text:
The invention relates to a functionalized compound of general formula (I):
A functionalized polynucleotide comprising at least one said compound and a labeled functionalized polynucleotide comprising at least one functionalized compound corresponding to the formula (I′).
A method for detecting a target nucleic acid using a said compound is described.
1. Functionalized compound of general formula (I):
in which:
W represents a nucleotide or nucleoside, or an analog of a nucleotide or nucleoside,
L represents a linker arm comprising a chain of at least four atoms in series, and L is a saturated or unsaturated hydrocarbon-based chain, optionally interrupted by at least one function chosen from amine, amide and oxy functions, and is grafted onto any position of a nitrogen-containing base of the nucleotide or nucleoside of W, and
R1 represents a linear or branched alkyl chain.
2. Compound according to
3. Compound according to
4. Compound according to
5. Compound according to
in which:
B represents a nitrogen-containing base,
R2 represents H or a protective group,
R3 represents H, F, OH, SH, NH2, OCH3 or OR5 in which R5 represents a protective group or an alkyl chain, and
R4 represents an H radical, a protective group or a mono-, di- or triphosphate group,
W being attached to L via B.
6. Compound according to
7. Compound according to
8. Compound according to
9. Functionalized polynucleotide comprising at least one functionalized compound according to
10. Functionalized polynucleotide according to
11. Functionalized polynucleotide according to
12. Labeled functionalized polynucleotide, characterized in that it comprises at least one functionalized compound of general formula (I′):
W-[-L-]n—CH2COR1 (I′)
in which:
W represents a nucleotide or nucleoside, or an analog of a nucleotide or nucleoside,
L represents a linker arm comprising at least four atoms, and L is a saturated or unsaturated hydrocarbon-based chain, optionally interrupted by at least one function chosen from amine, amide and oxy functions, and is grafted onto any position of a nitrogen-containing base of the nucleotide or nucleoside of W,
n represents an index equal to 0 or 1, and
R1 represents a linear or branched alkyl chain,
the alkyl ketone group of said functionalized compound being attached to a labeling reagent.
13. Polynucleotide according to
14. Polynucleotide according to
15. Polynucleotide according to
16. Polynucleotide according to
in which:
B represents a nitrogen-containing base,
R2 represents H or a protective group,
R3 represents H, F, OH, SH, NH2, OCH3 or OR5 in which R5 represents a protective group or an alkyl chain, and
R4 represents an H radical, a protective group or a mono-, di- or triphosphate group,
W being attached to L via B.
17. Polynucleotide according to
18. Polynucleotide according to
19. Compound according to
20. Polynucleotide according to
22. Method for detecting a target nucleic acid, characterized in that this target nucleic acid is brought into contact with at least one functionalized nucleotide as defined in
23. Method according to
24. Method for detecting a target nucleic acid, characterized in that this target nucleic acid is brought into contact with a functionalized polynucleotide according to
25. Method for detecting a target nucleic acid, characterized in that a labeled polynucleotide according to
26. Compound according to
27. Polynucleotide according to
28. Compound according to
The present invention relates to a novel nucleoside or nucleotide compound functionalized with an alkyl ketone group, to a polynucleotide comprising at least one nucleotide unit functionalized with an alkyl ketone group, before and after labeling, and also to the implementation and the uses of these products in particular for detecting nucleic acid sequences.
In the nucleic acids field, the synthesis of functionalized nucleotides has been described in particular in the diagnostic field and more particularly for preparing labeled nucleic acid probes which may be used for detecting a target nucleic acid.
Two main types of problem arise in producing these probes. Initially, the functionalized nucleotide must be incorporated into a polynucleotide. Next, the function carried by the nucleotide must be able to react with a tracer in a way which is specific and effective for using the polynucleotide as a detection probe.
Thus, EP-A-0 407 816 describes uracil derivatives modified at position 5 for producing probes via the chemical or enzymatic route. Still with the same aim, patents WO-A-86/06726 and EP-A-0 212 951 describe cytosine derivatives modified at position 4. Patent EP-A-0 254 646 describes an adenosine derivative modified at position 8.
Patent WO-A-92/00989 describes a specific use of modified nucleotides' for introducing proteins onto a polynucleotide.
Patent application WO-A-98/05766 by the applicant, poses the problem of the incorporation of functionalized nucleotides which may be incorporated using enzymatic reaction and in particular using enzymatic amplification techniques, no longer for preparing labeled probes, but directly for generating a labeled target. In this case, there is a greater need for sensitivity and the choice of the functionalized nucleotide is essential for producing the appropriate sensitivity.
A certain number of nucleophilic functions, such as amine and alkoxyamine functions, or electrophilic functions, such as the aldehyde function, are described which allow effective incorporation of the functionalized nucleotide in the course of amplification, but there nevertheless remains a need for a functionalized nucleotide which is even more effective, in particular in terms of ease of preparation, in terms of neutrality with respect to the enzymatic or chemical reactions and in terms of reactivity for the labeling of this nucleotide with a tracer after incorporation.
It has been found, by the applicant, that, surprisingly, the alkyl ketone function answers the abovementioned drawbacks.
In fact, while the prior art describes the use of R—CO— units in which R is an alkyl group, in the case of oligonucleotide synthesis (WO-A-93/22326), the aim of this function is to protect the exocyclic amine of the bases. At the end of synthesis, the amine is deprotected by the action of an alkali agent.
By way of illustration of this state of the art, the following documents may be mentioned. The article by K. K. Ogilvie and M. J. Nemer, Tetrahedron Letters, vol. 21, (1980) pages 4145-4148, discloses, as an intermediate of synthesis of a nucleotide, a nucleotide compound carrying a levulinoyl group attached to the hydroxyl function on the 3′ side of the pentose. The aim of introducing this group is to protect the OH function, and it is then immediately removed after said compound has been obtained. The article by C. T. J. Wreesmann et al., Nucleic Acids Research, vol. 11, No 23, (1983) pages 8389-8405, describes the production of a synthetic dinucleotide intermediate, the hydroxyl functions of which are protected by the levulinoyl group. In the presence of aqueous ammonia, the hydroxyl functions are released.
The alkyl ketone function as defined in the present invention is sufficiently stable to withstand this type of treatment and does not therefore represent a protective group, and its stability with respect to the various methods of chemical synthesis results in greater ease of preparation, in particular with respect to amine, aldehyde or alkoxyamine functions, as described in patent WO-A-98/05766.
Similarly, in the abovementioned patents (EP-A-0 407 816, WO-A-86/06726, EP-A-0 212 951, EP-A-0 254 646 and WO-A-92/00989), the functions described for functionalizing the nucleotides are chosen from functions which are reactive from a chemical point of view, such as amine and thiol functions, which are optionally protected. If these functions are protected, a deprotection step is necessary, which complicates the labeling step. If these functions are free, inhibition of the enzymatic reaction or of the side reactions in the case of chemical synthesis may occur.
This chemical stability of the alkyl ketone function should not make it a good candidate for a reaction for coupling to a label, and yet the present invention demonstrates that, surprisingly, this function is reactive with regard to labeling and that, in addition, the detection of the product after labeling is very sensitive.
Finally, the nucleotides carrying this function exhibit excellent neutrality with respect to enzymatic reactions since it is possible to completely replace a natural nucleotide with a nucleotide carrying this alkyl ketone function in an enzymatic reaction, without affecting the yield of this reaction and, even more surprisingly, in certain cases improving it.
Document WO-A-95/24185 describes a nucleoside modified with an alkyl ketone group, the alkyl component of which may comprise up to 20 carbon atoms. This compound is in particular intended for the synthesis of oligonucleotides which find an application in therapy, in diagnosis. The introduction of a group, in particular an alkyl ketone group, onto the pyrimidine nucleus of the nucleoside described is not directed toward functionalization in view of a subsequent reaction of said group, but is aimed at producing oligonucleotide analogs which, with respect to the natural oligonucleotides, exhibit properties which are advantageous in terms of their uses, such as greater ease of hybridization with a target nucleic acid or greater resistance to nucleases.
The object of the present invention is to describe a novel functionalized compound which comprises an alkyl ketone function and which has the formula (I) below:
The term "nucleotide analog" is intended to mean a nucleoside or a nucleotide, a nucleoside or a nucleotide carrying one or more modifications on one of the constituent elements of said nucleoside or nucleotide, for instance a modification of the deoxyribose or ribose sugar, in particular xylose, arabinose, sugars with an alpha configuration (FR 2 607 507), PNAs (M. Egholm et al., J. Am. Chem. Soc., (1992), 114, 1895-1897), sugar analogs such as 4′-thio-ribose or -deoxyribose, or sugars with a D or L configuration; a modification of the nitrogen-containing base; a modification of the phosphate or of its equivalent for the nucleotides and also all of the protective groups used in the chemical synthesis.
Advantageously, the compound corresponds to the formula (I) in which W has the general formula (II):
The nitrogen-containing base is chosen in particular from purines or pyrimidines, such as adenine, guanine, uracil, cytosine or thymine, or any other modified base allowing hybridization, for instance natural modified bases (such as 6-keto purine, xanthine, 5-methylcytosine or 2-aminopurine) or unnatural modified bases (such as thioguanine or 8-oxoguanine, deazapurine or azapurine), or analogs of bases such as universal bases (such as nebularin, nitroindole or nitropyrrole derivatives). Certain functions of the bases likely to interfere with the strategies of solid- or liquid-phase chemical synthesis may be protected with suitable protective groups.
Preferably, the nitrogen-containing base is adenine, uracil or cytosine.
The linker arm L is grafted onto any position of the nitrogen-containing base or of its analog. Preferably, the linker arm will be grafted onto a position which does not disturb hybridization. In particular, the linker arm will be attached to the amine at position 4 of cytosine, position 5 of uracil or the amine at position 6 of adenine.
The term "protective group" is intended to mean the groups conventionally used in the chemical synthesis of nucleosides, nucleotides and oligonucleotides (see, for example, Chemistry of Nucleosides and Nucleotides, Edited by Leroy B. Townsend, Plenum Press, New York and London and Protocols for Oligonucleotides and Analogs, Synthesis and Properties, Edited by S. Agrawal, Humana Press, Totowa, N. J.). Preferably, in the case of chemical synthesis, R4 is a 4,4′-dimethoxytrityl group and R2 is a 2-cyanoethyl-N,N-diisopropylphosphoramidite group and R3 is H or OR5, in which R5 is a protective group used in oligoribonucleotide synthesis.
Preferably, in the case of enzymatic synthesis, R4 is a triphosphate group, R2 is H and R3 is OH.
The phosphate groups are generally in the form of salts, and particularly, lithium, sodium or triethylammonium acetate salts.
The invention also relates to a functionalized polynucleotide comprising at least one functionalized nucleotide as defined above. It may be synthesized by chemical and/or enzymatic reaction. In the case of synthesis by enzymatic reaction, and in particular in the case of enzymatic amplification, the neutrality of the functionalized nucleotide with respect to enzymatic reactions allows the incorporation of several functionalized nucleotides.
The term "enzymatic reaction" includes all reactions in which at least one enzyme with activity which is related to a nucleotide is involved. It is thus intended to mean all reactions comprising at least one enzymatic step in which a nucleotide is used as the substrate for the enzyme, regardless of whether said nucleotide is transformed during this enzymatic step. By way of example, such reactions are chosen from those used in molecular biology techniques such as transcription, ligation, elongation and cleavage and more particularly in amplification techniques (see, for example, the article by E. Winn-Deen, Journal of Clinical assay, vol 19, p21-26, (1996)).
Thus, the enzymes which have activities which are related to nucleotides may, in particular, be selected from the following nonexhaustive list: DNA-dependent DNA polymerases, such as the DNA polymerase I Klenow fragment of E. Coli, TAQ polymerase, the T7, T4 or T5 DNA polymerases, eukaryotic cellular polymerases or viral polymerases; RNA-dependent DNA polymerases, such as AMV (avian myoblastosis virus) or MMLV (Moloney murine leukemia virus) polymerases; RNA polymerases, such as the T7, T3 SP6, N4, or PBSII RNA polymerases, or E. Coli RNA polymerase; enzymes with nuclease activity, such am restriction endonucleases or Rnase H; or polyA polymerases, replicases such as Q-beta-replicase, terminal transferases or ligases.
Heat-stable enzymes which have the enzymatic activities described above may also be used in the invention.
According to a preferred embodiment, techniques using a transcription step, such as NASBA (nucleic acid sequence based amplification), TMA (transcription mediated amplification) or post-PCR (polymerase chain reaction) transcription, as described in the articles by R. J. Lipshutz et al, Biotechniques, 19(3), p442-447, 1995 or M. Kozal et al, Nature Medecine, 2(7), p753-759, 1996, will be chosen for the synthesis of the functionalized polynucleotide.
The elements and conditions required for carrying out these enzymatic reactions so as to produce a polynucleotide are well known to those skilled in the art. The manual Current Protocols in Molecular Biology, edited by F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore J. G. Seidman, J. A. Smith and K. Struhl, John Wiley & Sons, 1996 volume 1 chapter 3, gives the methods for enzymatic manipulation of DNA and RNA. Similarly, the work "Molecular Methods for Virus Detection", edited by D. L. Wiedbrand and D. H. Farkas, Academic press, San Diego, 1995 in particular in chapters 8, 9, 12, 13, 14, 15 and 16, gives examples for the enzymatic amplification techniques.
The term "chemical synthesis" is intended to mean all methods, both in solid phase and in liquid phase, in which a suitably protected nucleotide monomer reacts with another nucleotide monomer or polymer via a coupling reaction.
Chemical synthesis methods are given, for example, in "Methods in Molecular Biology, volume 20, Protocols for oligonucleotides and analogs", edited by S. Agrawal, Humana Press, Totowa, N.J., 1993 and "Methods in Molecular Biology, volume 26, Protocols for oligonucleotides conjugates", edited by S. Agrawal, Humana Press, Totowa, N.J., 1994.
The term "polynucleotide" is intended to mean a chain of at least 2 nucleotide monomers. Preferably, if the polynucleotide is synthesized by the chemical route, it is less than 300 nucleotides, and advantageously less than 150 nucleotides, in size. Preferably, if the polynucleotide is synthesized by enzymatic reaction, it is less than 20 kb, and advantageously less than 10 kb, in size.
The two routes of synthesis, chemical synthesis and enzymatic synthesis, can be combined in order to prepare a polynucleotide.
The invention also relates to a labeled functionalized polynucleotide comprising at least one functionalized compound of general formula (I′):
W, L and R1 advantageously satisfy the definitions given above to describe preferred functionalized compounds of the invention.
The term "labeling reagent" is intended to mean a tracer which directly or indirectly generates a detectable signal capable of reacting with the alkyl ketone function.
The following is a nonlimiting list of these tracers:
Preferably, the tracer is a fluorescent compound with low steric hindrance, such as fluorescein, dansyl, chromophores of the IR (Li-COR Inc, Lincoln Nebr., USA), CY5 and CY3 (Randolph J. B. and al, Nucleic Acids Res., 25(14), p2923-2929, 1997) type and derivatives thereof. The term "low steric hindrance" is intended to mean a molecular weight of less than 1000 g/mol.
In order to react with the alkyl ketone function, this labeling reagent must carry a nucleophilic function capable of reacting with an alkyl ketone function, such as alkoxyamine or hydrazine functions.
Preferably, the function chosen is alkoxyamine, which may be introduced by any direct or indirect means. The term "direct means" is intended to mean a covalent bond between the tracer, or a molecule carrying the tracer, and the alkoxyamine function. The term "indirect means" is intended to mean complexation systems of the metal/chelate type or affinity systems, i.e. haptens which can be detected by a specific antibody or a protein, such as the biotin/avidin or streptavidin pairing or the sugar/lectin pairing. In this case, the tracer is carried by the antibody or the protein and the alkoxyamine function is carried by the hapten.
In particular, the labeling reagent has the formula:
The invention also relates to a solid support to which a nucleotide, a nucleoside or a polynucleotide according to the invention is attached by covalence.
In order to perform this attachment, a nucleotide, a nucleoside or a polynucleotide comprising an alkyl ketone group is reacted with a solid support on which there is an alkoxyamine or hydrazine function, preferably an alkoxyamine function.
In a first embodiment, the polynucleotide is preformed and the final reaction consists in grafting, at a predetermined position on the support, the polynucleotide. In one particular embodiment, the polynucleotides are synthetic oligonucleotides (synthesized via the chemical route) which are short in size (less than 50 bases), and in a second particular embodiment, the polynucleotides are more than 50 bases in size and are prepared using enzymatic methods such as enzymatic amplification.
In a second embodiment, the nucleoside or nucleotide is added via successive steps (chain extension) on the support in order to obtain, at the end of the synthetic cycle, a polynucleotide grafted to a predetermined position on the solid support.
A preferential use of these grafted supports is the production of biochips for gene analysis.
By way of illustration, examples of these biochips are given in the publications by G. Ramsay, Nature Biotechnology, 16, p40-44, 1998; F. Ginot, Human Mutation, 10, p1-10, 1997; J. Cheng et al, Molecular diagnosis, 1(3), p183-200, 1996; T. Livache et al, Nucleic Acids Research, 22(15), p2915-2921, 1994; J. Cheng et al, Nature Biotechnology, 16, p541-546, 1998 or in patents U.S. Pat. No. 4,981,783 (Augenlicht), U.S. Pat. No. 5,700,637 (Southern), U.S. Pat. No. 5,445,934 (Fodor), U.S. Pat. No. 5,744,305 (Fodor), U.S. Pat. No. 5,807,522 (Brown).
The invention also relates to a method for detecting a target nucleic acid in a sample, in which this target nucleic acid, optionally pretreated, is brought into contact with at least one functionalized compound corresponding to formula (I′), in the presence of the elements and under conditions required for producing a polynucleotide of the invention, so as to produce a functionalized polynucleotide; in labeling said polynucleotide with a labeling reagent and then in detecting said labeled polynucleotide. The above-mentioned elements and conditions are well known to those skilled in the art.
The term "pretreatment" is intended to mean the various steps for treating the sample in order to make the target nucleic acid accessible, such as for example lyzing, fluidifying, concentrating.
Preferably, the functionalized polynucleotide is obtained via an enzymatic amplification reaction which acts on the target nucleic acid which serves as a matrix, and which is capable of incorporating the functionalized nucleotide.
Advantageously, the enzymatic amplification technique is NASBA (nucleic acid sequence based amplification), TMA (transcription mediated amplification) or post-PCR (polymerase chain reaction) transcription, as described in the articles by R. J. Lipshutz et al, Biotechniques, 19(3), p442-447, 1995 or M. Kozal et al, Nature Medecine, 2(7), p753-759, 1996.
The labeled polynucleotide may be detected qualitatively and/or quantitatively in homogeneous or heterogeneous phase. A preferential detection mode consists in attaching the labeled polynucleotide to a solid support via a hybridization reaction between the labeled polynucleotide and another polynucleotide, itself attached to the solid support, and then revealing the presence of the labeled polynucleotide after a washing step. This revelation is carried out directly by reading, such as for example with a scanner or a camera if the tracer is a fluorescent molecule.
The detection method is particularly useful when a multitude of polynucleotides are attached to the solid support at a predetermined position so as to form a "DNA chip".
Specifically, the density of the polynucleotides attached to the solid support imposes considerable steric constraints during the hybridization and the labeled polynucleotide according to the invention allows good sensitivity of detection. Examples of these chips are given, for example cited in the publications and patents mentioned above.
The detection method can be used for sequencing, profiling the expression of messenger RNAs or screening mutations, or diagnosing infectious or genetic diseases.
A fragmentation step can take place in order to promote the hybridization of the labeled polynucleotide on the DNA chip, before, together with, or after the labeling step.
The invention also relates to a method for detecting a target nucleic acid in a sample, in which this target nucleic acid is brought into contact with a functionalized polynucleotide of the invention, the labeling reagent is reacted and the presence of the target nucleic acid is detected.
Another means of carrying out the invention is to react the functionalized polynucleotide and the labeling reagent, before the hybridization with the target nucleic acid. This target nucleic acid may have been amplified using an enzymatic amplification technique.
Finally the invention relates to the method for detecting a target nucleic acid, according to which a labeled polynucleotide of the invention is available for use, the nucleic acid is brought into contact with the labeled polynucleotide and the presence of the target nucleic acid is detected.
The term "solid support" as used herein includes all materials on which it is possible to immobilize a polynucleotide for use in diagnostic tests and in separation processes. Natural or synthetic materials, which may or may not be chemically modified, may be used as a solid support, in particular polysaccharides such as cellulose-based materials, for example paper, cellulose derivatives such as cellulose acetate and nitrocellulose, dextran; polymers such as polyvinyl chlorides, polyethylenes, polystyrenes, polyacrylates, polyamides, or, copolymers based on monomers of the styrene type, unsaturated carboxylic acid esters, vinylidene chloride, dienes or compounds with nitrile functions (such as acrylonitrile); vinyl chloride/propylene copolymers, vinyl chloride/vinyl acetate copolymers; natural fibers such as cotton and synthetic fibers such as nylon; inorganic materials such as silica, quartz, glasses, ceramics; latexes, i.e. colloidal aqueous dispersions of any water-insoluble polymer; magnetic particles; metallic derivatives, gels etc.
The following examples make it possible to illustrate some of the advantages of the invention without, however, limiting the scope thereof.
The attached FIGURE represents the transcription yields measured as a function of the nucleotide used.
I.1. Synthesis of uridine(C5)—C9-methylketone 1
The 2′,3′-OH positions are protected by reacting acetone in acid medium according to the protocol described in "Nucleic acid chemistry, Editors Townsend-Tipson, Wiley-Interscience, John Wiley & Sons, p 765-766 (1978)".
Synthesis of the Methyl Ketone Chain
The methyl ketone unit is introduced onto the arm by peptide coupling between propargylamine and 6-oxo-heptanoic acid.
6-Oxoheptanoic acid (2.75 g, 18.16 mmol) is solubilized in 40 ml of anhydrous THF. The solution is placed at 0° C. under an argon atmosphere. N-methyl-, morpholine (2 ml, 18.16 mmol) is then added, followed by propargylamine (1.25 ml, 18.16 mmol) 15 min later, and the mixture is allowed to return to room temperature. After reaction for 30 minutes, the precipitate is filtered and evaporated to dryness.
The residue obtained is taken up in dichloromethane and is washed with a 0.1 N aqueous sodium hydroxide solution, then with a 1 N aqueous hydrochloric acid solution and finally with water saturated with sodium chloride.
After drying over Na2SO4, the dichloromethane is evaporated off and the residue is chromatographed on silica gel (eluent: ethyl acetate). The product is thus obtained in the form of a white powder (2.6 g, 14.4 mmol; 80%).
The methyl ketone chain was characterized by proton NMR, carbon 13 NMR and mass spectrometry.
Introduction of the methyl ketone chain onto the nucleoside: Heck coupling (Hobbs, J. Org. Chem., 1989, 54, 3420-3422)
The mixture is left under argon for 10 minutes. Tetrakis triphenylphosphine palladium (134 mg, 0.116 mmol) is then added. After reaction for 3 hours, the DMF is evaporated off, followed by coevaporation with acetonitrile. The residue is taken up in ethyl acetate and the organic phase is washed with an aqueous solution saturated with sodium chloride. After drying over Na2SO4 and evaporation, the residue is chromatographed on silica gel (eluent: ethyl acetate/methanol: 90/10).
After evaporation, the methyl ketone nucleoside is obtained in the form of a whitish powder (340 mg., 0.73 mmol, 60%). The product was characterized by proton NMR, carbon 13 NMR and mass spectrometry. The methyl ketone nucleoside correctly protected for the introduction of the triphosphate in 5′ is thus obtained.
Production of the Uridine(C5)—C9-Methylketone Triphosphate Nucleoside 1
Phosphorylation: Eckstein, J. Org. Chem., 1989, 54, 631-635
The methyl ketone nucleoside (46 mg, 0.1 mmol) is dissolved in anhydrous pyridine and evaporated twice. 100 ml of pyridine/300 ml of dioxane and a freshly prepared solution of 2-chloro-4H-1,2,3-dioxaphosphorin-4-one (1 M) in dioxane (130 μl; 130 mmol) are then added under argon, the mixture is left to stir for 20 minutes, and then a 0.5 M solution of tributylammonium pyrophosphate in anhydrous DMF (320μl, 0.16 mmol) and, simultaneously, 130 μl of tributylamine are then added. After 30 minutes, 2 ml of 1% iodine solution are added to a pyridine/water (98/2: v/v) mixture.
After stirring for 20 minutes, the excess iodine is eliminated with a 5% aqueous NaHSO3 solution and the stirring is continued for 10 minutes. The mixture is evaporated to dryness and an extraction is carried out with a water/dichloromethane mixture. The aqueous phase is evaporated off and then C18 reverse phase chromatography (flash) is performed (eluent: water/methanol 1/1). The fractions containing the product are evaporated and the counter-ion exchange is carried out by passing over Dowex, Na+ resin. The protected triphosphate is thus recovered (0.03 mmol; 30%).
The protected triphosphate (0.03 mmol) is taken up in 15 ml of milli-Q water to which 15 ml of a 25% aqueous TFA solution are added. The solution is stirred for 15 minutes, then evaporated and coevaporated twice with water.
The product is taken up in 10 ml of milli-Q water and neutralized with 0.1 N sodium hydroxide, to pH 8. After the evaporation, the protected triphosphate is purified on C18 reverse phase (H2O then HMO/MeOH, 1/1). The counter-ion exchange is carried out by passing over cation exchange resin (Dowex Na+). The fractions containing the product are evaporated and assayed. 0.021 mmol (70%) of the uridine(C5)-C9-methylketone triphosphate nucleoside 1 is thus recovered, and this nucleoside was characterized by proton NMR, carbon 13 NMR and phosphorus 31 NMR.
I.2. Synthesis of Adenosine(N6)—C10-Methylketone 2
Synthetic Pathway
The introduction of diaminobutane at position 6 of the adenosine is carried out by substitution of the triazolo group carried by the protected intermediate nucleoside. The methyl ketone function is then introduced by peptide coupling at the nucleoside level. After deprotection of the silyl group in 5′, phosphorylation is carried out by the Eckstein method. After deprotection, the expected triphosphate is obtained, which is characterized by proton NMR and phosphorus 31 NMR.
Synthesis of the Protected Triazolo Nucleoside
Preparation of adenosine 2′,3′-Isopropylidene
(Nucleic Acid Chemistry, part 2, Editors Townsend, Tipson, Wiley Interscience, John Wiley & Sons p. 768, (1978))
Ethyl orthoformate (12.44 ml, 74.8 mmol) is added dropwise, under argon, to a suspension of adenosine (5 g, 18.7 mmol) in acetone (10 ml) containing APTS (para-toluene-sulfonic acid) (3.9 g, 20.6 mmol). After reaction overnight, 110 ml of water containing 1.86 ml of aqueous ammonia at 27% are added. After stirring for 30 minutes, the reaction mixture is evaporated until white crystals appear. After 12 h at +40° C., a white precipitate is obtained which is recrystallized in water. 4.17 g (13.5 mmol, 72%) of product is obtained in the form of a white powder. This intermediate was characterized by proton NMR.
Synthesis of amidine (Bartlett and Humphreg, J. Chem. Soc., 1967, 1664-1666)
Thionyl chloride (39.96 g, 24.5 ml, 0.338 mol) is added dropwise to N—N′-diformylhydrazine (12 g, 0.136 mol) in DMF (270 ml) at 10° C. The mixture becomes yellow. Stirring is maintained for 2 days. The precipitate obtained is filtered and washed with DMF then with ether. After drying under a vacuum, the amidine is obtained with a yield of 95% (28 g, 0.130 mol).
Preparation of the triazolo intermediate (Samano, Miles, Robins, J. Am. Chem. Soc., 1994, 116, 9331-9332)
The adenosine isopropylidene (1 g, 3.2 mmol) and the amidine described above (1.4 g, 6.5 mmol) are stirred into pyridine (15 ml) at 100° C. under argon for 48 h. The pyridine is then evaporated off and coevaporated with toluene. The oil obtained is then taken up with ethyl acetate and this organic phase is washed with water saturated with NaCl. After drying over Na2SO4 and evaporation, the triazolo nucleoside is obtained in the form of a white powder with a yield of 60% (700 mg, 1.9 mmol). It was then characterized by proton NMR.
Protection in 5′ of the Triazolo Derivative
The triazolo nucleoside (1 g, 2.8 mmol) is solubilized in 20 ml of pyridine. TBDMS-Cl (tert-butyldimethylsilyl chloride), (462 mg, 3 mmol) is added at 0° C. under argon. Stirring is maintained for two hours then the pyridine is evaporated off. The residue thus obtained is chromatographed on silica gel (eluent: CH2Cl2/methanol 95/5). After evaporation, the totally protected intermediate adenosine is obtained in the form of a white powder (1.25 mg, 2.6 mmol, 93%). This nucleoside was characterized by proton NMR, carbon 13 NMR and mass spectrometry.
Introduction of the Diaminobutane Chain onto the Protected Intermediate Adenosine
The triazolo adenosine (1.25 mg, 2.64 mmol) is solubilized in 10 ml of acetonitrile. Diaminobutane (2.7 ml, 25.4 mmol) is then added and the mixture is stirred at 50° C. under argon. After 5 hours, the solvent is evaporated off and the residue is taken up in ethyl acetate. This organic phase is washed with water saturated with NaCl. After drying over Na2SO4 and evaporation by chromatography on silica gel (eluent: CH2Cl2/MeOH: 8/2, then CH2Cl2/MeOH: 8/2 in the presence of 2% aqueous ammonia).
After evaporation, the product is obtained in the form of an oil (1 g, 2 mmol, 80%).
The aminated nucleoside was characterized by proton NMR, carbon 13 NMR and mass spectrometry.
Coupling to the Methyl Ketone Chain and Deprotection in 5′
6-Oxo-heptanoic acid (288 mg, 2 mmol) is dissolved in 5 ml of anhydrous THF. The solution is placed at 0° C. under argon. N-Methylmorpholine (223 p1, 2 mmol) is then added, followed by isobutyl dichloroformiate (258 μl; 2 mmol) 5 min later. After 15 minutes, the aminated nucleoside is added. After 2 hours, the THF is evaporated off and the residue is taken up with ether. This is washed with a 1 N aqueous NaOH solution and then with water saturated with NaCl. After drying over Na2SO4 and evaporation, an oil is obtained. The desilylation is carried out by taking the oil up in 10 ml of THF, to which TBAF (3.25 ml of a 1M solution in THF) is added. After one hour, the solvent is evaporated off. The residue is solubilized in dichloromethane and washing is carried out with water saturated with NaCl. After drying and evaporation, the residue is chromatographed on silica gel (eluent: CH2Cl2, then CH2Cl2/MeOH: 95/5).
After evaporation, 870 mg of the nucleoside carrying the methyl ketone function and deprotected in 5′ (1.7 mmol, 85% over 2 steps) is obtained. It is carried by proton NMR, carbon 13 NMR and mass spectrometry.
Phosphorylation and Production of the Adenosine-(N6)—C10-Methyl Ketone Nucleotide 2
The adenosine-(N6)—C10-methyl ketone protected in 2′, 3′ by isopropylidene is dissolved in anhydrous pyridine and is evaporated twice. 500 μl of pyridine, 1.5 ml of dioxane and a freshly prepared solution of 2-chloro-4H-1,2,3-dioxaphosphain-4-one (1 M) in dioxane (650μl, 0.65 mmol) are then added under argon. Stirring is allowed to proceed for 20 minutes and then a 0.5 M solution of tributylammonium pyrophosphate in anhydrous DMF (1.6 ml, 0.8 mmol) is added simultaneously with 650 Ml of tributylamine.
After 30 minutes, 10 ml of 1% iodine solution is added to a pyridine/water (98/2, v/v) mixture. After 20 minutes, the excess iodine is destroyed with a 5% aqueous NaHSO3 solution and stirring is maintained for 10 minutes, the mixture is evaporated to dryness and water/dichloromethane extraction is performed. The aqueous phase is evaporated off and then reverse phase (C18) purification is carried out (eluent: H2O/MeOH). The fractions containing the product are evaporated and the counter-ion exchange is carried out by passing over Dowex, Na+ resin. The protected triphosphate (0.28 mmol, 56%) is thus recovered.
0.035 mmol of protected triphosphate are taken up in 17.5 ml of milli-Q water, to which 17.5 ml of a 25% aqueous TFA solution are added. The mixture is stirred for 15 minutes and then evaporated and coevaporated twice with water, and the product is taken up in 10 ml of milli-Q water and neutralized with 0.1 M sodium hydroxide, to pH 8. After evaporation, a C18 purification is carried out (eluent: H2O; H2O/MeOH). The fractions containing the product are evaporated and assayed. 0.022 mmol (63%) of the adenosine-(N6)-C10-methyl ketone triphosphate 2 are thus obtained-
I.3. Synthesis of Cytidine-(M4)—C10-Methyl Ketone
This nucleotide was prepared in two ways:
II. 1. Synthetic Schemes
The oxyamine chain is introduced onto the fluorescein in three steps: the first step is nucleophilic addition of 1,3-diaminopropane to fluorescein isothiocyanate (FITC). After reverse phase purification, the oxyamine unit protected in the form of Fmoc is introduced by peptide coupling. The free oxyamine is generated by deprotection in basic medium.
Introduction of the 1.3-diaminoprogane Chain:
Diaminopropane (585 p1, 6.96 mmol) is added to 20 ml of anhydrous DMF. Fluorescein isothiocyanate (FITC) (500 mg, 1.16 mmol), solubilized in 7 ml of anhydrous DMF, is then added, dropwise, under argon. Stirring is maintained for 15 minutes after the FITC has been added. The mixture is evaporated to dryness and coevaporated twice with water. The residue is reverse phase (C18) chromatographed: (eluent: H2O/MeOH: 1/1).
The product is thus recovered in the form of an orange powder (420 mg, 0.93 mmol, 80%). It is characterized by proton NMR, carbon 13 NMR and mass spectrometry.
Introduction of the Protected Alkoxyamine Group:
Carboxyalkoxyamine protected with Fmoc: HOOC—CH2—ONH-Fmoc (473 mg, 1.5 mmol in 10 ml of anhydrous DMF) is solubilized. It is placed at 0° C. under argon. N-Methylmorpholine (166μl, 1.5 mmol) is then added and, after 15 minutes, the fluorescein carrying the diaminopropane chain (350 mg, 0.75 mmol) is added. After reacting for 1 hour, the DMF is evaporated off to dryness. The residue obtained is chromatographed on silica gel (CH2Cl2/MeOH: 85/15 (solid deposit)). The protected fluorescein-alkoxyamine is thus obtained in the form of an orange powder (227 mg, 0.3 mmol, 40%). It is characterized by proton NMR, carbon 13 NMR and mass spectrometry.
Production of the fluorescein-alkoxyamine Label 4:
The fluorescein protected with fmoc (100 mg, 0.13 mmol) is solubilized in 2 ml of anhydrous DMF. Pyridine (20μl, 0.2 mmol) is then added. After 15 minutes, the mixture is evaporated to dryness and then C118 reverse phase purification is carried out (eluent: H2O/CH3CN: 1/1 then CH3CN). After evaporation, the product is obtained in the form of an orange powder (49 mg, 0.09 mmol, 70%). This fluorophore was characterized by proton NMR, 13C NMR and mass spectrometry.
The reactivity was tested at the nucleoside level and at the nucleotide level:
The reaction is carried out in the presence of 1.1 eq. of fluorophore-ONH2 (4) with respect to the methyl ketone compound. The reaction is rapid and selective. At the nucleoside level, the adducts were characterized by proton NMR and mass spectrometry.
IV.1. Description of the Main Steps
The transcriptions were carried out on a PCR target (fragment of the 16 S RNA of Mycobacterium tuberculosis (Mtb)) (Troesch A. et al, J. Clin. Microbiol., 37(1), 49-55, 1999) or a fragment of HIV reverse transcriptase (Kozal M. J. et al, Nature Medecine, 2(7), 753-759, 1996) using T7 RNA polymerase and various ratios between the functionalized nucleotide and the natural nucleotides, while at the same time keeping the total concentration of each nucleotide at 1 mM. This ratio between the functionalized nucleotide and the corresponding natural nucleotide is expressed as a percentage and the ratios used are in general 0, 30, 70 and 100%. The point 0% is used as a transcription control since, in this case, there is no functionalized nucleotide and the transcription reaction comprises the 4 natural nucleotides. When the ratio is 100%, this means that the functionalized nucleotide represents 100% of the nucleotide studied (the other three nucleotides required for the transcription reaction naturally being the natural nucleotides). This ratio of 100% for a functionalized nucleotide is the most significant test of the neutrality with respect to an enzymatic reaction since the enzyme must incorporate this nucleotide in order to function correctly. The incubation time for the transcription reaction is 1 h at 42° C.
The transcriptions are analyzed by polyacrylamide gel electrophoresis under denaturing conditions (6% acrylamide, 7 M urea, 1×TBE). The volume loaded is 5 μl and the migration takes place for 45 min at 150 V. The transcripts, natural or functionalized with the methyl ketone function, are visualized under a UV lamp after staining with ethidium bromide.
The amount of transcripts produced in each reaction is determined by UV assay, after purification, of an aliquot derived from a transcription reaction.
Enzymatic Digestion
The transcripts are purified on microcon-50 filters (Amicon, Beverly, Mass.) in order to remove the excess of unincorporated nucleotides. They are then hydrolyzed according to the protocol described in patent application WO 98/05766 using P1 nuclease (Boehringer reference 2362251,2U, 2 h at 37° C.) and alkaline phosphatase (Boehringer-Mannheim reference 713023, 1U, 1 h at 37° C.). The digestions are carried out on 4×1014 transcript copies. The nucleoside composition is determined by reverse phase HPLC, by comparing with nucleoside standards composed of a mixture of natural nucleosides.
The HPLC conditions are as follows:
The transcripts are labeled using various proportions of fluorophore. The reaction time is 30 min at room temperature. The labeling is carried out on the transcripts generated from an Mtb target and/or HIV target, obtained by incorporation of 100% of a methyl ketone nucleotide. Initially, the labeled transcripts are analyzed by polyacrylamide gel electrophoresis and visualized under UV, before and after staining with ethidium bromide.
Before hybridizing on the DNA chip, the labeled transcripts are cleaved at ˜65° C. for 30 min using imidazole and manganese chloride (MnCl2) each at a concentration of 30 mM.
Hybridization on a DNA Chip
After cleavage, the fragments obtained are hybridized, detected and analyzed on a DNA chip (Affymetrix, Santa Clara, Calif., USA) according to the protocol supplied by the manufacturer.
The "myco" chips are designed for resequencing the 213-415 region of the "Genbank" M20940 sequence of the 16S RNA of Mycobacterium tuberculosis (Troesch A. et al, J. Clin. Microbiol., 37(1), 49-55, 1999). Those termed "HIV PRT 440" are designed for resequencing the RT (reverse transcriptase) and protease regions of the HIV virus (Kozal M. J. et al, Nature Medecine, 2(7), 753-759, 1996).
In the case of the Mtb transcript, 1014 copies are hybridized on the chip.
In the case of the HIV transcript, 5×1012 copies are hybridized on the chip.
IV. 2. Results
Incorporation of the uridine(C5)—C9-methyl Ketone Nucleotide 1 (U—COCH3)
The results in the table above show that the uridine-methyl ketone nucleotide (1) is very well incorporated by T7 RNA polymerase. This allows the production of activated transcripts capable of reacting with a label carrying the alkoxyamine function.
Evaluation of the Incorporation by HPLC
The peak which corresponds to the U-methyl ketone nucleoside (retention time 26.2 minutes) is indeed observed in the chromatographic profile of the hydrolyzate of the transcripts containing 100% of U-COCH3 (1) and obtained from the Mtb target, which means that the nucleotide is not modified during the transcription and enzymatic digestion steps. The absence of the peak corresponding to the natural uridine (retention time 14.97 minutes) is also noted.
This shows that the U-methyl ketone nucleotide is correctly incorporated and can be used at 100% in a transcription reaction.
Evaluation of the Labeling by Polyacrylamide Gel
The labeling was carried out on the crude transcripts generated from an Mtb target, using 100% of the U-COCH3 nucleotide (1). The amount of fluorophore compared to the number of reactive sites was varied. These ratios are: 2, 5, 10, 15, 20 and 50 equivalents of flurophore-ONH2. 10 equivalents are suitable for intensive and selective labeling.
On a gel, before staining with ETB, representing labeling using 5, 10 and 15 eq. of the fluorophore, labeling is observed which is more or less intense depending on the number of equivalents of fluorophore used, but there is a visible band whatever the amount of labels used. The appearance of new bands is also noted at high doses of fluorophore. This is certainly due to a high density of the label on the target.
Hybridization and Analysis of the Results
The Mtb and HIV transcripts generated from corresponding targets, using 100% of UTP-COCH3 (1), underwent the treatments described above. During the cleavage step, a "blocking" agent (acetone or glutaraldehyde) was used in order to avoid saturating the chip with the excess of fluorophore-ONH2 which adsorbs to the surface of this chip. The hybridization is carried out using the "Gene Chip fluidics Station" for hybridization (800101 Affymetrix, Santa Clara, Calif.), the suitable chips and buffers and the protocol supplied by the manufacturer.
The base call percentage, mean signal intensity, median intensity and background noise parameters were calculated using the software provided by the manufacturer (GenChip sequence analysis system, reference 900135, Affymetrix, Santa Clara, Calif.). The results are given in the two tables below. The signal intensity is expressed in RFU (fluorescence units of the manufacturer)
Mtb Transcripts:
HIV Transcripts
In both the Mtb and HIV cases, very effective labeling with intensities greater than 1000 Rfu and resequencing (base call) percentages close to 100% are observed. These results show that the reaction between the alkoxyamine and methyl ketone, demonstrated at the monomer stage, is therefore specific and effective between RNA sequences containing methyl ketone functions and the fluorescent label carrying the alkoxyamine function. Specifically, a sufficient amount of labeled and detectable transcript fragments are obtained with only 2 equivalents of alkoxyamine label per methyl ketone function.
The cytidine 3 was incorporated at 100% into RNAs transcribed from Mtb, as described in Example III. The cleavage and labeling method is also carried out as indicated above.
Here again, very effective labeling is observed. The signal intensity is greater than 6000 Rfu and the resequencing is 94%. As in the previous example, this result demonstrates that the reaction between the alkoxy-amine and methyl ketone is specific and effective between RNA sequences containing methyl ketone functions and the fluorescent label carrying the alkoxyamine function.
1. Comparison Between the Alkoxyamine and Methyl Ketone Function on the U Nucleotide.
Uridine carrying an alkoxyamine chain at position is prepared according to the method described in Example 4 of patent application WO-A-98/05766. The synthesis of the fluorescein aldehyde (FLUO-CHO) label is described in Example 18 of application WO-A-98/05766.
It is important to note that the alkoxyamine function requires permanent protection with the tert-butoxycarbonyl (BOC) group. This function is deprotected at the same time as the isopropylidene in 2′,3′ after phosphorylation in the presence of 50% trifluoroacetic acid. This percentage of TFA is necessary to obtain a deprotection of the alkoxyamine close to 90%. In order to obtain total deprotection, more concentrated acid solutions are necessary. In these solutions, the triphosphate degrades very rapidly to diphosphate and monophosphate.
In the case of the methyl ketone function, no deprotection is necessary and only 25% of TFA is used for total deprotection of the isopropylidene in 2′,3′ after phosphorylation. No degradation is observed with this percentage of TFA.
The methyl ketone chain is sufficiently hydrophobic even after deprotection; it thus contributes to good separation of the nucleotide triphosphate when it is purified by reverse phase HPLC. This separation is more difficult in the case of the alkoxyamine chain.
Incorporation and Post-Labeling of the UTP-Alkoxyamine
The reactions for transcription, enzymatic hydrolysis of the transcripts, HPLC analysis and post-labeling are carried out under the same conditions as those described in Example IV.
The FIGURE shows the transcription yields measured in terms of the amount of amplicons produced (on the y-axis in copies/microliters) as a function of the 3 nucleotides used.
In this example, the incorporation of the two nucleotides, alkoxyamine (U-ONH2) and methyl ketone (U-COCH3 compound 1), is studied.
The uridine-methyl ketone 1 at 100% has no effect on the transcription yield. This yield is lower when the same nucleotide carrying the alkoxyamine function is incorporated.
The resequencing percentage using the uridine-alkoxyamine is lower than that obtained with its homolog carrying the methyl ketone function.
2. Comparison Between the Aldehyde and Methyl Ketone Function on the U Nucleotide.
The uridine with an aldehyde chain (U—CHO) is prepared according to the following scheme:
The chain is synthesized by peptide coupling between 4-aminobutyraldehyde diethylacetal and 4-pentynoic acid. Heck coupling produces the protected nucleoside. Phosphorylation followed by deprotection in acid medium correctly produces the expected nucleotide. The products were characterized by 1H NMR and 13C NMR.
Here again, it is necessary for the aldehyde function to be protected during the coupling and phosphorylation steps. The deprotection of the aldehyde is carried out at the same time as that of the isopropylidene (25% of TFA); however, the purification and desalination of the deprotected product are more difficult compared to those of the nucleotide-methyl ketone (U—COCH3, compound 1). The yield from the phosphorylation step is 20%.
The HPLC analysis of the transcripts containing the UTP-aldehyde, hydrolyzed using the enzymatic technique described in Example IV, shows that the nucleotide-aldehyde is transformed. This transformation is certainly due to the aldehyde function interacting with components of the buffers during the transcription or enzymatic hydrolysis of the transcripts.
The post-labeling reaction using the fluorescein-alkoxyamine shows no result after analysis of the mycobacterium transcripts on gel and on DNA chips. This shows that transformation of the aldehyde has occurred during the transcription step, which shows the advantage of the methyl ketone function compared to the aldehyde.
3. Comparison between the Amine and Methyl Ketone Function on the U Nucleotide.
The uridine carrying an amine chain at position 5 (U-NH2) is prepared according to the method described in Example 3 of patent application WO 98/05766. It is necessary to protect the amine function with the Boc group in this case as well.
The transcription yields obtained with 100% of U-NH2 are much lower than when the methyl ketone function is used and do not allow efficient resequencing on the DNA chip both in the case of Mtb and in the case of HIV.
4. Comparison Between the Amine and Methyl Ketone Function on the C Nucleotide.
The cytidine carrying an amine chain at position 4 (C—N N2) is prepared according to Example 16 of patent application WO-A-98/05766.
This nucleotide was incorporated into fragments of the 16S RNA of Mycobacterium tuberculosis through transcription reactions carried out on a PCR target according to the protocol described in Example IV.
The HPLC analysis of the transcripts containing the C—NH2, hydrolyzed using the enzymatic technique described in the example above, shows that the aminated nucleotide is correctly incorporated and is intact.
The yields from transcription with various percentages of C—NH2, also determined as indicated in Example IV, are given in the table below.
It is important to note that, with 100% of C—NH2, the amount of amplicons obtained is 20 times less than that obtained with transcription containing 100% of natural nucleotide.
The labeling reagent used in the post-labeling reaction was fluorescein isothiocyanate obtained from Sigma-Aldrich (St Quentin Falavier, France).
Fluorescein isothiocyanate (FITC)
The labeling of the transcripts obtained by incorporation of the C—NH2 were labeled with FITC, hybridized on the DNA chip intended for the identification of these Mtb targets, and the signals were analyzed according to the protocol described above. The results obtained with and without blocking agent are given in the table above
If the diamine chain is not added after the labeling, the surface of the chip is completely saturated with the 100 eq of label and the signals cannot be exploited. This saturation is certainly due to the interaction of the isothiocyanate function with the exocyclic amine functions of the bases.
The post-labeling addition of 1000 eq of diamine, having the same structure as that carried by the cytidine, appears to confirm this hypothesis since the surface of the chip is no longer saturated, and 86% resequencing is obtained. The signal intensity is low compared to that obtained with transcripts containing the methyl ketone and labeled with fluorescein-alkoxyamine.
In addition to the inhibitory effect of the C-NH2 during the transcription, the resequencing percentage when the C-NH2 is used is lower than that obtained with its homolog carrying the methyl ketone function (C-COCH3, compound 3).
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4.0(18 reviews)
From: Hill
Lifetime limited warranty
Average Customer Review5.0 (121 reviews)
Create an online reviewand share your thoughts with other customers
Things I liked:it works very well
Things I would have changed:nothing
What others should know:i am 62 years old i can pump 3000 lbs but it takes a while i have to rest
Things I liked:Straight forward set up and easy assembly. Reliable and accurate pressure gauge, always agrees with the gauge on my rifle.
Things I would have changed:Nothing that I have found.
What others should know:Works as advertised.
Things I liked:Overall this Pump is really Top, and works even better than advertised, i had one and sold to my friend, he has 2 Diana P1000, he is continuously filling his both Dianas up to 300 bar with this Hill pump, and till now the hill pump is works very good,,, last year i bought 3 and this year i have my order placed for 5 more,,,1 for myself and rests for my friends,,
Things I would have changed:no thing,,, it is perfect...
What others should know:you need to let the pump for some time after pumping until the pump will cool down. if you repair it each month( cleaning & re-lubing parts and o-rings) this pump will late for years ,,,,
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Compatible with Hill Pump MK3, Up to 3300 PSI
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Keeping your driving teen safe while on the road
When The Who performed "Baba O'Riley" during Madison Square Garden's Sandy Relief concert, at least Pete Townshend looked his age. But when Roger Daltrey started singing about "teenage wasteland," well, something wasn't right, and we're not just talking about his waxed chest.
If you've seen the statistics on teens and driving, you can't help but be shocked by that wasteland. Every day in the U.S., seven teenagers are killed in car accidents; annually, a quarter of a million or more end up in the emergency room. The highest risk comes during the first six months they have their license, and it's especially risky when more kids are in the car and at night.
So, if you remember when The Who had hair -- both on Townshend's pate and Daltrey's chest -- and you've got a teenager asking for the keys, here's how to keep him or her safe.
1. Provide driving lessons -- and consider that you might not be the best choice for teacher!
2. Provide opportunities to practice with an adult onboard (in empty parking lots, empty streets, then gradually in higher-traffic zones).
3. Inspire your child to be a careful driver. How? By example: When you drive, don't talk on the phone, text, speed or drive without your seat belt. (Just developed and hopefully available soon, the CAP, or cellphone accident preventer, blocks a driver's cell, but not passengers'.)
4. Insist on check-ins to help your new driver resist peer pressure that puts your teen and friends in danger. And make it clear: Breaking rules means no driving.
Mehmet Oz, M.D. is host of "The Dr. Oz Show," and Mike Roizen, M.D. is Chief Medical Officer at the Cleveland Clinic Wellness Institute. For more information go to www.RealAge.com.
The Herald-Dispatch welcomes your comments on this article, but please be civil. Avoid profanity, obscenity, personal attacks, accusations of criminal activity, name-calling or insults to the other posters. Herald-dispatch.com does not control or monitor comments as they are posted, but if you find a comment offensive or uncivil, hover your mouse over the comment and click the X that appears in the upper right of the comment. If you do not want your comment to post to your personal Facebook page, uncheck the box below the comment.


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What's the only thing better than one adorable baby? Two adorable babies! (Or three or four or any number up until, say, 12. Then you really have to stop and take a look at your life) Katherine Heigl and husband Josh Kelley are clearly in agreement with us, seeing as how they've just adopted a second child. "Yes, they have adopted," their rep confirmed to E! Online today, while People reports that the family has welcomed a second daughter. Anyway, regardless of the baby's gender…squeeeeeeeee!
No word on the baby's age or name yet either, nor whether their latest child was adopted in the U.S. or internationally, though the National Enquirer is allegedly claiming Heigl and Kelley are the proud parents of a baby girl from Louisiana. Really, all we know about Heigl Baby #2 is so far is that 1) she will definitely be enjoying/constantly pestering a big sister (the One For The Money star adopted daughter Naleigh back in 2009) and 2) the world will probably have forgotten all about her mom's starring role in Killers before either of her kids are old enough to see it. Haha, just kidding! We will never let the world forget about Katherine Heigl's sexy spy rom-com with Ashton Kutcher! Her children's children's children will know about Killers!
[Photo: Splash News Online]


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Assistant Baseball Coach
Assistant Coach Jake Carlson
Phone: (620) 870-8005
Jake Carlson enters his 1st year with the Hawks. Carlson returns to the WJCAC conference after spending the previous two years at Blinn College.
Coach Carlson begins his 10th season at the collegiate level. Carlson started his coaching career as the Volunteer Assistant at Nicholls State University and was promoted to a full-time Assistant in his 2nd season. After Nicholls State, Carlson made stops at Coffeyville Community College, Odessa College and Blinn College. Carlson served as an associate scout for five years with the Kansas City Royals.
A native of Manhattan, KS, Carlson prepped at Manhattan High School. After high school, Carlson attended Butler County Community College and as a Sophmore was an all-conference outfielder. After Butler, Carlson transferred to Nicholls State and finished the remaining two years of his collegiate playing career. Carlson has a Bachelor of Science degree from Nicholls State University.


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It's only the first day, and we'll have to wait for the numbers over a longer time horizon; but if these lines and these stories are any indication, the Deferred Action for Childhood Arrivals (DACA) program inaugurated yesterday was a wild success.
Thousands of young illegal immigrants packed government centers across the nation Wednesday and sought help from volunteers on the first day they could apply to legally stay and work in the United States under a new federal initiative.
Under President Obama's most ambitious immigration program — officially called the Deferred Action for Childhood Arrivals — illegal immigrants between the ages of 15 and 31 who were brought to America before the age of 16 and have no criminal record are able to apply to remain in the U.S. for at least two years and work legally […]
"There was a lot of enthusiasm and hope," said Mary Meg McCarthy, the executive director of the Heartland Alliance's National Immigrant Justice Center, a Chicago-based immigration advocacy organization that helped organize the event at Navy Pier.
I like to report on things that involve enthusiasm and hope for a change. This is a very good story. So far, my fears that the $465 application fee would dampen the enrollment has been unfounded; the immigrants are finding the money. (If you have a million immigrants take this up – and the Migration Policy Institute lists 1.7 million as eligible – that's $465 million for the government. Does anyone think that the application process will cost that much?) I guess it's true that you cannot put a price on two years of relief and certainty, more than these kids have had in their entire lives.
It's also good to see Democratic officials working hard to help DREAM-eligible immigrants navigate the application process. Ultimately, that's what politics should be about, improving people's lives. And while this does not confer a path to citizenship, it does confer improvement. I'm sure there are political overtones to aiding the Hispanic community, considering their growth as a voting bloc. These kids are not eligible voters, however. And yet lots of Congressional Democrats are at the forefront of providing what amounts to social work for the DREAM-eligibles. I've seen more action aimed at getting these kids enrolled for the DACA program than I have seen enrolling, say, people with pre-existing conditions into the temporary high-risk pools in the Affordable Care Act.
Meanwhile, on the Republican side, Jan Brewer issued a vindictive executive order attempting to stop state compliance with the deferred action program. This ultimately is a toothless directive that will not stop anyone from anything; it just restates existing law in Arizona around granting driver's licenses and other benefits to non-citizens. But it shows the cruelty that has characterized a substantial segment of the Republican Party against a group of 1 million or more kids that know no other country than the US.
All you have to do is to look at these pictures to understand the importance of this program, which offers a glimmer of hope and two years of relief to these people. More from The Washington Post and the Las Vegas Review-Journal. Here's a representative story:
Because his well-intentioned mother smuggled him into the United States at the age of 2 on a commercial airliner, (Luis) Avila has been forced to forgo the sort of sacrosanct privileges that come with the coming of age: He can't drive to the movies on a Friday night because he can't get a license; he can't flip burgers because he can't legally work; he can't open a bank account because he doesn't have a valid form of U.S. identification.
The only piece of identification he does have is a card issued to him by the Mexican consulate. It has the name of his birthplace, Chilpancingo, Guerrero. It's a town he can barely pronounce, much less spell […]
"I've been waiting years for this. I can hardly believe it," said an emotional Avila, whose first revelation that he was different from the rest of his class came in the fourth grade, when he was asked to write down the name of his hometown and he couldn't answer.
This is enormous in the Hispanic press as well. A good day all around. I wish it had come sooner and these kids were on their second work permits already.


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Pi Solutions practice at Systech couples Pi Profit Analytics™ with its proven methodology and past experience to provide rapid increase profitability to clients in different industries.


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Rocky Mountain Research Station Publications
RMRS Online Publication - Journal
Articles, External Publications, and Special Reports
Resistance to invasion and resilience to fire in desert shrublands of North America
Brooks, Matthew L.; Chambers, Jeanne C. 2011. Resistance to invasion and resilience to fire in desert shrublands of North America. Rangeland and Ecology Management. 64: 431-438.
Settlement by Anglo-Americans in the desert shrublands of North America resulted in the introduction and subsequent invasion of multiple nonnative grass species. These invasions have altered presettlement fire regimes, resulted in conversion of native perennial shrublands to nonnative annual grasslands, and placed many native desert species at risk. Effective management of these ecosystems requires an understanding of their ecological resistance to invasion and resilience to fire. Resistance and resilience differ among the cold and hot desert shrublands of the Great Basin, Mojave, Sonoran, and Chihuahuan deserts in North America. These differences are largely determined by spatial and temporal patterns of productivity but also are affected by ecological memory, severity and frequency of disturbance, and feedbacks among invasive species and disturbance regimes. Strategies for preventing or managing invasive plant/fire regimes cycles in desert shrublands include: 1) conducting periodic resource assessments to evaluate the probability of establishment of an altered fire regime; 2) developing an understanding of ecological thresholds associate within invasion resistance and fire resilience that characterize transitions from desirable to undesirable fire regimes; and 3) prioritizing management activities based on resistance of areas to invasion and resilience to fire.
Keywords: Chihuahuan Desert, ecological resilience, ecological resistance, Great Basin Desert, Mojave Desert, Sonoran Desert
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Welcome to Nationwide Towing & Transport Pty Ltd
We provide towing & transport services 24 hours a day, 7 days a week for:
|Anything, Nationwide, Anytime – Safely
Nationwide Towing & Transport successfully operates as one of Australia's leading towing and transport companies. We have provided towing and transport services for over 25years. We complete over 300,000 tows per year, servicing a broad range of clients from one off transportation or relocation jobs through to contracted transport solutions, servicing major organizations such as Auto clubs, Police, Municipal Councils, Insurance and large scale private Equipment hire organizations.
Today Nationwide Towing & Transport operates out of offices in Victoria , Queensland, and Western Australia. We are a progressive company that is well established and growing every day.
Our goal is to further expand our services and expertise across the country.
We are a 'Can Do Company' and live by our motto – "Anything, Nationwide, Anytime – Safely".


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Dansko OlgaSKU: #8167324
Product Information
- Spruce up those grey and dreary days with the sleek and classy look of these Dansko® boots.
- Nappa leather upper features a modern ruching on the shaft.
- Pull-on construction offers a secure fit.
- Man-made lining and footbed are odor resistant and antimicrobial.
- Supportive, removable PU footbed that will never flatten out.
- Heel is designed for high-impact shock resistance.
- Durable rubber outsole.
- Imported.
- Dansko Footwear Size Chart
- Measurements:
- Heel Height: 1 1⁄4 in
- Weight: 13 oz
- Circumference: 13 3⁄4 in
- Shaft: 9 in
- Product measurements were taken using size 39 (US Women's 8.5-9), width Regular. Please note that measurements may vary by size.


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Fallout Tactics: Brotherhood of Steel
Random Encounter FAQ by PDillinger
Version: 1.1 | Updated: 04/06/01 | Printable Version
/------------------------------------\ < FALLOUT TACTICS RANDOM ENCOUNTER FAQ > \------------------------------------/ Version 1.1 April 6th, 2001 By : "Samurai" firstname.lastname@example.com Well, I believe that I've found most if not all of what's out there. If you find something that I don't have feel free to let me know. You can contact me at firstname.lastname@example.com Also, I created this FAQ to confirm all random encounters. I will not tell any of the funny things that happen. Some of what happens or what people say is very funny, so wander around looking for them yourself. There are some great items you can get or steal in these encounters, but I am not listing them yet. Play the game to see this stuff. I will list the encounter name as found on the map and a description of what it is. These are minor spoilers and you have been warned. TABLE OF CONTENTS -Updates -Finding Random Encounters -List Of Random Merchants 1. Hermit 2. Trader 3. Farmer 4. Merchant -List Of Encounters 1. The Pipboy 2. B1000 3. "" (The Eveready Bunny) 4. L33tists 5. Brahmin Poker 6. Amelia 7. Deathclaw Liberation 8. CPF vs. PFC 9. Canadian Invasion 10. Sixth Sense 11. Komodo Man 12. Four Horseman 13. Titanic 14. Invasion Recreationists 15. Bazaar Day 16. Brahmin Tipping 17. Uprising 18. Brothers Grimm 19. Coppertops 20. Phil 21. Mir 22. Pitch Black 23. Reaver Dance 24. Gas Station 25. Brahmin Armor 26. Morte -Shameless Plug /-------\ < UPDATES > \-------/ 1.0 Origional 1.1 Fixed a few errors Added Brahmin Armor Added the link to get the latest version of the FAQ /-------------------------\ < FINDING RANDOM ENCOUNTERS > \-------------------------/ I have found that these encounters don't seem to be quite random. They do happen in different places, but the grid on the map that they happen in seems to be pre-determined. So the only way that you will find these encounters is to explore. Whenever you come across a random encounter, try going through the same square again. Almost every random encounter I had, I was able to get another in the same square. Also, some random encounters ( "Deathclaw liberation", and "Pitch Black" come to mind ) are dangerous. Others are one time only events. If you watch it once you can't see it again. So I would recommend saving if possible before exploring any encounter. /----------------\ < RANDOM MERCHANTS > \----------------/ These encounters seems to be the same as any other except they always have someone that is willing to trade (for ring pulls) and they have a larger circle on the map (unlike the gas station) 1. Hermit 2. Trader 3. Farmer 4. Merchant /-----------------\ < RANDOM ENCOUNTERS > \-----------------/ One Time only events (They don't revert to normal after the encounter) are denoted with a * 1. The Pipboy * The guy from all of the skill/perk pictures. He's small but he carries a BIG gun. 2. B1000 Part brahmin, part terminator. 3. "" (The Eveready Bunny) This one is not labeled on the map. It is full of invisible People who seem to think that this bunny keeps going and going. 4. L33tists A clan of frag fanatics. 5. Brahmin Poker First dogs playing poker, now mutated cows. 6. Amelia She must have been REALLY lost. We're no where near the Atlantic. 7. Deathclaw Liberation * See pen filled with dethclaws. See wacko decide to set them free. See wacko run. Run wacko run. 8. CPF vs. PFC "Canadian Peoples Front vs. Peoples Front of Canada" 9. Canadian Invasion Guarding our future against those pesky Canadians. 10. Sixth Sense "I see dead brahmin." 11. Komodo Man * See what happens when a post apocalyptic crocodile hunter decides to molest a mutated komodo dragon. 12. Four Horseman The four horseman of the post-apocalypse. 13. Titanic The sketch he drew looked a bit different in the movie. 14. Invasion Recreationists See all the action of the invasion of Canada! 15. Bazaar Day Mmmm... Diablo armor... *drools* 16. Brahmin Tipping The greatest sport of the 22nd century 17. Uprising Those civilians could have been armed. 18. Brothers Grimm * Help a brother out! 19. Coppertops Fallout, meet the Matrix. Matrix, meet fallout. 20. Phil Bike, bike, bike, all day long. Delivering Nuka-Cola while I song this song. 21. Mir Yup. It's the space station all right. 22. Pitch Black It's dark and filled with danger. Have fun. 23. Reaver Dance They don't get any more dead. 24. Gas Station How is she still alive after all of these years? Hmmm... 25. Brahmin Armor I'll let you guess from the title. 25. Morte This seems to be the last encounter. I only got it after getting all of the others. I even got it about 5 times before I quit looking for random encounters. It's a dead man walking. Well, thanks about it. Enjoy... Also if you find something not on here e-mail me at email@example.com. /--------------\ < SHAMELESS PLUG > \--------------/ Visit www.motorcitysamurai.com I will post the latest version of this FAQ at http://www.motorcitysamurai.com/FOT_Random_FAQ.txt as well as http://www.gamefaqs.com This document Copyright 2001 Paul R. Dillinger You can freely distribute this document as long as no commercial transaction or modification is involved.


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Swagbucks vs Inbox Dollars. They are two of the leading online rewards clubs which offer opportunities to make money online through completing paid surveys, reading emails, playing games, searching and shopping online amongst other things, but which website is best? They both have a lot of similarities, but also differences. So rather like I did with Amazon MTurk or Microworkers, which is best? and Amazon MTurk vs Inbox Dollars, which is best? I thought that I would compare and contrast Swagbucks and Inbox Dollars, with a view to deciding which one is preferable for those who wish to make money online.
Swagbucks and Inbox Dollars Similarities
Both are website-based rewards clubs where you can make money online.
Both pay you to do paid surveys, play games, read emails, watch videos, shop online.
Both give you coupons that you can print off and save money with.
There is a Swagbucks toolbar and an Inbox Dollars toolbar that you can install and use to enhance your make money online experience.
Swagbucks and Inbox Dollars Differences
One big difference is that with Inbox Dollars, when you make you make money online it is in dollars and once you reach $30, they pay you cash in the form of a check, whereas with Swagbucks you earn virtual money which you then have to spend in their online shop, or exchange for amazon giftcards, or PayPal money.
Swagbucks is a bigger organization than Inbox Dollars and their number of users is larger than Inbox Dollars.
Swagbucks have an active official Twitter account too, which you can use to keep contact with them and get info on Swag codes. Inbox Dollars have no active official Twitter account.
Swagbucks generally is more interactive and community orientated, whereas Inbox Dollars users are more a collection of individuals with no forum or blog to link them up, like with Swagbucks.
Some of the Swagbucks paid surveys pay slightly better than the Inbox Dollars ones, in my experience.
Inbox Dollars give you $5 when you join, Swagbucks give you 50 swagbucks.
You do get more emails from Inbox Dollars than Swagbucks. My advice would be to do what I do and don't use your main email address when you join any of these sites. I have a specific email account that I set up specifically for use with all these make money online websites.
In my wife's opinion, Swagbucks has slightly more desirable gifts and rewards in their offers than Inbox Dollars does. I will have to take her word on that one.
I personally prefer Inbox Dollars to Swagbucks on balance, I like the fact that when you make money online, it is in dollars. I find the Swagbucks concept of giving you virtual money which you then have to exchange a little gimmicky, although I guess it doesn't really matter much in the long run, as you can convert the Swagbucks to Amazon gift cards or PayPal money, if you wish. My wife, on the other hand, is a huge fan of Swagbucks (although she still does Inbox Dollars as well, of course), so I guess that makes us a balanced couple!


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Nov 4th 2013 new
(quote) John-184825 said: After digressions that took this topic off course, I return to the original subject matter with some related questions.Career, relationships, losses, achievements, failures, responsibilities, the Holy Spirit , and life in generalall will reveal selfAs I take stock at certain milestonesand self examine to ascertain goals for ministry, relationship, career, etc.,up pops the good,the bad, and the ugly
Over the years, how has deeper insight about yourself contributed to your ability to have a fuller social life?
Over the years, how has deeper insight about yourself contributed to your ability to have a fuller social life?
So, over the years, any insight gained has enabled me to make others laugh, cry, and shake their heads in disbeliefI am a "basket case" or as certain friends here in the South say (in affection, at least that is why they tell me!)"You are a mess, just a mess!" And that, really is the true state of affairswhich is why I have no problem with the first part of our Mass that you mentioned, John, I am a "mess" without HimI know for sure after looking at what I bring to the table.It is ALL G.R.A.C.Egifts received at Christ's expense.
This self awareness has not applied in a "dating world" for meas a widow coming out of many years in married lifebut my social lifeas in my opportunities for conversation and contact with others in my community or profession is not encumbered by the fears and self consciousness of youthI enjoy mixing, sharing, and just socializing,,,and He covers me despite myself and all that awareness :-) Thank, God!


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I think Volquez could take Lohse...
Hairston draws a two out 12 pitch walk.
Bruce got ahead 3-0, took a couple called strikes and then grounded out. Lohse is at 93 pitches.
After 5 innings:
Reds 2, Cards 0
Volquez 55 pitches, 34 strikes
Lohse 93 pitches, 63 strikes.
Nice 12 pitch AB there by Hairston to draw the walk.
A healthy Reds team is a strong Reds team"
Games are won on run differential -- scoring more than your opponent. Runs are runs, scored or prevented they all count the same. Worry about scoring more and allowing fewer, not which positions contribute to which side of the equation or how "consistent" you are at your current level of performance.
Singles by Schumaker and Ludwick. First and second with only one out for Pujols. I have a bad feeling about this one.
One out.
Too bad the offense fell asleep because now we're about to be behind.
He just made Ankiel look stupid - that changeup is just N-A-S-T-Y
Ankiel K's. Big out.
Still loaded for Glaus.
Still got a bad feeling.
two HUGE k's by EV
Pure filth!
The Sox traded Bullfrog the only player they've got for Shottenhoffen. Four-eyes Shottenhoffen a utility infielder. They've got a whole team of utility infielders.
Board Moderators may, at their discretion and judgment, delete and/or edit any messages that violate any of the following guidelines: 1. Explicit references to alleged illegal or unlawful acts. 2. Graphic sexual descriptions. 3. Racial or ethnic slurs. 4. Use of edgy language (including masked profanity). 5. Direct personal attacks, flames, fights, trolling, baiting, name-calling, general nuisance, excessive player criticism or anything along those lines. 6. Posting spam. 7. Each person may have only one user account. It is fine to be critical here - that's what this board is for. But let's not beat a subject or a player to death, please.


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American Home Shield December Newsletter
Ask the Experts
The guest bedroom in our new house is in desperate need of a paint touch-up, but I have no record of the paint that was used. Is there any hope for us, or do we have to repaint completely?
- Cindy, Augusta, Ga.


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All posts by Jason Cohen
A Proto-Punk Revival At Woodward Theater
Cincinnati is just one of the 11 stops on the 50th anniversary tour of Pacific Northwest garage-rock legends the Sonics.Read more
Rosanne Cash at Music Hall
She joins the Pops for a weekend of collaboration.Read more
Flushed With Success?
The Cincinnati Cyclones' logo was as dated as the name "Firstar Center." Not anymore.Read more
The Glitzy Hire: Tommy Tuberville
Can Tommy Tuberville lead UC to the Promised Land of major-conference money?Read more
How I (Finally) Learned to (Stop Whining and) (sort of) Tolerate (Cincy's Infamous) Chili
A halfhearted (but sincere!) defense of the ethnic junk food America loves to hate.Read more
Recipe: A Cincinnati Short-Timer's Goetta
Back in 2009, we asked Jason Cohen to share his personal recipe for our other notorious local delicacy.Read more
Pardon our Slush
In 1996, the then–Riverfront Coliseum put on "what many believe was the worst Frozen Four ever," notes Miami RedHawks website Blog of Brotherhood.Read more
The Money Speaks
The Science Guy and the Creation Guy debate.Read more
He's Goetta Have it
How one man fell in love with our homemade mystery meat, and why he still can't get enough.Read more
The Rookie
The Washington Nationals have a history of scavenging the Reds for players and front office personnel. Now they've come for William Howard Taft. The infamously portly Cincinnati native has joined George Washington, Thomas Jefferson, Abraham Lincoln, and Teddy Roosevelt in the Presidents Race, D.C. baseball's less delicious version of Milwaukee's Racing Sausages. We caught up with the nation's 27th president via e-mail (he doesn't have an iPhone).Read more


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Time for a Drink: Marmalade Sour
While the practice of serving cocktails at breakfast is mostly relegated to history books and weekend brunches, putting breakfast-table ingredients into the cocktail shaker is a practice that's only picking up steam. Drinks containing eggs or egg whites are now fairly common at craft-cocktail bars; here's one that takes another familiar morning food and uses it to very good effect. More


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Department of Planning, Public Policy and Management
Department of Political Science
Environmental Studies Program
Ph.D. (1999) Political Science, Yale University
M. Phil (1994) and MA (1993) Political Science, Yale University
B.A. (1990) Mathematics and Government, Cornell University
Environmental politics and policy
Community-based conservation
Community-based forestry
Environment and development
Community-government relations
American political development
Political institutions
Federal forest management
Forest Service
Pacific Northwest and the American West
Resource Colloquium PPPM 690 (Fall 2007, Sp. 2008)
Environmental Politics (US)-PS 497/597
(Sp. 2006).
Intro. to Environmental Politics and Policy (US)-PS 297 (Sp. 2005)


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It is an indisputable fact that prisoners continue to use contraband cell phones to stay in contact with their criminal associates on the outside. But so far the federal government hasn't been willing to allow state prison officials to block those cell phone signals despite the capacity of technology to do so.
The case of former state prison Capt. Robert Johnson, who in 2010 was shot six times, underscores the necessity of keeping the pressure on the feds — specifically the Federal Communications Commission.
The "hit" on Mr. Johnson was believed to have been initiated by cell phone from the Lee Correctional Institute in Bishopville, where he worked. His lawsuit to have cellular providers held liable for his assault is currently on appeal.
The FCC has yet to acquiesce to the requests for jamming authority by prison officials across the nation. That's a pity. The technology has been improved so that jamming can be done very precisely in an area — like a prison — without interfering with other cell phone calls, including emergency calls.
So for now people like Mr. Johnson remain vulnerable to inmates who manage to get cell phones. Last year, around 3,000 illegal cell phones were confiscated in state prisons.
Meanwhile, William Byars, director of the S.C. Department of Corrections, has overseen alternate measures to try to stem the flow of contraband cell phones in prisons.
For example, prison officials are trying to work more closely with local law enforcement agencies — usually county police. Now, staff at the prisons and in local law enforcement agencies are communicating with each other, and sharing information that might be helpful.
Local police are also patrolling the perimeters of prisons more often to stop people from tossing cell phones, drugs and other contraband over the fence to inmates.
Further, prisons are now equipped with new, portable machines that detect cell phones. People who enter or leave prisons continue to be screened. But these new machines allow officials to go cell-to-cell and screen for cell phones.
Although a federal judge dismissed his lawsuit against 20 cellphone companies and cellular tower owners, Mr. Johnson is optimistic about his appeal. He contends that these companies had the ability to block inmate calls. His main obstacle is a 1934 law that allows only federal agencies to jam public airwaves, and the FCC hasn't made an exception for state prisons.
His obstacle is South Carolina's obstacle. The progress being made with help from local police authorities and new screening equipment is to be applauded.
But being able to jam illicit cell phone signals in prisons could put contraband phone crimes out of business.
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text: I-Like-Electric-Motors 5pts Drivers who cannot drive should give up their keys. The solution to those people who have no business being behind the wheel is not any high tech electronic gadget. It's a simple innovation carried over from the earliest wagons and it is called the "passenger seat." I do not need a computer to take over my driving. No thank you.


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Two-Slot OpenVPX Development Platform Supports Both 3U and 6U Cards
Elma Electronic Systems, a leading supplier of embedded products and systems solutions built on open standards-based platforms like CompactPCI, ATCA, VME and VPX, has released a new two-slot VPX/OpenVPX test and development platform that accommodates both 3U and 6U boards via a shelf divider. The E-Frame Series Test Platform can connect multiple backplanes to efficiently simulate various fabric topologies, eliminating the need for costly custom backplanes and allowing high speed signals to be passed from one slot to the next.
The new E-Frame Series enables developers to power up one or more VPX blades under test and interconnect the J1 fabric connections to emulate the user's application. Signals from an external device can also be introduced through the J1 fabric connector or accessed on the J1 fabric connector using the provided SMA and SATA cable headers.
The use of a standard VPX RTM (rear transition module) plugged into the back provides access to the J0, J2, J3, J4, J5 and J6 connectors, while simultaneously accessing high speed signals in the J1 connector, routed out the side of the backplane. Each slot's J1 "A" channel is broken out into 16 SMA connectors and the "B", "C" and "D" channels into four SATA2 cable headers (12 total per slot).
Designed for ease of use, the test platform features a 1.6" card pitch that enables easy cable access to components on either side of the board under test. A built-in LED voltage monitor provides bus voltage compliance, and VNAs (vector network analyzers) and other probes can be used on the surface of the VPX card under test for enhanced testing capabilities.
The new E-Frame Series measures 9U x 42 HP (8.4") x 11.73" and weighs only 18 lbs. Located on the rear of the chassis, power input voltage is 97 VAC to 264 VAC auto-ranging and power input frequency is 47 Hz to 63 Hz. Total power consumption is 580 W. The test platform operates from 0°C to +50°C at altitudes of up to 6,000 feet. It withstands shock of up to 10 Gs at 11 ms and vibration to 1 G at 10 Hz to 330 Hz in non-condensing humidity from 5% to 95%.
Optional Elma computing products compatible with the E-Frame Series include 3U and 6U OpenVPX SBCs; secure, rugged, NAS and RAID storage solutions; fabric network switches; and FPGA-configurable I/O solutions.
Delivery for an E-Frame Series Test Platform is two to four weeks through Elma's Express Program.