all is sweetness and light and the two clearly get on well. How beautifully the violinist sings his phrases, yet he never lingers to make a point. Because his playing is so stylish, it ends up saying more by saying less, and the episodes in the finale are delightfully delineated with the minimum of fuss. Those who have been collecting this series of Great Violinists can compare this performance with one set down more than a decade later by Jascha Heifetz with the same conductor (Naxos 8.110960). In the Beethoven Concerto, it is possible to compare Szigeti directly with his post-war self, as he made a second recording with the same conductor, Bruno Walter. This 1932 performance wins on every score. Szigeti himself is in better trim, the recording is better balanced and even though Walter has the famous New York Philharmonic-Symphony for the later version, he actually does better with the pick-up British Symphony Orchestra. Not that Walter is the ideal accompanist, and he has changed tempo several times before Szigeti even enters with Beethovens broken octaves. Thereafter, though, the violinist takes charge. The greatest interpreters of this concerto, Kreisler, Busch, Huberman, Milstein, Menuhin, Kogan and Perlman, always touch us most at three points: during the G minor episode and then again after the cadenza in the first movement; and in the central Larghetto. Szigeti is of that exalted company, one of the few who can make the Larghetto sound really profound, rather than a mere intermezzo. In the finale, the soloists gently buoyant sense of humour and his sheer joy in making music prevail.
Tully Potter
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On this week's Capital Impact show, Bloomberg Government analysts take a look back at the first year of the Jobs Act, and discuss how energy companies could benefit from the government's green initiatives.
April 4, 2013
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Dorothy Robyn, the deputy undersecretary of defense for installations and environment, said DoD is taking the lead in helping clean energy companies clear the commercialization hurdle.
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Katherine Hammack, the assistant secretary of the Army for Installations, Energy and Environment, is the service's energy guru. She discussed the service's latest efforts to make strides in clean energy at the Association of U.S. Army 2011 conference.
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Sunday, September 14, 2008
© Copyright 2015 Gwinnett Daily Post
Ashley Nicole Dickerson and Mark Alan Bradford Jr. were married June 14 at the Bethlehem First United Methodist Church. The Rev. Ronnie Kendall officiated the ceremony.The bride is the daughter of Dr. and Mrs. Franklin Neal Dickerson of Loganville. She is a 2004 graduate of Loganville High School. She graduated summa cum laude from the University of Georgia with a bachelor's degree in early childhood education. She is employed by Margaret Winn-Holt Elementary. The groom is the son of Mr. and Mrs. Mark Alan Bradford Sr. of Loganville. He is a graduate of Loganville High School and received a bachelor's degree in accounting from Georgia State University. He is employed by Acuff Acounting and Tax Services Inc. Lesley Anne Dickerson of Loganville, sister of the bride, served as the bride's maid of honor. Bridesmaids were Creagh Andrews, Macall Dennis, Leah Holloway, Shannon Pierce, Erica Wagoner and Nicole Yardley. Mark Alan Bradford Sr. of Loganville, father of the groom, served as the groom's best man. Groomsmen were Robert Bradford, Nathan Boyd, Kevin Cash, Drew Dickerson, Kyle Fenton and Ryan Wagoner. After a honeymoon in Kiawah Island, the couple resides in Loganville.
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I Could Sing This Song For Hours
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I Could Sing This Song For Hours
Score 21 awesome dance moves in the Construction Site dance mini game.
To unlock this trophy/achievement you need to get a Awesome score 21 times (a perfect score) in the dance event in the Bricksburg Construction level.
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Crime Watch: December 27, 2013
A sixty-three-count superseding indictment in a conspiracy to defraud the IRS was unsealed December 23. The indictment alleges that Primetime Tax Services Inc. was a tax return preparation business with three storefronts in the Minneapolis area.
Dec 27 2013
Crime Watch: December 20, 2013
A San Jose, California, woman was convicted December 18 of a federal fraud charge for running a Ponzi scheme that bilked more than 250 victims out of approximately $9.5 million.
Dec 20 2013
Bernie Madoff Cried as His Empire Toppled
The name Bernard Madoff has become synonymous with investment scams after his firm defrauded investors of $20 billion in investments and $47 billion in fake profit his clients believed they had.
Dec 13 2013
Crime Watch: December 13, 2013
Rathana Ung, the former director and officer of Lim's Income Tax and Lim's Tax, Inc., was sentenced to twelve months and a day imprisonment, one year of supervised release, and ordered to pay $103,736 in restitution to the IRS.
Dec 13 2013
Crime Watch: December 6, 2013
Gary Mach was sentenced to sixteen months in prison, two months of house arrest, and eighteen months of probation and ordered to pay $270,725 in restitution to the IRS after previously pleading guilty to conspiracy to defraud the United States.
Dec 6 2013
Supreme Court Piles on Hefty Tax Shelter Penalty
In a new case decided by the US Supreme Court, the remnants of a tax shelter partially constructed by wily Texas billionaire Billy Joe McCombs – known informally as "Red" – collapsed like a house of cards.
Dec 4 2013
Bernie Madoff Employees on Trial as Assistant Scammers
The name Bernie Madoff probably rings a bell. It has also been known to set off a few whistles and sirens. His well-publicized crime masterminded the biggest Ponzi scheme ever recorded.
Nov 19 2013
Crime Watch: November 18, 2013
Chicago lawyer Gary J. Stern designed at least three tax-fraud schemes that helped hundreds of customers falsely claim over $16 million in improper tax credits and avoid paying income tax on at least $3.4 million.
Nov 18 2013
IRS Warns Taxpayers of Phone Scam
A phone scam in which taxpayers, especially recent immigrants, are told they owe money to the IRS and are threatened with arrest or deportation if they don't pay up has been occurring lately in nearly every state in the country.
Nov 3 2013
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Vernon Harrison of Montgomery, Alabama, was sentenced to 111 months in prison, three years of supervised release, and ordered to pay $82,791 restitution for his role in a stolen identity refund fraud scheme. Harrison is a former US Postal Service mail carrier.
Nov 1 2013
Crime Watch: October 25, 2013
A Los Angeles man was sentenced October 24 to sixty months in federal prison for defrauding professional football player Dwight Freeney in a multimillion dollar fraud scheme.
Oct 25 2013
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Randolph Scott of Doylestown, Pennsylvania, an attorney whose practice included estate and probate matters, was charged by Indictment on October 3, 2013, with defrauding a client's estate of more than $1.7 million.
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Beth Ann Pettyjohn of Englewood, Colorado, was sentenced to serve twenty-eight months in federal prison for failure to pay over employment tax. The judge also ordered her to pay $4,669,532.05 in restitution to the IRS as well as a $25,000 fine.
Sep 19 2013
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A couple from Iowa and a Las Vegas accountant were charged September 11 with conspiring to defraud the IRS of more than $700,000 by allegedly using nominee corporations and bank accounts to hide their income and other assets.
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David Haigler, who pleaded guilty in US District Court for the Middle District of Alabama September 5, 2013, to one count of theft of public funds and to one count of passing US Treasury checks with forged endorsements, faces twenty years in prison.
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The Justice Department announced August 20 that Teresa Marie Marty, Charles Tingler, and Victoria Tingler have been charged with conspiracy to defraud the United States and filing multimillion-dollar liens against government officials.
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Mark Goldberg, a Bronx-based tax preparer, pled guilty to charges related to his participation in a scheme to file fraudulent tax returns on behalf of numerous clients, falsely claiming more than $7 million in bogus deductions, including school tuition credits and expenses.
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Crime Watch: August 9, 2013
A former Washington State real estate developer and his long-time girlfriend were convicted August 4 on twenty-five counts of tax evasion and false statements related to their scheme to avoid paying taxes on more than $23 million in income.
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The Justice Department and IRS announced that John T. Hoang of Virginia pleaded guilty in federal district court in Washington, DC, to willfully aiding and assisting in the preparation of false income tax returns for tax year 2004.
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A former manager of an H&R Block Preparation store has been sentenced to twelve months and one day in federal prison for using the identities of former tax preparation clients to file false returns with the IRS seeking fraudulent income tax refunds.
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